tag:blogger.com,1999:blog-368400632024-03-19T05:47:54.086+00:00HyperlipidYou need to get calories from somewhere, should it be from carbohydrate or fat?Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.comBlogger973125tag:blogger.com,1999:blog-36840063.post-72035523226296661692024-03-19T05:47:00.000+00:002024-03-19T05:47:21.452+00:00Foie Gras (6) inflammatory mRNAs<p> Okay, time to look at this study</p><div><a href="https://pubmed.ncbi.nlm.nih.gov/26404455/" target="_blank">A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue</a></div><div><br /></div>It presented itself to me while I was looking for explanations as to why linoleic acid was or wasn't inflammatory in hepatocytes. Being "inflammatory" is determined, here, by the response of treating freshly isolated cells, in this case macrophages, to incubation with linoleic acid at 50 μmol/l for 12 hours, in addition to the normal culture medium. Some ferreting around on the internet suggests that the culture medium used, when fresh, contained glucose at 33 mmol/l. The concentration of LA, 50μM, is low and physiological for a FFA concentration under hyperglycaemia. The macrophages were assessed for expression of pro-inflammatory genes coding for these proteins:<br /><br />Monocyte Chemoattractant Protein-1 (MCP-1)<br /><br />Macrophage inflammatory protein-1 (MIP-1)<div><br /></div><div>F4/80 is a gene for the surface marker of macrophages<br /><div><br /></div><div>and good old TNFα and Il6 get thrown around too.</div><div><br /></div><div>We can ignore the anti-inflammatory gene expression Ym1 and Ym2.</div><div><br /></div><div>This is what they found for pro-inflammatory gene mRNA relative expression, linoleic acid is tall hatched bars. It's a log scale:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh14yIWcpPOzsvislNXx-p8Mv66-4UQa2v8lGukYQucPHINJBDJXa1c3u3BIirtMHK_s4R39-sqD38ICWbtCBBWBUXMUQHZtwVjejkEf8yiu-KBrRQqGdcegyWnq9OapIZf_GKDtzZJD3-Ajeyu3hiX9MFpWuya3oxlfeUnZlzdHunQw8_i7o1m7Q/s656/imflammatory%20cell%20culture.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="419" data-original-width="656" height="255" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh14yIWcpPOzsvislNXx-p8Mv66-4UQa2v8lGukYQucPHINJBDJXa1c3u3BIirtMHK_s4R39-sqD38ICWbtCBBWBUXMUQHZtwVjejkEf8yiu-KBrRQqGdcegyWnq9OapIZf_GKDtzZJD3-Ajeyu3hiX9MFpWuya3oxlfeUnZlzdHunQw8_i7o1m7Q/w400-h255/imflammatory%20cell%20culture.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br /><br /><br /><br /><div><br /></div><div>So we can say LA is pro-inflammatory in this model. No one in obesity research wants to find that the cardiological darling and cholesterol lowering PUFA are pro-inflammatory. I told you they were rank amateurs. Anyway, they developed another model, in-vivo this time, which got the correct results.</div><div><br /></div><div>They fed the mice the diets I <a href="https://high-fat-nutrition.blogspot.com/2024/03/foie-gras-5-aside-on-how-to-stay-slim.html" target="_blank">mentioned in the last post</a> for four weeks then looked at pro-inflammatory gene expression in both white adipose tissue and liver tissue immediately after euthanasia.</div><div><br /></div><div>This was much more satisfactory. In both tissues the high PUFA diet was not inflammatory, with a trend towards it being anti-inflammatory in adipose tissue:</div><div><br /></div><div>Adipose, from Fig 2:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhQyjfQrxoWvn8a4Wi80YxE-1YgMrzDK2friTBkGIrHTJGf8pu4ysXpvQtTDAQ2Dgmc3bOuKPw7PicXJ6tM1F57ElISBXEUy7zjI9TGsz4PRW8GH104xz-Rt5pWV_4tY4HfhhE1udpEsE-jZEoIFKk2BS8PJazFq2lBtXoD9kReJq351QTD-_mhvw/s1504/WAT%20infl%20labelled.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="472" data-original-width="1504" height="125" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhQyjfQrxoWvn8a4Wi80YxE-1YgMrzDK2friTBkGIrHTJGf8pu4ysXpvQtTDAQ2Dgmc3bOuKPw7PicXJ6tM1F57ElISBXEUy7zjI9TGsz4PRW8GH104xz-Rt5pWV_4tY4HfhhE1udpEsE-jZEoIFKk2BS8PJazFq2lBtXoD9kReJq351QTD-_mhvw/w400-h125/WAT%20infl%20labelled.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and liver from Fig 3:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEij_j31PdLw13gEpyM0YjVV8OcvVUDIkhFDsTCMccP8RaPH7rvrqGXrg7fHKzpZUVZJA_-GUVUGmwbrHLJuj5TH4mrrhUVw7i0wwXJXUpSnXH0EyxgZs0TLoSbC5OJBqLi5cAbiJzz72jhu4nSecEM_AKbaXsp_T8GcMunufVvq6s1u1vNj6G8ygQ/s1504/liver%20annotated%20pufa.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="475" data-original-width="1504" height="126" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEij_j31PdLw13gEpyM0YjVV8OcvVUDIkhFDsTCMccP8RaPH7rvrqGXrg7fHKzpZUVZJA_-GUVUGmwbrHLJuj5TH4mrrhUVw7i0wwXJXUpSnXH0EyxgZs0TLoSbC5OJBqLi5cAbiJzz72jhu4nSecEM_AKbaXsp_T8GcMunufVvq6s1u1vNj6G8ygQ/w400-h126/liver%20annotated%20pufa.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>However, there is another snippet of information also available in Fig 3. The activity of myeloperoxidase system tells us slightly more than the relative mRNA for inflammatory genes do in the bulk of the figures. Myeloperoxidase tells us whether the WAT cells in Fig2 are actually using the pro inflammatory genes to produce inflammation. This is what they found. The chart is split in two because the HF (high fat diet, something like D12492) was so activating it was an order of magnitude higher than all of the others, so needed it's own scale:</div><div><br /></div><div><br /></div><div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/a/AVvXsEiF_jwFQZM8cSuSRdUvzdJgHNM196e3qH9sa8aTGzLgtr7kRXoviBHxdEncFtPKfeof-54jqxhaMSexEpoSVaNJpbZRZsHSFHxgRq1FsNLQAv0fGuPvA92APZoGYPU3n6nUz--JHPaMmcYBdqFdh04in_HgEX9_JldooAFt-lVT59c12MPyRkMCxA" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img alt="" data-original-height="345" data-original-width="453" height="305" src="https://blogger.googleusercontent.com/img/a/AVvXsEiF_jwFQZM8cSuSRdUvzdJgHNM196e3qH9sa8aTGzLgtr7kRXoviBHxdEncFtPKfeof-54jqxhaMSexEpoSVaNJpbZRZsHSFHxgRq1FsNLQAv0fGuPvA92APZoGYPU3n6nUz--JHPaMmcYBdqFdh04in_HgEX9_JldooAFt-lVT59c12MPyRkMCxA=w400-h305" width="400" /></a></div><br /><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>I think we have to be very, very careful about what we mean by "inflammation". In fact I might just take a brief pause here and explain what I think "inflammation" means, before running through what is happening in this study. That may need some cellular physiology rather than mitochondrial physiology.</div><div><br /></div><div>Here's my conundrum:</div><div><br /></div><div>CVD is an "inflammatory" condition, sic.</div><div><br /></div><div>The most potent anti-inflammatory agents available to modern medicine are the corticosteroids.</div><div><br /></div><div>Corticosteroids make CVD much, much worse.</div><div><br /></div><div>Somebody, somewhere, has an odd idea about what "inflammation" might be.</div><div><br /></div><div>With apologies for the hiatus.</div><div><br /></div><div>Peter</div></div></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com0tag:blogger.com,1999:blog-36840063.post-87379005088967724922024-03-18T08:54:00.000+00:002024-03-18T08:54:12.927+00:00Foie Gras (5) An aside on how to stay slim<p> Another one-liner:</p><div><a href="https://pubmed.ncbi.nlm.nih.gov/26404455/" target="_blank">A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue</a></div><div><br /></div><div>Now, I like this paper. They found some weird stuff which is fully Protons compliant. That's not today's post. I just wanted to share this graph:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgO9wVG5yZ-jYlZU8kizOmCbTAN18cBE8C9baOSde3BcvmtZw298Bo5OCmLSjCZ058u_ajl0TS6dvdv28O82Dm5KwW5m968Nljgo9Pr826DRojFl2SyqnRv05CKjzOXZZX-gI8tU1NvhcoOCFr40l6F3cLt29h3uZdIkBUMmOebJL9Uy3GeLgWh8g/s1129/Weight%20gains%20LA.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="816" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgO9wVG5yZ-jYlZU8kizOmCbTAN18cBE8C9baOSde3BcvmtZw298Bo5OCmLSjCZ058u_ajl0TS6dvdv28O82Dm5KwW5m968Nljgo9Pr826DRojFl2SyqnRv05CKjzOXZZX-gI8tU1NvhcoOCFr40l6F3cLt29h3uZdIkBUMmOebJL9Uy3GeLgWh8g/w289-h400/Weight%20gains%20LA.png" width="289" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>These people are clearly rank amateurs rather than hardcore obesity researcher, they have utterly failed to "improve" the saturated fatty acid (SFA) diet to make it obesogenic. Want to stay slim? Cocoa butter. As good as chow, and who would want to eat chow? Though I'd personally choose beef fat.</div><div><br /></div><div>The linoleic acid diet is also not very fattening. Hmmmmm.</div><div><br /></div><div>Even without looking at Table 1 you absolutely know what has been done, both to get the low weight gain on LA and to eliminate the inflammatory changes in the liver (which are there in cell culture).</div><div><br /></div><div>Hard to decide whether to go through this study or start on the mitochondrial data from the hungry Italian rats. Or maybe go to Winks Meadow to see if the Green Lipped orchids are up and flowering yet. </div><div><br /></div><div>The sun is shining.</div><div><br /></div><div>Peter </div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com6tag:blogger.com,1999:blog-36840063.post-85249168149055858452024-03-18T05:42:00.001+00:002024-03-18T05:56:34.282+00:00Foie Gras (4) RERA quick one-liner from the Italian rats in<br /><br /><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663608/" target="_blank">Fat Quality Influences the Obesogenic Effect of High Fat Diets<br /></a><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3XlnXzfwxsse2AqIsgFTW9zT1NUaSJIpwFj2lf7RCF7hBR6070iyGr_Op9nK_zdm3aDlnwDyZznZiGrVkdR3tKTnhQSwkIiuxYMlpzanmA9jDred8vS8I-5eQ3apIpkEZuKwvvFvCgwGlxsH5Qb7ruFjRTPrDLfeW6-S0aC1d8stKV2Seibf_pg/s726/italian%20rq.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="517" data-original-width="726" height="285" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3XlnXzfwxsse2AqIsgFTW9zT1NUaSJIpwFj2lf7RCF7hBR6070iyGr_Op9nK_zdm3aDlnwDyZznZiGrVkdR3tKTnhQSwkIiuxYMlpzanmA9jDred8vS8I-5eQ3apIpkEZuKwvvFvCgwGlxsH5Qb7ruFjRTPrDLfeW6-S0aC1d8stKV2Seibf_pg/w400-h285/italian%20rq.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>These are the RERs of the rats on the last day of the study. The horizontal lines are the food quotients calculated from the macros of the diets. Under stable conditions 24h RER should equal FQ. These are not stable conditions</div><div><br /></div><div>It's clear that both groups of rats are catabolising protein for energy in excess of what is present in the diet. More so for the lard diet than the safflower/linseed. They are, undoubtedly, hypocaloric.</div><div><br /></div><div>The lard based diet rats fail to oxidise lipid despite it being in the diet because they are still sequestering lipid in to adipocytes. Basal lipolysis may be high but hyperinsulinaemia (not measured) from incipient metabolic syndrome is recycling some of that lipid back in to adipocytes. Where it's not being oxidised. The rats are cold and hungry.</div><div><br /></div><div>The safflower/linseed group have normal fat oxidation, ie equal to FQ, because fat is not being lost in to adipocytes, it's being lost by uncoupling so lipid oxidation on RER is normal. It may provide nothing but heat but the RER looks good. Of course these rats are warm and hungry.</div><div><br /></div><div>I like it.</div><div><br /></div><div>Peter<br /></div><div><br /></div><div>Edit, I don't like the rats being hungry! Just the RER data. End edit.</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com0tag:blogger.com,1999:blog-36840063.post-73409852104302606312024-03-18T04:40:00.002+00:002024-03-18T05:01:12.459+00:00Foie Gras (3) The Japanese mice<div><div><div>So the interesting question about the rats in the Italian study which were fed on the Safflower/linseed oil diet is:</div></div></div><div><br /></div><div>How calorically restricted were they?</div><div><br /></div><div>In the absence of a control group allowed ad lib high fat intake (or even one fed chow) we will have to look elsewhere to estimate this. Japan is a good start with this paper:<br /><br /></div><div><a href="https://pubmed.ncbi.nlm.nih.gov/30192428/" target="_blank">Voluntary Corn Oil Ingestion Increases Energy Expenditure and Interscapular UCP1 Expression Through the Sympathetic Nerve in C57BL/6 Mice</a></div><div><br /></div><div>Just give Bl6 mice a free choice to munch chow and/or drink corn oil and they will eat/drink progressively more corn oil over 4 weeks until they are stable at ~75% of their calories from corn oil, 25% from chow. Which gives us in the region of 38% of calories from linoleic acid. They are weight comparable to chow-only fed rats throughout, maybe slightly lighter:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjE73GT7jq5Dz-UlSJNijXQObpJWvMrglVl6WoF-W8N_qRXwvnTghZqn4tTOpY4IPQ3c4TZIksasCHiRxx23nJ5ZKQKIH6OXbY6ffHFjgD88RTfmrCKz24FVcF0We1UncK7kr0C_OOuH5pw6v3vAze9PPULMLCOwJ0lau0aLG0Aiu4cyEYrBEk5Ug/s515/ad%20lib%20CO%20weights.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="491" data-original-width="515" height="381" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjE73GT7jq5Dz-UlSJNijXQObpJWvMrglVl6WoF-W8N_qRXwvnTghZqn4tTOpY4IPQ3c4TZIksasCHiRxx23nJ5ZKQKIH6OXbY6ffHFjgD88RTfmrCKz24FVcF0We1UncK7kr0C_OOuH5pw6v3vAze9PPULMLCOwJ0lau0aLG0Aiu4cyEYrBEk5Ug/w400-h381/ad%20lib%20CO%20weights.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Under stable conditions (weeks six to eight) they feed themselves 35% extra total calories to maintain an identical bodyweight to the chow fed mice:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgzoe8vAweRKEgMf4bWlX6zj6gt28RzwCZsPeQPXpb75-iy1fEs2tfUuym-G-MVrsqXJFFX1WsvxDXkZP4HnaawEwF3TwbDt9Ijs6WocVcg5kX_iMKiAm-5adp_3e1iBzH4synW60kwkioP21MYnYUkFcnBwrylMHYYVL9pE74EC8t_nf3d_y-kuA/s651/ad%20lib%20CO%20calories.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="651" data-original-width="591" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgzoe8vAweRKEgMf4bWlX6zj6gt28RzwCZsPeQPXpb75-iy1fEs2tfUuym-G-MVrsqXJFFX1WsvxDXkZP4HnaawEwF3TwbDt9Ijs6WocVcg5kX_iMKiAm-5adp_3e1iBzH4synW60kwkioP21MYnYUkFcnBwrylMHYYVL9pE74EC8t_nf3d_y-kuA/w364-h400/ad%20lib%20CO%20calories.png" width="364" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This is because they were uncoupling, primarily in interscapular brown adipose tissue but (from other papers, a future post) they also do so in white adipose tissue.</div><div><br /></div><div>Back to<br /><br /><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663608/" target="_blank">Fat Quality Influences the Obesogenic Effect of High Fat Diets</a><br /><br />If we imagine the Italian rats had been offered ad-lib access to the safflower/linseed diet we could expect them to eat somewhere in the region of 380kJ x 135%, so around 500-530kJ/d.</div><div><br /></div><div>They got fat on just 380kJ/d. Like the lard fed rats. But by two weeks they were a bit less fat (p<0.05).</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjqAchDlDTnVfkPwFq6LUd5JdDb2F6nQdniitZt1tkaVhB7Yc-Bq0u4cOt4CYzmsjYFrYsavfiLkgXuZtgtbgGM8OJt7ALp5IQ-h5ISi1bxdvdyjn3nS9zQxxiaLUAs7UlIzsYV7pRTAY1AvTvjbaiHPljNTMxclYBAomc-5ABKF3pcRMreIr3NZw/s569/saff%2014%20days%20lipids.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="507" data-original-width="569" height="356" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjqAchDlDTnVfkPwFq6LUd5JdDb2F6nQdniitZt1tkaVhB7Yc-Bq0u4cOt4CYzmsjYFrYsavfiLkgXuZtgtbgGM8OJt7ALp5IQ-h5ISi1bxdvdyjn3nS9zQxxiaLUAs7UlIzsYV7pRTAY1AvTvjbaiHPljNTMxclYBAomc-5ABKF3pcRMreIr3NZw/w400-h356/saff%2014%20days%20lipids.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>How does this work?</div><div><br /></div><div>On day one of the safflower/linseed diet there is a marked increase in insulin sensitivity, by the standard Protons/polyunsaturate mechanism, with an associated hypocaloric episode as calories poured in to adipocytes but no additional food was provided. There is no significant increase in uncoupling immediately.</div><div><br /></div><div>But now we have an insulin sensitive liver and the standard response of the liver to ingress of excess non-carbohydrate calories is to signal, using FGF21, to BAT to induce uncoupling giving thermogenesis and calorie disposal. </div><div><br /></div><div>The time scale for onset of uncoupling could be estimated if we had daily food intakes and body weights, but we don't, so let's just guess at around a week.</div><div><br /></div><div>As uncoupling in white adipocytes kicks in they will become poorly able to respond to insulin with the correct ROS signal, so insulin signalling decreases and they release FFAs. At the same time as this suppressed insulin signalling occurs BAT will be disposing of bulk calories by thermogenesis and the caloric drive for the pancreas to secret insulin also drops, again assisting lipolysis.</div><div><br /></div><div>By two weeks there is active, on going weight loss from an obese baseline. Lipid is being lost at an excessive rate accentuated by hypocaloric eating and the liver is dealing with this excess, under hypoinsuliaemia, in part via BAT and in part by the peroxisomal <a href="https://high-fat-nutrition.blogspot.com/2024/03/foie-gras-1-peroxisomes.html" target="_blank">mechanism described in the previous post</a>, the cost of which is, in rats (and mice), of hepatic lipid accumulation.</div><div><br /></div><div>Would the lipid accumulation have occurred if the Italian rats had not been calorically restricted?</div><div><br /></div><div>No.</div><div><br /></div><div>Uncoupling made the Japanese rats hungry, they ate extra to stop pathological weight loss. The extra calories include some carbohydrate and would have slowed lipolysis by raising insulin secretion to a level where lipolysis did not overload the liver. There are several papers to cover this in future posts.</div><div><br /></div><div>Given long term ad-lib access to an uncoupling diet based on PUFA the rats would have eventually and gradually lost weight until they matched bodyweight with their non existent control group. Assuming these mice are anything to go by, who did it over a period of 10 weeks. From here:</div><div><br /></div><div><div><a href="https://pubmed.ncbi.nlm.nih.gov/20213045/" target="_blank">Prevention of diet-induced obesity by safflower oil: insights at the levels of PPARalpha, orexin, and ghrelin gene expression of adipocytes in mice</a></div><div><br /></div></div><div>I plotted the numbers for the body weights in Table 2, by eye, in PowerPoint. The dashed lines are the ones to follow. Obese on ad-lib lard, back to control (low fat, 35% of calories from sucrose) mouse weight on ad-lib safflower oil based diet giving LA at 35% of calories:</div><div><br /></div><div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0A5Z_xDrXyU9VBP7_CExqmc2_2b_rBDUgkZWNRsWfu673pOCY4nQVHZgZl8sp3tAWtv42TeNZFBKp6txdsnVV4yB5AFlp2RXYVDkAZed_U4C97BivIapXJbIV6L0nE38qbGzwqTxtBDm51D7KThyuO48QkxGyZVhYJzPBmanGLldklXDBVAmXWQ/s1397/dashed%20safflower.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="729" data-original-width="1397" height="209" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0A5Z_xDrXyU9VBP7_CExqmc2_2b_rBDUgkZWNRsWfu673pOCY4nQVHZgZl8sp3tAWtv42TeNZFBKp6txdsnVV4yB5AFlp2RXYVDkAZed_U4C97BivIapXJbIV6L0nE38qbGzwqTxtBDm51D7KThyuO48QkxGyZVhYJzPBmanGLldklXDBVAmXWQ/w400-h209/dashed%20safflower.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Okay, that will do for today.</div></div><div><br /></div><div>I would not, in any way, endorse drinking either varnish or safflower oil, even if they produce weigh normalisation by what are, to me, metabolically convincing/plausible mechanisms.</div><div><br /></div><div>Better not to make your adipocytes pathologically insulin sensitive, then you wouldn't need to address the obesity with potentially pathological double bonds in your food.</div><div><br /></div><div>Probably safflower oil and hepatic inflammation next.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com0tag:blogger.com,1999:blog-36840063.post-41858625520904389632024-03-16T08:25:00.002+00:002024-03-16T18:04:06.060+00:00Foie Gras (2) Lard fed rats<div>There is too much in the Italian rat study for a single post. Here's the easy bit discussing the rats fed the lard based diet.</div><div><br /></div>I guess everyone is familiar with this graph in Figure 1 of this paper from the Schwartz lab:<br /><br /><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248304/" target="_blank">Obesity is associated with hypothalamic injury in rodents and humans</a><br /><br />which provides this gem. These are the *daily* caloric intakes of rats on bog standard lab chow, in grey, or during the sudden onset of feeding lard based D12492, in black:<br /><br /><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjIchLoarERpYuAtLXQmycaCtT_PdSNwYvX6_y4IO_GcBHjJ7QmVkCAIFpSxCIP3Z6D1Bl2LjtSbK41IC7nwEIvzYn6y2lk5NV6Fi85BCf4itq4EGQ7vHrGt1_T9ahqEmXgK00PjbfrU8leWVJMl-h8WFXdfLmtwi0CgA-hgT_iOPIo7C6ALrYooA/s1129/Schwartz%20food%20intake.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1120" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjIchLoarERpYuAtLXQmycaCtT_PdSNwYvX6_y4IO_GcBHjJ7QmVkCAIFpSxCIP3Z6D1Bl2LjtSbK41IC7nwEIvzYn6y2lk5NV6Fi85BCf4itq4EGQ7vHrGt1_T9ahqEmXgK00PjbfrU8leWVJMl-h8WFXdfLmtwi0CgA-hgT_iOPIo7C6ALrYooA/w396-h400/Schwartz%20food%20intake.png" width="396" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>These are Long-Evans rats weighing 300-350g. I drag this up because Protons suggests that on day 1 of exposure to D12492 the rats immediately sequester approximately 70kcal of energy in to adipocytes (ignoring processing losses) while still maintaining the basal (when chow fed) intake of 75kcal which are needed to run the rat's metabolism.</div><div><br /></div><div>This immediate significant fat gain on day one (seen in section F of the same figure) raises adipocyte diameter, so increases basal lipolysis, so reduces hunger, such that on day two the extra food intake needed is lower and eventually, by day seven, excess food intake is no longer significantly increased and by day 14 it is normalised. Simple, yes?</div><div><br /></div><div>Now let's return to the current study of interest discussed by Tucker, which I think of as the "Italian" study:</div><div><br /></div><div><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663608/" target="_blank">Fat Quality Influences the Obesogenic Effect of High Fat Diets</a></div><div><br /></div><div>This used Sprague-Dawley rats weighing 250g, so probably more actively growing than the rats in the Schwartz lab study. They were measured as consuming 90kcal/d (380kJ/d) of chow assessed over the time before the study started. So we can plot the food intake of rats in the Italian study on a modification of graph H of the Schwartz study. I've left the chow fed line from the Schwartz study as an imaginary chow fed control line which was omitted by this group and I've renumbered the y axis to reflect the values of caloric intake actually reported in the Italian study:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjmvFXu06ewoJU2QIynmwr4CLATaKw9pnGaspbEJS68qrLWeVXhMROtmIITFJCaC7NQNJazBNRIxJpNZXUaUV0YFE2Rit53fihuckgU-dL6ffmjtG0gf1jijSJKwCjHRphzP6sZFwLS6ExDY101wBVfBYxXIxm7fwbnhAtfqI59aF0QWuYvh1l__g/s1129/fat%20type.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1120" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjmvFXu06ewoJU2QIynmwr4CLATaKw9pnGaspbEJS68qrLWeVXhMROtmIITFJCaC7NQNJazBNRIxJpNZXUaUV0YFE2Rit53fihuckgU-dL6ffmjtG0gf1jijSJKwCjHRphzP6sZFwLS6ExDY101wBVfBYxXIxm7fwbnhAtfqI59aF0QWuYvh1l__g/w396-h400/fat%20type.png" width="396" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>We know that the red line for the lard fed group is close to correct from the methods section:</div><br />"Rats were divided in two groups with the same mean body weight (250 ± 5 g) and were pair fed with 380 kJ <span style="color: red;">[90kcal]</span> metabolisable energy (ME)/day (corresponding to the spontaneous energy intake of the same rats, that was assessed <span style="color: red;">[on chow]</span> before the start of the experiment) of a lard-based (L) or safflower-linseed oil based (S) diet for two weeks."<div><br /></div><div>The Italian rats, without any access to the luxurious amounts of D12492 provided to the Schwartz rats, still got fat. </div><div><br /></div><div>If you view this from my perspective this is not surprising. The Italian rats still lost calories in to adipocytes but, without access to extra food, had an hypocaloric crisis. They ended up with a reduced percentage of protein mass in their carcass, despite an increased fat mass. Obese and sarcopaenic.</div><div><br /></div><div>However, by two weeks on a diet which sequesters lipid in to adipocytes at the cost of reduced growth, a few of the rats will have achieved a sufficient increase in basal lipolysis to normalise hunger, as per the Schwartz rats, at the "cost" of obesity. It is very simple to multiply 380kJ/d by 14 days and get 5320kJ of offered food over 14 days. The mean of the actual food intake over the study was measured as 5286kJ in total for the lard fed rats. We don't have individual data but any rat still hungry on day 14 will have eaten all of its 380kJ, but no extra, because none was on offer. Adequately obese rats will have, via increased basal lipolysis, not needed to eat all of the 380kJ offered, ie these rats will have slightly reduced the mean total energy intake, by 34kJ, probably in the last few days of the study.</div><div><br /></div><div>These rats are in a difficult position, they must maintain an adequate fat mass for increased basal lipolysis to offset increased insulin mediated lipid sequestration induced by the linoleic acid component of their diet. As they grow they will have to increase fat mass to maintain adequate basal lipolysis to function.</div><div><br /></div><div>Running on basal lipolysis derived FFAs at a time when you have access to dietary glucose is the basic definition of metabolic syndrome.</div><div><br /></div><div>If you keep adipocytes small by forcibly keeping insulin low (ie caloric restriction) you will completely side step increased basal lipolysis, side step insulin resistance, side step or delay many diseases and *increase* the duration of the miserable, hungry existence which will be your extended life.</div><div><br /></div><div><div><a href="https://pubmed.ncbi.nlm.nih.gov/25313149/" target="_blank">The Influence of Dietary Fat Source on Life Span in Calorie Restricted Mice</a><a href="#"></a><br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/33440166/" target="_blank">An isocaloric moderately high-fat diet extends lifespan in male rats and Drosophila</a></div><div><br /></div><div>As regards the generation of a fatty liver:<a href="https://pubmed.ncbi.nlm.nih.gov/33440166/" target="_blank"><br /></a><br /></div></div><div>There is no way in which you can transfer sufficient the FFAs from insulin sensitive but non-adequately distended adipocytes to hepatocytes as is needed to maintain a fatty liver. Caloric restriction is highly protective. Just ask any obesity researcher, the cure for fatty liver is hunger. Oops, I mean weight loss rather than hunger, in obesity doublespeak.</div><div><div><br /></div></div><div>Safflower/linseed oil next.</div><div><br /></div><div>Peter</div><div><br /></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com0tag:blogger.com,1999:blog-36840063.post-55597435000565244082024-03-13T11:58:00.002+00:002024-03-13T13:28:31.681+00:00Foie Gras (1) PeroxisomesJaromir <a href="https://twitter.com/mct4health/status/1767320286913860085" target="_blank">posted this link</a> on Twitter/X. It's excellent groundwork for further discussions relating to the study <a href="https://tuckergoodrich.substack.com/p/hello-can-we-have-your-liver-understanding" target="_blank">cited by Tucker</a> in rats and the hepatic lipidosis in mice <a href="https://twitter.com/CaloriesProper/status/1766913400037126252" target="_blank">mentioned by Bill Lagakos</a>.<br /><br /><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102918/" target="_blank">Fasting induces hepatic lipid accumulation by stimulating peroxisomal dicarboxylic acid oxidation</a><div><br /></div><div>which can be summarised in this picture, modified very sightly to remove their drug manipulations. Each step was validated by a set of experiments, it looks to have been a large part of several people's lives over several years:</div><div><br /></div><div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/a/AVvXsEhZAbnZM5myY2bs_WY3Q1YROkcNozbBovdLZcCrgFRaEnFJXNUBak2ZnagcKxjaA1UDjjlZdKqKwJFbOLuyxQ10g3QGYk4C0XyP4xwbXdVWvXNA-oXH5Cuic996ssx0l4uV_6XTQlR1tl3E_fyS9__FFWL-h1nzdAOQvw83jMz1o8O0OOnzuQ2HWQ" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img alt="" data-original-height="542" data-original-width="288" height="400" src="https://blogger.googleusercontent.com/img/a/AVvXsEhZAbnZM5myY2bs_WY3Q1YROkcNozbBovdLZcCrgFRaEnFJXNUBak2ZnagcKxjaA1UDjjlZdKqKwJFbOLuyxQ10g3QGYk4C0XyP4xwbXdVWvXNA-oXH5Cuic996ssx0l4uV_6XTQlR1tl3E_fyS9__FFWL-h1nzdAOQvw83jMz1o8O0OOnzuQ2HWQ=w213-h400" width="213" /></a></div><br /><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This looks very, very much like a protective mechanism, put in place within hepatocytes (which are the final port of call for FFAs when the system is overloaded from excess lipolysis, think of ethanol or fructose acting on adipocytes) to protect them from potentially lethal damage from excess beta oxidation derived ROS.</div><div><br /></div><div>It's executed by peroxisomes. Anyone who is still shamefully unaware of Dave Speijer's ideas of the role of peroxisomes in the protection of LECA (last eukaryote common ancestor) from ROS damage should go and read <span> </span><br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/24864314/" target="_blank">How to deal with oxygen radicals stemming from mitochondrial fatty acid oxidation<br /></a><br />Ultimately, when the liver cells are flooded with FFAs the peroxisomes respond* by producing DCAs, di-carboxylic acids. This is basically taking a FFA such as palmitic acid and sticking, by a specific process, a second carboxyl group on to the omega end. This is used a signal to increase peroxisomal oxidation to offload calories without generating the high delta psi which would damage mitochondria. Shortening DCAs ends up with the dicarboxylic acid succinate (HOOC.CH<sub>2</sub>.CH<sub>2</sub>.COOH) which is exported to mitochondria where it increases the NADH:NAD+ ratio resulting in the generation of inhibitory metabolites which divert FFAs from beta oxidation to stored triglycerides, fatty liver.</div><div><br /></div><div>All that is missing from the story is the role of ROS.</div><div><br /></div><div>*I have absolutely no data on this but, if you expect the signal for peroxisomes to generate dicarboxylic acids by omega oxidation is going to be anything other than mitochondrial derived ROS, then I cannot help you.</div><div><br /></div><div>Peroxisomal beta oxidation generates hydrogen peroxide (well duh). And we can guesstimate how much H<sub>2</sub>O<sub>2</sub> is being produced in hepatocytes under these circumstances. Enough to replace the missing insulin when a rat is being fasted. Never forget that insulin is merely a superficial overlay over the ROS signal. If you want to get damaging levels of FFAs out of the vicinity of mitochondria you need to divert them to be stored as inactive triglycerides. That requires activation of the "insulin" signalling pathway, mediated by H<sub>2</sub>O<sub>2</sub>. Without insulin all you need are the ROS. You can buy hydrogen peroxide in a bottle (here working on adipocytes) which, at an extracellular concentration of ~1mmol, will act to provide what <a href="https://www.jbc.org/article/S0021-9258(19)42932-3/pdf" target="_blank">we describe as peak insulin signalling</a>:</div><div><br /><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgo8sbv-tihObZdTpiVFpKIaJrbI4Tt4pB7TrhCMuxZ5kPAYo9s3Epbae9-3yqlTtXYlkOqET0HufBO7NSkWFQM42E-iihWbSdFEIR3AxGFxzRA1d-1dOoqHS7nFK5sfq8EJblVA/s1600/glucose+H2O2.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="682" data-original-width="825" height="330" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgo8sbv-tihObZdTpiVFpKIaJrbI4Tt4pB7TrhCMuxZ5kPAYo9s3Epbae9-3yqlTtXYlkOqET0HufBO7NSkWFQM42E-iihWbSdFEIR3AxGFxzRA1d-1dOoqHS7nFK5sfq8EJblVA/s400/glucose+H2O2.png" width="400" /></a></div><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /></div><div>Equally, you can evolutionarily generate these insulin equivalent levels of H<sub>2</sub>O<sub>2</sub> from peroxisomes whenever FFAs are too high and insulin is too low. It's a safety net.</div><div><br /></div><div>Which can go wrong.</div><div><br /></div><div>It does go wrong, spectacularly so, in cats.</div><div><br /></div><div>Just imagine a slightly more obese than normal domestic moggy, fed on chicken fat and starch (better known as Crapinabag). It never goes without eating for more than a few hours because a) there is dry "kibble" available at all times and b) the cat is hungry all the time c) whenever it eats, a proportion of its food is lost in to its adipocytes, keeping it hungry. That's why it's fat.</div><div><br /></div><div>Now drop its level of insulin to basal fasting levels for three days. Maybe a road traffic accident, getting shut in a garden shed, having an acute infection, anything which stops it eating. It becomes hypoinsulinaemic. You can even do it by being too aggressive when putting your porky cat on a weight loss diet, without needing complete fasting.</div><div><br /></div><div>It will do lipolysis. It has a huge fat mass. It will *really* do lipolysis. The hepatocytes are going to get massively overloaded with FFAs. The peroxisomes will kick in and protect the liver by signalling conversion of dangerous FFAs to harmless inactivated triglycerides. Fatty liver. There are limits to this protection. Evolution has not anticipated a cat with a body condition score of 9/10. The liver becomes so overloaded with lipid that cellular damage takes over and we are in to hepatic lipidosis. You can get the flavour of it here</div><div><br /></div><div><a href="https://www.msdvetmanual.com/digestive-system/hepatic-diseases-of-small-animals/feline-hepatic-lipidosis" target="_blank">Feline Hepatic Lipidosis</a></div><div><br /></div><div>Basically, after years of continuous enhanced insulin signalling from a largely carbohydrate and LA diet insulin is suddenly withdrawn and basal lipolysis can take over. With a vengeance. FFAs cannot be recycled to adipocytes because there is too little insulin. Hepatocytes have to take over. They use peroxisomal ROS to signal lipid sequestration in the absence of insulin.</div><div><br /></div><div>There can be so much hepatic lipid sequestration that severe hepatocellular damage occurs and then it's up to the ICU clinician to try to save the cat's life. This is done by oesophageal tube feeding for anything up to a month. If the cat survives, recovery can be complete. The current advice is to feed protein based foods, the worry being insulogenic carbohydrate food might worsen hepatic lipid storage. Quite why no one has considered acipimox is beyond me, or peripheral low dose insulin infusion to target adipocytes rather than hepatocytes as an anti-lipolytic... But then I'm totally out of ICU work nowadays.</div><div><br /></div><div>Anyhoo.</div><div><br /></div><div>I hope people get the concept that fasting can trigger hepatic lipidosis. Bill Lagakos commented that he has seen chow fed mice develop the mouse equivalent of hepatic lipidosis by extended fasting. I completely believe him.</div><div><br /></div><div>It puts us in a better position to understand what is happening in Tucker's cited study of safflower oil inducing fatty liver, which I was not expecting. I had been edging towards these concepts for weeks but the information from Jaromir and Bill have been extraordinarily helpful.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com4tag:blogger.com,1999:blog-36840063.post-7092699657240773892024-03-10T19:31:00.000+00:002024-03-10T19:31:18.677+00:00Foie gras from safflower oil hiatus<p>Tucker discussed a paper in some detail here <a href="https://tuckergoodrich.substack.com/p/hello-can-we-have-your-liver-understanding" target="_blank">on his substack</a>. I fell for it hook line and sinker. It's been pulling me around for weeks. How badly? I've had a copy of Gold's book The Deep Hot Biosphere since late February and I'm only on page 10.</p><p>Here's the paper:</p><p><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663608/" target="_blank">Fat Quality Influences the Obesogenic Effect of High Fat Diets</a></p><p>I *think* I understand what is going on but am not quite certain enough to hit "publish" of the third version of my blog post about it. I have been back through so many layers of references that some interesting studies have come up and I think I'll write about one of these next while I continue to mull over the fatty livers. I'm not in an "ignore the blog" phase. It's just the study which has me hooked is very, very complicated and, to me, very, very unexpected. So I can't leave it alone.<br /><br />More when I can.</p><p>Peter</p><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg1Dt7MD8f0uwfgE0PC7V5lshM1FN1we5A8pc89IjOUzu3XANFWj6hLOJT2EFmJETLSXcZgZ7Thc7JJjcfYH6zX6n0GFhynhy-VfTtuKuea8BHpEFWXB14V_kbv0BSg-aSucYcz4eiehJRahuPXLj0YigIt_r7F5Xm3rr2IY-N5ayBhkw5-dhm7cw/s1100/Livers%20safflower%20linseed.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="605" data-original-width="1100" height="220" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg1Dt7MD8f0uwfgE0PC7V5lshM1FN1we5A8pc89IjOUzu3XANFWj6hLOJT2EFmJETLSXcZgZ7Thc7JJjcfYH6zX6n0GFhynhy-VfTtuKuea8BHpEFWXB14V_kbv0BSg-aSucYcz4eiehJRahuPXLj0YigIt_r7F5Xm3rr2IY-N5ayBhkw5-dhm7cw/w400-h220/Livers%20safflower%20linseed.png" width="400" /></a></div><p><br /></p><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com6tag:blogger.com,1999:blog-36840063.post-72870266075821651812024-02-21T18:19:00.002+00:002024-02-21T18:19:55.892+00:00Electrochemistry (2) Superoxide<div>I keep trying to get back to simple things like weight loss/gain/insulin/LA but the electrochemistry won't leave me alone.</div><div><div><br /></div><div>Someone, possibly Jaromir (mct4health) or Brad Marshall, pointed out that the pyruvate dehydrogenase complex (PDC) is a significant source of ROS.</div><div><br /></div><div>So I was rootling around through papers on PDC and read, in a now-lost paper, that the decarboxylase component of the complex converted pyruvate to acetate but that a similar effect could be achieved by reacting isolated pyruvate with a source of free radicals to give acetate and carbon dioxide. It was probably H<sub>2</sub>O<sub>2</sub> they were working with. The decarboxylation is a lot quicker with the enzyme but a chemical source of ROS will get the job done.</div><div><br /></div><div>Anyway, the significance only dawned on me weeks later and I hadn't saved the paper. It's gone.</div><div><br /></div><div>There are, it turns out, many papers looking at pyruvate decarboxylation using H<sub>2</sub>O<sub>2</sub>, other ROS or RNS. It's a generic trait. I went through this first paper in some detail:</div><div><br /></div><div><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925109/" target="_blank">Reaction rate of pyruvate and hydrogen peroxide: assessing antioxidant capacity of pyruvate under biological conditions</a></div><div><br />Here's a simplified version of their Figure 1 which is the basic reaction. An electron from the H<sub>2</sub>O<sub>2</sub> attacks the alpha carbon of the pyruvate to give the completely unstable 2-hydroperoxy-2-hydroxypropanoate which spontaneously degrades to acetate and CO<sub>2</sub>:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipm4RedbB4MupQYO90Zmc8d4djUZjYI0CoGwwbnI-tmay0Joy_A8vALC-9shckpQ0mu1167O6GOKSD6rMzEK022GOdzjz3_7N4YgP1njHRWHNZ6SqMh0GaKILIXDKxvVnbvzTLW6AuZ5pfoQvSUSsqLpdEeJR4akSXlpuF7l_HAJOhyphenhyphenTLIETovzw/s1477/pyruvate%20h2o2%20reaction.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="633" data-original-width="1477" height="171" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipm4RedbB4MupQYO90Zmc8d4djUZjYI0CoGwwbnI-tmay0Joy_A8vALC-9shckpQ0mu1167O6GOKSD6rMzEK022GOdzjz3_7N4YgP1njHRWHNZ6SqMh0GaKILIXDKxvVnbvzTLW6AuZ5pfoQvSUSsqLpdEeJR4akSXlpuF7l_HAJOhyphenhyphenTLIETovzw/w400-h171/pyruvate%20h2o2%20reaction.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><br />"The reaction of pyruvate and H<sub>2</sub>O<sub>2</sub> produces acetate, carbon dioxide (CO2) and water; its transition intermediate has been recently confirmed..."</div><div><br /></div><div>Essentially the H<sub>2</sub>O<sub>2</sub> is providing an electron which destabilises the alpha carbonyl group and the molecule then rearranges itself in to the decarboxylation products.</div><div><br /></div><div>Now look at Nick Lane's slides in the <a href="https://high-fat-nutrition.blogspot.com/2024/01/electrochemistry.html" target="_blank">last post</a>. First we need this bit:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJawx1qcXpKumqIPxehL-LjblnvjfvROMGgRTX_1h190vbr51XuztkatFF15uHxHFmwRDaFhKIyz9l8syEkX93o0NENydaQ7kO7V4bkUO-0bOJgZe5mkl2kAgk-bbMg3MurAGCpfY_RgqJWEEdCbbYy3lOpj1cajgvab05whMGGr4Uz6WHzsp-hg/s1066/activating%20CO2.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="525" data-original-width="1066" height="198" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJawx1qcXpKumqIPxehL-LjblnvjfvROMGgRTX_1h190vbr51XuztkatFF15uHxHFmwRDaFhKIyz9l8syEkX93o0NENydaQ7kO7V4bkUO-0bOJgZe5mkl2kAgk-bbMg3MurAGCpfY_RgqJWEEdCbbYy3lOpj1cajgvab05whMGGr4Uz6WHzsp-hg/w400-h198/activating%20CO2.png" width="400" /></a></div><br /><div><br /><br /><br /><br /><br /><br /><br /><br /><br />in which CO<sub>2</sub> accepts a geochemical derived electron to become a bound CO molecule and a bound oxygen anion. This lets us re write this line</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEid7cGqJU-dDG-RAiOjLQxqR4wMP5eEF8snIbRMurzsfe_W1RFk5OBBBDbGLOPKvO3GKooV8YVs-J1DCCVrlU1rIIZQu-BlB5VRSPRPNMKSgSs-XyLT0mZRxhadlRYRvQYaHQxZGn9u7Xeoc9ztzqhHlvNSUHMZq7O4XG9N499MbFQr2JqCA6Fgbw/s1504/acetyl%20to%20pyruvate%20CO2.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="337" data-original-width="1504" height="90" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEid7cGqJU-dDG-RAiOjLQxqR4wMP5eEF8snIbRMurzsfe_W1RFk5OBBBDbGLOPKvO3GKooV8YVs-J1DCCVrlU1rIIZQu-BlB5VRSPRPNMKSgSs-XyLT0mZRxhadlRYRvQYaHQxZGn9u7Xeoc9ztzqhHlvNSUHMZq7O4XG9N499MbFQr2JqCA6Fgbw/w400-h90/acetyl%20to%20pyruvate%20CO2.png" width="400" /></a></div><br /><div><br /><br /><br /></div><div><br /></div><div>(in which the activated CO<sub>2</sub> derivatives are highlighted with red ovals) in to the much simpler form of:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh3ZpVQ_eAKcgaw8quQlTNsQDuYP7P4fT7Vffahtk9TNTnzKw8V_0EtwNxAIU1VXE1TxCZ2VjHpDg0yloKH36dHTVG9GRgyhivU0unkLf71_HvnnQKPXATGo0x25cWDbM_fVYG8glftJ0mbUxVIxEzhDCWkRxaiKE6pb40fXHKvu39cG460UkFIog/s1504/summary%20nick%20lane%20acetyl%20to%20pyruvate.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="685" data-original-width="1504" height="183" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh3ZpVQ_eAKcgaw8quQlTNsQDuYP7P4fT7Vffahtk9TNTnzKw8V_0EtwNxAIU1VXE1TxCZ2VjHpDg0yloKH36dHTVG9GRgyhivU0unkLf71_HvnnQKPXATGo0x25cWDbM_fVYG8glftJ0mbUxVIxEzhDCWkRxaiKE6pb40fXHKvu39cG460UkFIog/w400-h183/summary%20nick%20lane%20acetyl%20to%20pyruvate.png" width="400" /></a></div><br /><div><br /><br /><br /><br /><br /><br /><div><br /></div><div><br /></div><div>Ultimately we can convert an acetyl group and CO<sub>2</sub> to pyruvate using a geochemical electron from the origin of life scenario<span style="font-size: 13.3333px;">.</span></div><div><br />We can do the exact opposite and convert pyruvate to acetate and CO<sub>2</sub>, again using a donated electron, this time from H<sub>2</sub>O<sub>2</sub>.</div><div><br /></div><div>Pyruvate is stable. You can buy it in tablet form as a metabolic nutritional "supplement". It won't convert to vinegar in the jar. If you were to carbonate a bottle of vinegar it would stay as "fizzy vinegar" long term without converting to pyruvate. Exactly as you could mix H<sub>2</sub> and CO<sub>2</sub> in aqueous solution and they would remain stable without a hint of formate formation. Until you add an electron.</div><div><br /></div><div>Then the reaction moves. The change in energy is quite small and you could push the reaction one way or the other way depending on the relative concentrations of acetate, CO<sub>2</sub> or pyruvate. The electron is what makes it happen, in either direction.</div><div><br /></div><div>This is not unique to the reaction of pyruvate with hydrogen peroxide.</div><div><br /></div><div>A little more grubbing around suggests that peroxinitrite is even better:</div><div><br /></div><div><a href="https://pubmed.ncbi.nlm.nih.gov/9250413/" target="_blank">Peroxynitrite-mediated decarboxylation of pyruvate to both carbon dioxide and carbon dioxide radical anion</a><br /><br /></div><div>"... oxygen consumption studies confirmed that peroxynitrite mediates the decarboxylation of pyruvate to free radical intermediates. Comparing the yields of acetate and free radicals estimated from the oxygen uptake studies, it is concluded that pyruvate is oxidized by both one- and two-electron oxidation pathways..."</div><div><br /></div><div>No one seems to have looked at superoxide but you can bet your bottom dollar it does the same. In fact, for the similar reaction of alpha ketoglutarate to succinate, superoxide will do the job:</div><div><br /></div><div><a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000690" target="_blank">The Tricarboxylic Acid Cycle, an Ancient Metabolic Network with a Novel Twist</a><br /><br />"The significance of KG [alpha ketoglutarate], a metabolite that can detoxify H<sub>2</sub>O<sub>2</sub> and O<sub>2</sub><sup>-</sup> with the concomitant formation of succinate in this process is also discussed."</div><div><br /></div><div>So what?</div><div><br /></div><div>The interconversion of metabolites of the TCA appears to be quite possible mediated by nothing other than the availability of spare electrons and the relative concentrations of the core reactants. From Nick Lane's doodles the essential component for the actual dissipation of energy in the sections of the TCA at the origin of life are actually mediated by the availability of electrons. No enzymes required.</div><div><br /></div><div>The availability of electrons from geochemical sources is what drove the conversion of CO<sub>2 </sub>and protons to core metabolites of the essential parts of the TCA. No electrons, no conversion of anything in to anything else.</div><div><br /></div><div>Today the electrons come from all sorts of places, reduced ferredoxin, NADH, FADH2 etc. But originally, in the beginning, I suspect that the first source of free electrons to replace the geochemical source might have been superoxide.</div><div><br /></div><div>We know that LUCA used oxygen despite the anoxic conditions of the early Earth. She had superoxide dismutase, catalase and a <a href="https://www.sciencedirect.com/science/article/pii/S1631069108002278" target="_blank">precursor of haemoglobin</a> which stored (precious) oxygen. So LUCA actively controlled oxygen availability, superoxide dismutation and hydrogen peroxide catabolism to oxygen and water. Presumably for a very specific purpose.</div><div><br /></div><div>All that is needed to convert pyruvate to acetate is a free electron. Electrons are continuously being placed on to ferredoxin by our prototypical membrane bound hydrogenase. Once the cellular supply of ferredoxin has been largely converted to reduced ferredoxin then a) trying to move more electrons to ferredoxin gets harder and b) there is enough reduced ferredoxin to be worth activating metabolism and growth c) this can be initiated by transferring electrons on to stored oxygen (possibly derived from radiolysis or photolysis of water) and allow the superoxide generated to perform the process of converting one metabolite to another.</div><div><br /></div><div>Ferredoxin (and even ATP, once evolved) could then be used for the more obscure reactions that might require more complicated metabolite interconversions, possibly not amenable to simple ROS mediated methods.</div><div><br /></div><div>We are still using superoxide today, from reverse electron transport through complex I (directly comparable to that of the prototypical hydrogenase), as the core control of metabolism (pax NOX enzymes). The above speculative narrative would start with superoxide as the actual catalyst at first, one step removed from the origin of life, rather than as a signal. It will be interesting to see whether the modern production of superoxide has any residual enzymic function per se or whether it is now merely a signal/mediator, working through modification of functional sulphydryl groups on proteins which now perform their essential redox catalysis deep within their active sites.</div><div><br /></div><div>I find it a fascinating idea.</div><div><br /></div><div>Peter</div><div><br /></div><div><br /></div><div>Addendum. These papers were formative of the above ideas but are a bit like excess baggage to the core principle. I enjoyed them so here they are:</div><div><br /></div><div>You can do other interesting things with sources of electrons and core members of the modern TCA. If you would like to decarboxylate oxaloacetate to malonate just add ROS:</div><div><div><br /></div><div><a href="https://pubmed.ncbi.nlm.nih.gov/27493727/" target="_blank">Malonate as a ROS product is associated with pyruvate carboxylase activity in acute myeloid leukaemia cells</a><br /><br />"We have shown that malonate can be formed from oxaloacetate by chemical conversion under the influence of hydrogen peroxide..."<br /><br />The ROS in this paper which convert oxaloacetate to malonate appears to come from the pyruvate carboxylase enzyme. This is their previous paper which they cited above:<div><div><br /></div><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2621336/" target="_blank">Metabolomic Profiling of Drug Responses in Acute Myeloid Leukaemia Cell Lines</a><br /><br />"However, in vitro treatment of oxaloacetate and pyruvate confirm that these conversions are in fact induced by hydrogen peroxide as shown in Figure S4."<br /><br />"In addition, previous reports have established that ROS mediate the non-enzymatic conversions including that of α-ketoglutarate into succinate [24]-[26]."<br /><br />This is the alpha-ketoglucarate paper:<br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/16781453/" target="_blank">Nonezymatic formation of succinate in mitochondria under oxidative stress</a></div></div><br />"The occurrence of nonenzymatic oxidation of KGL in mitochondria was established by an increase in the CO<sub>2</sub> and succinate levels in the presence of the oxidants and inhibitors of enzymatic oxidation. H<sub>2</sub>O<sub>2</sub> and menadione as an inductor of reactive oxygen species (ROS) caused the formation of CO<sub>2</sub> in the presence of sodium azide and the production of succinate, fumarate, and malate in the presence of rotenone. These substrates were also formed from KGL when mitochondria were incubated with tert-BuOOH at concentrations that completely inhibit KGDH. The nonenzymatic oxidation of KGL can support the TCA cycle under oxidative stress..."</div><div><br /></div><div>Okay, that will do!</div></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com16tag:blogger.com,1999:blog-36840063.post-1611557901722074022024-01-31T13:34:00.010+00:002024-02-02T05:40:35.955+00:00ElectrochemistryPassthecream posted this link in comments which is a nice listen to Nick Lane explaining his ideas to a non biochemistry audience. I enjoyed it.<div><br /></div><div><iframe allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen="" frameborder="0" height="315" src="https://www.youtube.com/embed/FLaTU-t1CQM?si=-G9KYOJDbSMSRNYa" title="YouTube video player" width="400"></iframe></div><div><br /></div><div>Youtube recalled that I watched it and suggested this similar item</div><div><br /></div><div><iframe allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen="" frameborder="0" height="315" src="https://www.youtube.com/embed/vBiIDwBOqQA?si=r_O_ydK9cv9l4POY" title="YouTube video player" width="400"></iframe></div><div><br /></div><div>which also has some core concepts in it. This few minutes really caught my eye as it's something I've been think about for a long time. It's a reiteration of his ideas from pages p133 to 140 of Transformer with all of the doodles merged in to two slides</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><iframe allowfullscreen='allowfullscreen' webkitallowfullscreen='webkitallowfullscreen' mozallowfullscreen='mozallowfullscreen' width='320' height='266' src='https://www.blogger.com/video.g?token=AD6v5dyE0iDmBcIeT8AJyBjp4onn2iCvhW6JQAcUNIm81TPurdXQX0vARHyxVt8-l_0IPpz9u_3evUbsn3Q' class='b-hbp-video b-uploaded' frameborder='0'></iframe></div><br /><div>Although he does specify a negatively charged surface in the book, this doesn't get fitted in to the brief overview he presents in the talk. But this negative charge is fundamental to the chemistry being discussed. I've snapshotted the two slides and added in the supply of electrons needed for each step of the reaction, with a different colour for each electron or pair of electrons, all coming from the charged surface.</div><div><br /></div><div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgFibAY4TKbyDw4qFRBy4mXue0xbfhh8R4R69jQwwb9AlESAw4HTd6ikG4jsja_B4jAICFvrZ3tIT5t5umPmYVAlBLOTjJN7sWMMCht1zrBxgUXBCm9RwqvEUbaFsggA80dXN8GEjDTjHrdo6kk1nCKRc3VDWxsirOUhmUepssTlDeppvEw43F4eQ/s1504/NL%20slide%202.png" style="clear: left; float: left; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="937" data-original-width="1504" height="199" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgFibAY4TKbyDw4qFRBy4mXue0xbfhh8R4R69jQwwb9AlESAw4HTd6ikG4jsja_B4jAICFvrZ3tIT5t5umPmYVAlBLOTjJN7sWMMCht1zrBxgUXBCm9RwqvEUbaFsggA80dXN8GEjDTjHrdo6kk1nCKRc3VDWxsirOUhmUepssTlDeppvEw43F4eQ/w320-h199/NL%20slide%202.png" width="320" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div></div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjnsB96Tkl1UMu1P5XxnsIWtcMrh_Lx8OIyLe606uwPoWuBPZRqmTiCxgDvwVET0TBUsOeQaJztfILygaEkAALtWcn46YNY8mqO0vjXKZtF_UqpF0E5wkmvU4DlRiOTpohonP_eAKt6a1WCo6sLr5Mcd6d1bSrNEid1rC8Ku-YZqcKtQ_rSQ_ghzQ/s1504/NL%20slide%201.png" style="clear: left; float: left; margin-bottom: 1em; margin-left: 1em;"><img border="0" data-original-height="937" data-original-width="1504" height="199" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjnsB96Tkl1UMu1P5XxnsIWtcMrh_Lx8OIyLe606uwPoWuBPZRqmTiCxgDvwVET0TBUsOeQaJztfILygaEkAALtWcn46YNY8mqO0vjXKZtF_UqpF0E5wkmvU4DlRiOTpohonP_eAKt6a1WCo6sLr5Mcd6d1bSrNEid1rC8Ku-YZqcKtQ_rSQ_ghzQ/w320-h199/NL%20slide%201.png" width="320" /></a></div><br /><blockquote style="border: none; margin: 0px 0px 0px 40px; padding: 0px; text-align: left;"><blockquote style="border: none; margin: 0px 0px 0px 40px; padding: 0px; text-align: left;"></blockquote></blockquote><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><div><br /></div><div>Aside. The two red ovals pick out a single, bound, negatively charged oxygen atom. If you're trying to keep the charges balanced it is helpful to realise that they are the same moiety illustrated in two places on different slides. End aside.<div><div><br /></div><div>This is pure electrochemistry. I picked up a paper years ago which was looking at origin of life reactions driven by an external voltage. You can drive the sort of reactions Nick Lane is describing with a tenth of a 1.5 volt battery's potential. The beauty of vents is that they supply the battery.<br /><blockquote style="border: none; margin: 0px 0px 0px 40px; padding: 0px; text-align: left;"><blockquote style="border: none; margin: 0px 0px 0px 40px; padding: 0px; text-align: left;"><br /></blockquote></blockquote><div>A subgroup of industrial chemical engineers is well aware of this phenomenon and they are amazed that electrochemistry for organic carbon based molecular synthesis has never been commercialised. This abstract gives the flavour of their frustration<br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/31823612/" target="_blank">A Survival Guide for the "Electro-curious"</a></div><div><br /></div><div>Understandable.</div><div><br /></div><div>Peter</div></div></div></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com31tag:blogger.com,1999:blog-36840063.post-89623749379651136952024-01-28T05:21:00.000+00:002024-01-28T05:21:25.038+00:00Life (40) Proton pumpingOkay. Time to finish the complex I series. Under conditions of a cell surface membrane which is partially permeable to protons and (less so) to hydroxyl ions there can be a proto-metabolism based on the ingress of protons driving both carbon fixation and energy generation, with neutralisation by OH<sup>- </sup>ions. This is dependent on having a partially permeable membrane to both of these ions. Subsequently, by using the simultaneous impermeability to (larger, less permeant) Na<sup>+</sup> ions, combined with the above ability to neutralise protons with OH<sup>-</sup>, a Na<sup>+</sup>/H<sup>+</sup> antiporter can establish a Na<sup>+</sup> potential to drive a proto-ATP synthase. Koonin's group discussed it here:<div><div><br /></div><div><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2359735/" target="_blank">Evolutionary primacy of sodium bioenergetics</a><br /><br />As the protocell membrane becomes progressively more impermeable to both H<sup>+</sup> and OH<sup>- </sup>then running a Na<sup>+</sup>/H<sup>+</sup> antiporter becomes progressively more difficult. At the same time this makes proton pumping potentially advantageous. This is how I am guessing that proton pumping may have developed.</div><div><br /></div><div>If we start from that neat doodle from</div><div><br /></div><div><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577180/" target="_blank">Redox-induced activation of the proton pump in the respiratory complex I</a></div><div><br /></div><div>looking like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgvVC553-519Sdci9LCdUCQrwq6Q0VHQdPUz3cmcxaWMhmPkBODZjcfzFPiz42-_1aQ9t1pSu7c573Gnkz8nGEWW7lZYMh0MOQqrq12ZLjb2g6rOX2XeqKdGc0pjIyKEa-AJbD-qK8sAoTatRSN1Z3wY4Zq9JChVaNJqo6cSuXxbDvYr-JP23vEYg/s820/Amino%20acids%20in%20Nqo8.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="820" data-original-width="629" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgvVC553-519Sdci9LCdUCQrwq6Q0VHQdPUz3cmcxaWMhmPkBODZjcfzFPiz42-_1aQ9t1pSu7c573Gnkz8nGEWW7lZYMh0MOQqrq12ZLjb2g6rOX2XeqKdGc0pjIyKEa-AJbD-qK8sAoTatRSN1Z3wY4Zq9JChVaNJqo6cSuXxbDvYr-JP23vEYg/w306-h400/Amino%20acids%20in%20Nqo8.png" width="306" /></a></div><br /><div><br /></div><div><br /><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>we can reverse model it back to a simpler NiFe hydrogenase in a proton semi-permeable membrane and need just four images to sum it up:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi0sIDAfDECwWhpV4EEPshDjMPHqaJhz0geu2UlGEQJrEK_rTx7wQ4C9GWlq4A7LE_tk16B4S-05iPnZjflpz4cMvK4FoP8dbY9uC0LqUCTythJR0bRwFE7nPfzsBJWPC-85jQ7Qz4UC1eW2YqkfixcZ1SIiH9W4Kvm8khpNgSTKvCWFNhyphenhyphenE06_Lw/s939/Last%201.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="939" data-original-width="920" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi0sIDAfDECwWhpV4EEPshDjMPHqaJhz0geu2UlGEQJrEK_rTx7wQ4C9GWlq4A7LE_tk16B4S-05iPnZjflpz4cMvK4FoP8dbY9uC0LqUCTythJR0bRwFE7nPfzsBJWPC-85jQ7Qz4UC1eW2YqkfixcZ1SIiH9W4Kvm8khpNgSTKvCWFNhyphenhyphenE06_Lw/w393-h400/Last%201.png" width="393" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This has the ocean at pH 6 protonating acidic amino acids in a channel from the ocean to the FeS cluster. There is also a side chain of acidic amino acids in contact with the NiFe cluster which are non-protonated because they are contiguous with the cytoplasmic fluid of pH10.</div><div><br /></div><div>A molecule of hydrogen arrives at the NiFe cluster and is split in to a pair of electrons and a pair of protons:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg82olfUQpvpBTAYuWqUeRf9knMmStmtV4uHt9F5c6va_mrAl6H1Irspb_OQqVj2QJ5ayb1icLh7rt12Uik22dvsHPaKoBe1CuMq-gkXrj3UFlmM1dPTZ3YT-QMV-lOu766nKZh6iSiQYJpOU9NU0Uuw3QrhHtcpIhOJsQBe_Ic97sIS5g3EmuAsw/s1041/Last%202.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1041" data-original-width="920" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg82olfUQpvpBTAYuWqUeRf9knMmStmtV4uHt9F5c6va_mrAl6H1Irspb_OQqVj2QJ5ayb1icLh7rt12Uik22dvsHPaKoBe1CuMq-gkXrj3UFlmM1dPTZ3YT-QMV-lOu766nKZh6iSiQYJpOU9NU0Uuw3QrhHtcpIhOJsQBe_Ic97sIS5g3EmuAsw/w354-h400/Last%202.png" width="354" /></a></div><br /><div><br /></div><div> </div><div><div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The electrons hop on to the FeS cluster and thence to ferredoxin (accompanied by their ability to do work) to give reduced ferredoxin, Fd<sup>2-</sup>, while the protons go to the waiting carboxylates of the amino acids on the route to the pH10 cytoplasm:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi06bV4qnQbYp7BavfRnF_JHYFntDO28EahQBUFsnChHg0bbDVmSp1k35fmiw6Ot-Dyn-EJ_vZCuQskpBY__LW_Dl0ehVb2ng3qOvYWk4LHMrK97pcR1jM4mcjyAGvGopyyQJldX_JAcNpEW6_CP9lEemrIujEcsL_t7mgwxiYukz44Bh5tIITCKQ/s1295/Last%203.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1139" data-original-width="1295" height="351" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi06bV4qnQbYp7BavfRnF_JHYFntDO28EahQBUFsnChHg0bbDVmSp1k35fmiw6Ot-Dyn-EJ_vZCuQskpBY__LW_Dl0ehVb2ng3qOvYWk4LHMrK97pcR1jM4mcjyAGvGopyyQJldX_JAcNpEW6_CP9lEemrIujEcsL_t7mgwxiYukz44Bh5tIITCKQ/w400-h351/Last%203.png" width="400" /></a></div><br /><div><br /><br /><br /><br /><br /> </div></div></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which then leaves the complex ready for the next hydrogen molecule to come along after the protons on the cytoplasmic route's amino acids have been deprotonated by the pH10 cytoplasm:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhertps7I0Qm3P2iiFkMri9fad0oaOjFTirUeOi12HsoIAKvUQx6yrAbwUgfn11o53G8UvZRmafD3QYKUFG2TCnQUSC2w7gymD6ekdNrsLNXMHLK_AgJL_v65Iw-Hs8hg3i28nYWMZcX7NA-EesZQwQ9KurVhkyYUt2ztgaFeyzsieuv1FU8EvLFw/s962/Last%204.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="939" data-original-width="962" height="390" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhertps7I0Qm3P2iiFkMri9fad0oaOjFTirUeOi12HsoIAKvUQx6yrAbwUgfn11o53G8UvZRmafD3QYKUFG2TCnQUSC2w7gymD6ekdNrsLNXMHLK_AgJL_v65Iw-Hs8hg3i28nYWMZcX7NA-EesZQwQ9KurVhkyYUt2ztgaFeyzsieuv1FU8EvLFw/w400-h390/Last%204.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The cost of this manoeuvre being a small fall in the intracellular pH, to be neutralised by the same alkaline vent fluid which supplied the molecular hydrogen.</div><div><br /></div><div>That seems quite simple.</div><div><br /></div><div>If we consider what might happen if the availability of hydroxyl ions is curtailed by progressively rising impermeability of the cell membrane to both H<sup>+</sup> and OH<sup>-</sup> then the process must halt. With the evolution of soluble hydrogenases, and especially of electron bifurcation, then Fd<sup>2-</sup> might become more plentiful but molecular hydrogen less so.</div><div><br /></div><div>We can consider what the immediate advantage might be to a cell to consume Fd<sup>2-</sup> and regenerate molecular hydrogen by running this complex in reverse.</div><div><br /></div><div>So now I've set the intracellular pH to pH7 and left the ocean fixed at pH6. It's a big ocean.</div><div><br /></div><div>In this scenario all of the amino acids in the complex would be protonated:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjGGT0AALCYbOOWd0d1ZKwOOBfcKsbQ-JEv6wQD_gTI14wN3u0dUl2v7TMKWPm5_HBrbdw7CmcH9KSsu7fejZnjcAUI4hnXdV1e4kcpCeP3bKKX1zwqpgLC8nNlLQOhswyeKPS1M0wgnaKMipjKSOn74W1_u6Yjg3qXvOf85IF8OtXpt1_DeKRGfQ/s939/Last%205.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="939" data-original-width="920" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjGGT0AALCYbOOWd0d1ZKwOOBfcKsbQ-JEv6wQD_gTI14wN3u0dUl2v7TMKWPm5_HBrbdw7CmcH9KSsu7fejZnjcAUI4hnXdV1e4kcpCeP3bKKX1zwqpgLC8nNlLQOhswyeKPS1M0wgnaKMipjKSOn74W1_u6Yjg3qXvOf85IF8OtXpt1_DeKRGfQ/s320/Last%205.png" width="314" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div> </div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>If we allow a Fd<sup>2- </sup>molecule to place a pair of electrons on to the FeS cluster:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiUdiynmsVoTqP0XaXLVnwOeSeqVZmle-5gFf-ZRgqaglOv83Uthu52ot8dh0_-k_Sqc6D7XYnHPUyGoQ94pqJDC_SmpvLUs-QQuWvUykuwUw6i0j_ATzDWxpCdYjpLqBmERFvgbb4-J5-sTTxZyaSO1SBZferGWMDc1sEGBqjmvnTnBF9TfJfFBw/s1295/Last%206.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1056" data-original-width="1295" height="326" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiUdiynmsVoTqP0XaXLVnwOeSeqVZmle-5gFf-ZRgqaglOv83Uthu52ot8dh0_-k_Sqc6D7XYnHPUyGoQ94pqJDC_SmpvLUs-QQuWvUykuwUw6i0j_ATzDWxpCdYjpLqBmERFvgbb4-J5-sTTxZyaSO1SBZferGWMDc1sEGBqjmvnTnBF9TfJfFBw/w400-h326/Last%206.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>these can combine with a pair of protons to form molecular hydrogen. These protons should come from the (very slightly) more acidic ocean channel:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjtBA056mJnz22BlKOizHSVJO1SiJOGCsg-zlb-mSrSHQpfyasvcbgohvqAGvTOKBUFkAaijxOXVnhIdtq0daKiOajkU4Qi5yt5ZsQ58HVWerfXOd_P7cx9dKluL705raW469zvsOU1ONq2N1IyQlX4X6Puh3PgNOX867HTtVudAkbquzTF1IZcQw/s1114/Last%208.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1114" data-original-width="920" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjtBA056mJnz22BlKOizHSVJO1SiJOGCsg-zlb-mSrSHQpfyasvcbgohvqAGvTOKBUFkAaijxOXVnhIdtq0daKiOajkU4Qi5yt5ZsQ58HVWerfXOd_P7cx9dKluL705raW469zvsOU1ONq2N1IyQlX4X6Puh3PgNOX867HTtVudAkbquzTF1IZcQw/w330-h400/Last%208.png" width="330" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This reaction is exothermic and needs no proton gradient. It leaves us with a deficit of protons in the oceanic pH channel:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg7vpfHGIvzhFlyMJ2lJw0KlquOqvneOcVULgMyd-VuKwNTkQLV7rW2_tEReGo_Aqh2BB3G2v5ygXp8lBvKuMeDWD8ajc6lrJkihhDyppyGagdTrO58gMxQEtCt6iZN_AWFMoeCCzTUe8X-5dpKULaRieG7ySHmkY5THavNOjSo34oO4RJ-p1NPgA/s1041/Last%209.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1041" data-original-width="920" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg7vpfHGIvzhFlyMJ2lJw0KlquOqvneOcVULgMyd-VuKwNTkQLV7rW2_tEReGo_Aqh2BB3G2v5ygXp8lBvKuMeDWD8ajc6lrJkihhDyppyGagdTrO58gMxQEtCt6iZN_AWFMoeCCzTUe8X-5dpKULaRieG7ySHmkY5THavNOjSo34oO4RJ-p1NPgA/w354-h400/Last%209.png" width="354" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which you would expect to be replenished from the bulk ocean. But we have a certain amount of free energy available from the high energy Fd<sup>2-</sup> molecule used to make the molecular hydrogen. All that is needed is an electrostatic/conformational change comparable to the "Doohickey" function of the last several posts and it becomes simple to take two protons from the cytoplasmic influenced amino acids and put then on to the oceanic side using the energy available from Fd<sup>2- </sup>oxidation:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi8ZqjWqiDd3bBBoQ5vXyQliwEH1pfggd43cOQO3ZA8bv10glzRSnNsD8j26jsWnAw7rWbRwWfau0IcpI3isDDbSDAfOvhxdnYGp9XAKn7Je_i7qGGtSqrFEcvcb0TuWlMaie7mMf9iXN77oAjrNRQZvo-HVKo8eWUSgBQN_U3HkK85NprLR6pE3g/s1197/Last%2010.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="939" data-original-width="1197" height="314" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi8ZqjWqiDd3bBBoQ5vXyQliwEH1pfggd43cOQO3ZA8bv10glzRSnNsD8j26jsWnAw7rWbRwWfau0IcpI3isDDbSDAfOvhxdnYGp9XAKn7Je_i7qGGtSqrFEcvcb0TuWlMaie7mMf9iXN77oAjrNRQZvo-HVKo8eWUSgBQN_U3HkK85NprLR6pE3g/w400-h314/Last%2010.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>What might be the immediate advantage of doing this?</div><div><br /></div><div>The pH7 environment on the cellular side will allow spontaneous re-protonation of the acidic residues in the complex:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgmyTsE-MG8fB3gh4Ll5le6WyFfXx92dNVH26Q0atJsNqJ_YtvPb3GJq_7q8IRo-WonvXdjB0zFzDKAvKDVcG1YjKWGgOz3uBtjUznqm1JH3bUQtrYzJKIx3p2gNaX5K6zbtDGPwNFfhjvFmkGeLcdnWE39HHOq8BWNh9EmbxxA2gp9EM1MEAWduA/s1062/Last%2011.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="939" data-original-width="1062" height="354" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgmyTsE-MG8fB3gh4Ll5le6WyFfXx92dNVH26Q0atJsNqJ_YtvPb3GJq_7q8IRo-WonvXdjB0zFzDKAvKDVcG1YjKWGgOz3uBtjUznqm1JH3bUQtrYzJKIx3p2gNaX5K6zbtDGPwNFfhjvFmkGeLcdnWE39HHOq8BWNh9EmbxxA2gp9EM1MEAWduA/w400-h354/Last%2011.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which will clearly leave a very small and very localised area of higher pH, here designated as pH8 for illustrative purposes only:</div></div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgu6bfyHMD5UyM4Wqbw9us_lMLp0nBXZ4GhrYxQVtGqDsXt_v4sVfR1pyYBURhVj5AUf2fK3I0nC8Y7wGruZscefyGs9KbSHzaQfUGztoau9ZtU0UulP581vwKPZHfVQo4ik5gpDDJI5kv9qQZaKlCTBbNayjK1ZOHnCC11H_VxXlEr7uqU-bPB5A/s1062/Last%2012.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="939" data-original-width="1062" height="354" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgu6bfyHMD5UyM4Wqbw9us_lMLp0nBXZ4GhrYxQVtGqDsXt_v4sVfR1pyYBURhVj5AUf2fK3I0nC8Y7wGruZscefyGs9KbSHzaQfUGztoau9ZtU0UulP581vwKPZHfVQo4ik5gpDDJI5kv9qQZaKlCTBbNayjK1ZOHnCC11H_VxXlEr7uqU-bPB5A/w400-h354/Last%2012.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>We have now produced a very localised accentuation of the progressively feebler pH gradient resulting from the cell membrane becoming progressively more opaque to OH<sup>- </sup>ions.</div><div><br /></div><div>As cell energetics are highly Na<sup>+</sup> dependent, as per the introduction to this post, establishing a small area of accentuated pH gradient will allow the immediate advantage of facilitating the struggling Na<sup>+</sup>/H<sup>+</sup> antiporting process, at the cost of allowing the loss of the newly developed localised area of pH 8 (as shown) back down to pH7 (not shown). Like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnUBlnXRzd5TCfR8Cp3SZHzyHkteOCiW4I3MEojkn3rpp5wxSeLsKQOwMSkgn01SCD7unFNHp2GhyOMjEK9oTtwOx3nmIlYo9-GNAxa7KUr2PnsO8onp5zmgeM5UklMwzanGG646K0wo77793_eaOekz3H4yL5vgMzKwPU0jOjRKLgsDSWyr2Ugg/s1410/Last%2013.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1410" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnUBlnXRzd5TCfR8Cp3SZHzyHkteOCiW4I3MEojkn3rpp5wxSeLsKQOwMSkgn01SCD7unFNHp2GhyOMjEK9oTtwOx3nmIlYo9-GNAxa7KUr2PnsO8onp5zmgeM5UklMwzanGG646K0wo77793_eaOekz3H4yL5vgMzKwPU0jOjRKLgsDSWyr2Ugg/w400-h320/Last%2013.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which is fine except a simple "monogenetic" antiporter is actually pretty useless at low membrane potentials, as in:</div><div><br /></div><div><a href="https://www.sciencedirect.com/science/article/pii/S0014579301024176" target="_blank">Mrp‐dependent Na+/H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters</a></div><div><br /></div><div>So it would be better to have the ancient ancestor of the modern MRP ultra-low proton gradient antiporter instead. Here we have several protons each "kicking" another inward channel to finally </div><div>"kick" a Na<sup>+</sup> ion out of the cell:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipuecaxfDT4JXBGAkklmr5U3ocMJosWzw-WIlzHMQAXz3CZAK93dMzFdJ48HSDKTGqPO5NOrwlN4fvSvnLL26cSl2ZBSzU3Jsz-uLEf7jhtqYVhBcJdSsxoyn1moR2B1Kh1Q4GyiWon_YDCHFa4quDOWhxibTwEot7wNXK9bJ63aJl737yqrDWgg/s1410/Last%2014.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1410" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipuecaxfDT4JXBGAkklmr5U3ocMJosWzw-WIlzHMQAXz3CZAK93dMzFdJ48HSDKTGqPO5NOrwlN4fvSvnLL26cSl2ZBSzU3Jsz-uLEf7jhtqYVhBcJdSsxoyn1moR2B1Kh1Q4GyiWon_YDCHFa4quDOWhxibTwEot7wNXK9bJ63aJl737yqrDWgg/w400-h320/Last%2014.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>At this point having MRP snuggle up to a membrane bound hydrogenase to access a better pH gradient is starting to look vaguely like a complex I precursor, but not quite. All we have is a small improved localised pH gradient, no gross expulsion of protons, and the sole use is to generate a Na<sup>+</sup> ion gradient. But that would be advantageous, immediately.</div><div><br /></div><div>Now let's worsen matters still further and drop the intracellular pH to 6.5, where even the mighty MRP antiporter is in trouble. We can get extracellular protons to the half way inward mark, and intracellular Na<sup>+</sup> to the half way outward mark but there is insufficient pH drive to complete their respective journeys. Stalemate:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgRZPdrGSBWVef6fQwCYhsM65YSXApJue57kz_rakxAwED1-qe8da5MX7MfPciIlfGZPhlXPKnTbS6awb6F1CW2RoeW-fFA7DrQFbVu9DyWckiZBf7mTBKY38TCbxkPAHESt591uUcPtPPKhaDCAl80mPmfl7nJ3F0sZIT4COofmmPo_aabQogJdA/s1410/Last%2015.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1052" data-original-width="1410" height="299" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgRZPdrGSBWVef6fQwCYhsM65YSXApJue57kz_rakxAwED1-qe8da5MX7MfPciIlfGZPhlXPKnTbS6awb6F1CW2RoeW-fFA7DrQFbVu9DyWckiZBf7mTBKY38TCbxkPAHESt591uUcPtPPKhaDCAl80mPmfl7nJ3F0sZIT4COofmmPo_aabQogJdA/w400-h299/Last%2015.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Now if we just think about that energy input from "wasting" a Fd<sup>2-</sup> molecule we can have a conformational/electrostatic change in the green outlined amino acid (modern day aspartate D72 in the original diagram) like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhPb9GAKMUcPytZlxe6P7uQNNq9pQ5nsVSfUIX5MLUvGobaoAyHy0qfUPGvGaIlCggyJgT659Xz4iyniZe1Vz5FqFnyh-bnX_EBkfJuzB0ZUlmd9YGLBYQF6nDFA-wW_b-nK4v73i6gblzL9B-Yvi9UgLsGhIYpOrvW0Zf9jStk2I-Zx2xsm9w4BQ/s1410/Last%2016.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="962" data-original-width="1410" height="272" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhPb9GAKMUcPytZlxe6P7uQNNq9pQ5nsVSfUIX5MLUvGobaoAyHy0qfUPGvGaIlCggyJgT659Xz4iyniZe1Vz5FqFnyh-bnX_EBkfJuzB0ZUlmd9YGLBYQF6nDFA-wW_b-nK4v73i6gblzL9B-Yvi9UgLsGhIYpOrvW0Zf9jStk2I-Zx2xsm9w4BQ/w400-h272/Last%2016.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>giving a "push" to help the struggling MRP antiporter:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjHf4tmCl_lqEWlI2-m1ZoxT-5E7JpbX0X8PSJnzEuKLb7OSDWMAptxmLJA4XVE0KrOLcYVOyHxsQ1WvZHCeKjGnwlKeDsWvFrL0vHBGyJS0FD4MSVAXeidPL-ZNo2n6sP_mQP9mP6woPxdFox3yw3qtKvaomU2-mbW2pet8PDP-b3D346gQ8VbPA/s1410/Last%2017.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1095" data-original-width="1410" height="311" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjHf4tmCl_lqEWlI2-m1ZoxT-5E7JpbX0X8PSJnzEuKLb7OSDWMAptxmLJA4XVE0KrOLcYVOyHxsQ1WvZHCeKjGnwlKeDsWvFrL0vHBGyJS0FD4MSVAXeidPL-ZNo2n6sP_mQP9mP6woPxdFox3yw3qtKvaomU2-mbW2pet8PDP-b3D346gQ8VbPA/w400-h311/Last%2017.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>by providing a "kick" which the pH gradient can't manage alone. As it stands there need be no outward proton translocation, just a push to the MRP antiporter. In fact the localised pH gradient would be lost on Na<sup>+</sup> antiporting but the cell would have bought a better Na<sup>+</sup> gradient for ATP synthase in return:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgt53mYO8p18zUUJdr03Vh_iV5OnbDTnIk6O6xjtHH3HqmAAMZEYVAPGEVkrFHU2zQnlUuK7Y5BV0BYkomdcPIsrxTmtghbuY6b2-tEBuZopzeLl_HncrPahXJc_-R4h02_h1pzBwzg6GeopVeWEJxaUUf2mdHCQIEzX8wb-eOzhlpgzz4Ew98UEg/s1410/Last%2019.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1095" data-original-width="1410" height="311" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgt53mYO8p18zUUJdr03Vh_iV5OnbDTnIk6O6xjtHH3HqmAAMZEYVAPGEVkrFHU2zQnlUuK7Y5BV0BYkomdcPIsrxTmtghbuY6b2-tEBuZopzeLl_HncrPahXJc_-R4h02_h1pzBwzg6GeopVeWEJxaUUf2mdHCQIEzX8wb-eOzhlpgzz4Ew98UEg/w400-h311/Last%2019.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>In this last image I've suggested that blocking off access to the ocean would be an incremental advantage too as it might make a conformational change in the "kicking" acidic amino acid more effective at facilitating MRP antiport completion.</div><div><br /></div><div>None of this is a proton pump. But as the cell membrane become essentially impermeable to protons there develops an advantage to running MRP in reverse. All you have to do is attach the kicking-complex the wrong way round to MRP and you could kick a Na<sup>+</sup> in to the cell and two H<sup>+</sup> out of the cell, then start of using protons in ATP synthase. Or completely drop the module which translocates Na+ and just use the "kick" to push two protons outwards. Or, given a power source like the NADH:CoQ couple, kick four protons out wards, as in complex I. Notice the "kicker" is on the opposite end of the MRP antiporter derivative here and the Na+ module has been abandoned/replaced:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgLlUcUFNAMvjGkC_I80xXAzHQOd6BnMOQAWfIyrrTCLD3wTJM_qVPwtSyux9c8OfQ3m8EMoR9pXztpRh9ZvyaNymFa6mBefhMN1D3j-g-qgy-JIOMd5BJbm41E5wh6cDJ5nzumdJoK0WoL7JftoE2j4u95tFEYNHqiAMEHSTNYBucZzNRUUANIZw/s1283/Last%2020.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="800" data-original-width="1283" height="250" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgLlUcUFNAMvjGkC_I80xXAzHQOd6BnMOQAWfIyrrTCLD3wTJM_qVPwtSyux9c8OfQ3m8EMoR9pXztpRh9ZvyaNymFa6mBefhMN1D3j-g-qgy-JIOMd5BJbm41E5wh6cDJ5nzumdJoK0WoL7JftoE2j4u95tFEYNHqiAMEHSTNYBucZzNRUUANIZw/w400-h250/Last%2020.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Given a less potent power source such as the Fd<sup>2-</sup>/H<sup>+</sup> couple you can just drive out one proton, as Ech does:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjvwVwCtHr7masmT2G0zP0FDBfiFo7ifQKYUJOwqiUFY-HCBb1hg3nkLZpEP0g35Q4GwBwGrXZNdYfosaGdl84shjs3YXyBXmmHx0_vn8K7uyi59Cl6D8vEtsDNlHmgAIMTM-n1jx_peLtDm-y6vqhwpEnBCfVqYfsc87KvBOGvbytD1fHPeRWEzw/s1185/Last%2021.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="843" data-original-width="1185" height="285" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjvwVwCtHr7masmT2G0zP0FDBfiFo7ifQKYUJOwqiUFY-HCBb1hg3nkLZpEP0g35Q4GwBwGrXZNdYfosaGdl84shjs3YXyBXmmHx0_vn8K7uyi59Cl6D8vEtsDNlHmgAIMTM-n1jx_peLtDm-y6vqhwpEnBCfVqYfsc87KvBOGvbytD1fHPeRWEzw/w400-h285/Last%2021.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>You can also, if you're Pyrococcus furiousus living at 100degC, still pump Na<sup>+</sup> ions (it's not easy to build a proton tight membrane at 100degC, so Na<sup>+</sup> energetics are retained) by flipping one proton channel round, pushing a proton outwards and allowing this proton back inwards to antiport a Na<sup>+</sup> ion outwards:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgF0_Z9kiQWIoHuesrx9ohSlmEltbYxnwbPycycL00VNhGzZGmDBI1Wcz0nPSomTaevEq43m1sOhdi6UaY278JI9JHObRk-k9F-b8FLm7ibIkwPQ5LSIAxSDpYZLXg2zX-9o0qpMv5qgqXLmLLGAHdZQn4aTU0hEp4Pdbgc9atjxs-2xTB_OTE0ag/s1139/Last%2022.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1012" data-original-width="1139" height="355" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgF0_Z9kiQWIoHuesrx9ohSlmEltbYxnwbPycycL00VNhGzZGmDBI1Wcz0nPSomTaevEq43m1sOhdi6UaY278JI9JHObRk-k9F-b8FLm7ibIkwPQ5LSIAxSDpYZLXg2zX-9o0qpMv5qgqXLmLLGAHdZQn4aTU0hEp4Pdbgc9atjxs-2xTB_OTE0ag/w400-h355/Last%2022.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which is a proton-neutral technique to establish a Na<sup>+</sup> ion gradient.</div><div><br /></div><div>All you have to do is to develop a "kicker" for MRP and the world is your oyster. There are many derivatives of this type of pump with various subunits arranged in various orders. It's a molecular Lego set. All that is needed is for each step during its development to be continuously advantageous.</div><div><br /></div><div>The concept that modern derivatives might be the best guide as to where and how life began fascinates me and has been laid out by Nick Lane's group here:</div><div><br /></div><div><a href="https://nick-lane.net/wp-content/uploads/2023/10/Harrison-et-al-Life-as-a-Guide-AREES-online-2023.pdf" target="_blank">Life as a Guide to its
Own Origins</a></div><div><br /></div><div>It makes a lot of sense to me.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com41tag:blogger.com,1999:blog-36840063.post-2246799027233676522023-12-22T08:13:00.003+00:002023-12-22T14:31:54.310+00:00Life (39) NuoHThis paper is almost completely dedicated to the function of NuoH and the CoQ binding pocket, AKA the Doohickey.<br /> <br /><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577180/" target="_blank">Redox-induced activation of the proton pump in the respiratory complex I</a><div><br /></div><div>Here is Figure 1, it's the inset we're interested in:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEixE_mWKxcuwRfZoen8QgUWlC8pTzLJF9kQrGHO_lewKyj8g5UK02pf0CTC_8ySUCkhgA3TlnDvzHrNhlQvorMAW707tC3fcBL8bcSGFMGR6TAiyV3WiJT2W7MXYfNZ4DRdS8T0NDVawABO7jjKBIYxkKlIe6ZQ0T7aeWyvbC2mHh2pre20k3Ob-g/s752/Complex%20I%20plus%20inset%20NuoH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="597" data-original-width="752" height="318" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEixE_mWKxcuwRfZoen8QgUWlC8pTzLJF9kQrGHO_lewKyj8g5UK02pf0CTC_8ySUCkhgA3TlnDvzHrNhlQvorMAW707tC3fcBL8bcSGFMGR6TAiyV3WiJT2W7MXYfNZ4DRdS8T0NDVawABO7jjKBIYxkKlIe6ZQ0T7aeWyvbC2mHh2pre20k3Ob-g/w400-h318/Complex%20I%20plus%20inset%20NuoH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and here is the inset. I've taken the liberty of inserting arginine R216 as shown in Figure 6 (left panel) and as mentioned in the text section "Electrostatic coupling elements". Which is what we want to know about.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgvVC553-519Sdci9LCdUCQrwq6Q0VHQdPUz3cmcxaWMhmPkBODZjcfzFPiz42-_1aQ9t1pSu7c573Gnkz8nGEWW7lZYMh0MOQqrq12ZLjb2g6rOX2XeqKdGc0pjIyKEa-AJbD-qK8sAoTatRSN1Z3wY4Zq9JChVaNJqo6cSuXxbDvYr-JP23vEYg/s820/Amino%20acids%20in%20Nqo8.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="820" data-original-width="629" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgvVC553-519Sdci9LCdUCQrwq6Q0VHQdPUz3cmcxaWMhmPkBODZjcfzFPiz42-_1aQ9t1pSu7c573Gnkz8nGEWW7lZYMh0MOQqrq12ZLjb2g6rOX2XeqKdGc0pjIyKEa-AJbD-qK8sAoTatRSN1Z3wY4Zq9JChVaNJqo6cSuXxbDvYr-JP23vEYg/w306-h400/Amino%20acids%20in%20Nqo8.png" width="306" /></a></div><br /><div><br /></div><div><br /><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>To make sense of this it's easiest to break it down in to three sections, each representing a specific process. We can start with the aspartate D139 which is protonated and hydrogen bonded to histidine H38, like this. I've faded the rest out:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEglQ6ySjQ0UxzjQIENd7zEzuZD8TE1YWtsldPmVFe3PRsbczrfHKkti245Ol1ebH1mBh8xJAGZe5mGn7_0-F4AUQgaH4qGeanyVtPflL6y7agc_5kYq2RdrUWokwyUikIOUACO0oebbsUBdc_cygbK-xCSwkbAPSPYB-nVjNgxKla0jtlx6F7Jaow/s939/H38%20and%20D139.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="939" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEglQ6ySjQ0UxzjQIENd7zEzuZD8TE1YWtsldPmVFe3PRsbczrfHKkti245Ol1ebH1mBh8xJAGZe5mGn7_0-F4AUQgaH4qGeanyVtPflL6y7agc_5kYq2RdrUWokwyUikIOUACO0oebbsUBdc_cygbK-xCSwkbAPSPYB-nVjNgxKla0jtlx6F7Jaow/w278-h400/H38%20and%20D139.png" width="278" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Two electrons are delivered to CoQ from NADH and nothing happens. A few picoseconds later one electron on CoQ "steals" a proton from histidine H38 (along with a second proton, for the second electron, taken from the Tyrosine Y87 just visible at the top of the image. I've left this out for clarity) to form reduced CoQ2H:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhqkqE1ihDSB0ax9fveSoRtQKxLrrkOx1zg3CXfhaUFrXhIulj9rY-VF0X7dfeRTdMSeay_kNcT0U2fsgVTbW8UDPUvr2dbkiAiLP3KisQjZHz5yECgzRt7DbpVkF9dzwZL26LJmanpXt4r81mRJuj35iFxvY5yHMnmTWv6LCAvUdZ52iWwCjrAPg/s860/H38%20proton%20loss.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="860" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhqkqE1ihDSB0ax9fveSoRtQKxLrrkOx1zg3CXfhaUFrXhIulj9rY-VF0X7dfeRTdMSeay_kNcT0U2fsgVTbW8UDPUvr2dbkiAiLP3KisQjZHz5yECgzRt7DbpVkF9dzwZL26LJmanpXt4r81mRJuj35iFxvY5yHMnmTWv6LCAvUdZ52iWwCjrAPg/w304-h400/H38%20proton%20loss.png" width="304" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Histidine H38 immediately replaces its lost proton by "stealing" it from aspartate D139. This aspartate becomes negatively charged and alters the protein conformation to move itself downwards (in the diagram)</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIv85h4C_dRPZHbwddv0UeQjHV6EpJB1r_tHliVG3Yb3KSuxA0sZcChMxNQ5URJLEPZgKHAhyphenhyphen7PCqCtPeg-gzNK2geL_SNrphoqcpnw-T4O3ZE1IP2mzQ9vaPYybgDa0Yg8wk9Xh5tDx9M4itXIisFGjTheu4pVQwiS6N570RdEyL25jlzRK4jcg/s854/proton%20transfer.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="854" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIv85h4C_dRPZHbwddv0UeQjHV6EpJB1r_tHliVG3Yb3KSuxA0sZcChMxNQ5URJLEPZgKHAhyphenhyphen7PCqCtPeg-gzNK2geL_SNrphoqcpnw-T4O3ZE1IP2mzQ9vaPYybgDa0Yg8wk9Xh5tDx9M4itXIisFGjTheu4pVQwiS6N570RdEyL25jlzRK4jcg/w305-h400/proton%20transfer.png" width="305" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>taking an area of negative charge with it:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhp_IwK9IT_cOUwqFUhAXm10wuCvzmyilI1X6jQiecDsB3OMRD455Pf8IzXagaVXrcx-aY3iS7MdV6IHeE7t-eZdTyzsSPBlzjzx8emwV8lF4cKc7CZ5AQ-z09g9HDxyQ4vI0fYBrgG-i3q2qwv3fr2QvFXYHZnGvpQF08_rEIURaQ53P-YVJ6n2A/s850/negative%20charge%20movement.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="850" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhp_IwK9IT_cOUwqFUhAXm10wuCvzmyilI1X6jQiecDsB3OMRD455Pf8IzXagaVXrcx-aY3iS7MdV6IHeE7t-eZdTyzsSPBlzjzx8emwV8lF4cKc7CZ5AQ-z09g9HDxyQ4vI0fYBrgG-i3q2qwv3fr2QvFXYHZnGvpQF08_rEIURaQ53P-YVJ6n2A/w306-h400/negative%20charge%20movement.png" width="306" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Now we can move on to step two and add in some more important amino acids. These red circles are all glutamates and the blue circles are all arginines:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgeM6rrbTR16cPugFtc9g5Y2sz79TKSm6lveYaEMldqxph3r_n2HwQGRTcVDeMPb-MugRrWt8-BrRufBTisXmt-hdCwqQ9rhsj2Pxw_r1qaLPut4OIvrPLyvYRIA0RXhYxkHxy_-cIcBYAmjMPQdVK5KH46vpiupx-4ti_bNF9jdu7-PMku7UaFXw/s852/Step%202%20start.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="852" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgeM6rrbTR16cPugFtc9g5Y2sz79TKSm6lveYaEMldqxph3r_n2HwQGRTcVDeMPb-MugRrWt8-BrRufBTisXmt-hdCwqQ9rhsj2Pxw_r1qaLPut4OIvrPLyvYRIA0RXhYxkHxy_-cIcBYAmjMPQdVK5KH46vpiupx-4ti_bNF9jdu7-PMku7UaFXw/w306-h400/Step%202%20start.png" width="306" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The combination of change to surrounding protein shape with the localisation of the negative charge on aspartate D213 forces the combination of the arginines with the glutamates in to electrostatically bonded pairs shown as green ovals. The dotted green oval is my guess, the two solid ones are specified in the paper:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhyuvxnpZ8Ku0V_AjgVNeL-A4c29n7OAUTpb-jxAWs6toXcpk4LDAmKiGDppdzccbvpwmp1UXi_xuTThUEu6q7m4o8Hto4rOl688BJpGhmnszaqtoD0dDoX2OB_qrXpHQozYl26C_yhwR42whfUareRAmpHvSRlJSKZ8GtolKBU_AFev1p5m4mBdw/s856/green%20ovals.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="856" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhyuvxnpZ8Ku0V_AjgVNeL-A4c29n7OAUTpb-jxAWs6toXcpk4LDAmKiGDppdzccbvpwmp1UXi_xuTThUEu6q7m4o8Hto4rOl688BJpGhmnszaqtoD0dDoX2OB_qrXpHQozYl26C_yhwR42whfUareRAmpHvSRlJSKZ8GtolKBU_AFev1p5m4mBdw/w305-h400/green%20ovals.png" width="305" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which repositions the polar amino acids like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhGpEjnRhpZwAWzkEAo4Mr07E02gh6cO1aBdFNEfvy-sFHhh1ux7vn-hDgMKqCfnHkTp7QYhFxCaitGxRLnq-YRRvedqmwKwUGyg47oJpGhyxb1YkCQoXRQWe5wBJMjD1z_Fr4uJuEhBKLfgjydeaHn6T8JKaS7fiLxlw7skUk1ZzpNU7zND8z-uQ/s854/bonded%20green.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="854" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhGpEjnRhpZwAWzkEAo4Mr07E02gh6cO1aBdFNEfvy-sFHhh1ux7vn-hDgMKqCfnHkTp7QYhFxCaitGxRLnq-YRRvedqmwKwUGyg47oJpGhyxb1YkCQoXRQWe5wBJMjD1z_Fr4uJuEhBKLfgjydeaHn6T8JKaS7fiLxlw7skUk1ZzpNU7zND8z-uQ/w305-h400/bonded%20green.png" width="305" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Quite how this rearrangement forms a proton channel is unclear (or whether protons are simply transferred from amino acid to amino acid without a water channel forming, there doesn't appear to be a water channel modelable, yet) but the paper suggest it does so and the negative charge zone encourages protons to transfer from the bulk solvent of the cytoplasm to the centre of the complex:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0anSEjfFZPIWrz-BxtTB0oRBn84pxv6lqlYt00BTDccX2pIkR5D6oYOllfQw2D5cwRJr9z0F1JoSea5xIi9Outf0XUk_hgat2RndRA4z6HT7GctsU0e9pdddh8lADVU_-lZOup-NmSmKoTjoHQ8zHTyLSXhrmVrRLieNg5S6fKzwEpzHkN5S7vQ/s866/protons%20enter.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="856" data-original-width="866" height="395" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0anSEjfFZPIWrz-BxtTB0oRBn84pxv6lqlYt00BTDccX2pIkR5D6oYOllfQw2D5cwRJr9z0F1JoSea5xIi9Outf0XUk_hgat2RndRA4z6HT7GctsU0e9pdddh8lADVU_-lZOup-NmSmKoTjoHQ8zHTyLSXhrmVrRLieNg5S6fKzwEpzHkN5S7vQ/w400-h395/protons%20enter.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The final step involves these amino acids, mostly glutamates with an aspartate D72 at the end of the chain:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiKyglIOtpaPHvn1NBXNZRO9Yf0HHGNTMKlva6S0HAcwsZEFgqtV6zkv-4Ui3NELycguzDWA1G9nI6Qck5_A-A73wCpt6VHyErQiHSZlL2LSr87nA3yMleVPJVdLh-_1sPSacANcmNQm65p-MXln7tT3KonZBLh1NIEfKRL6BeF7m90l9Itw_q3Wg/s883/last%20step%20start.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="883" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiKyglIOtpaPHvn1NBXNZRO9Yf0HHGNTMKlva6S0HAcwsZEFgqtV6zkv-4Ui3NELycguzDWA1G9nI6Qck5_A-A73wCpt6VHyErQiHSZlL2LSr87nA3yMleVPJVdLh-_1sPSacANcmNQm65p-MXln7tT3KonZBLh1NIEfKRL6BeF7m90l9Itw_q3Wg/w295-h400/last%20step%20start.png" width="295" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The conformation change in the protein structure moves these amino acids towards the source of protons </div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuVNLLWh9LSye6wYtGaz-2WPdhnJE5cDp5G5D_gK6-1XGyp_m3tg0dKLTjOoX4YpqlGKdhSNC3ahdozSJ4TuKMmu2nvf23WVmnotcHiyW4ngrpxu_kj8bE4VW1PIMSAuiPogRP1DwTTmIe3k7lzsmcFKU_R1Ls9he-cikP3ldWdYQrUMALnw-ryQ/s875/reposition%20acid%20residues.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="875" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuVNLLWh9LSye6wYtGaz-2WPdhnJE5cDp5G5D_gK6-1XGyp_m3tg0dKLTjOoX4YpqlGKdhSNC3ahdozSJ4TuKMmu2nvf23WVmnotcHiyW4ngrpxu_kj8bE4VW1PIMSAuiPogRP1DwTTmIe3k7lzsmcFKU_R1Ls9he-cikP3ldWdYQrUMALnw-ryQ/w298-h400/reposition%20acid%20residues.png" width="298" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and puts them in to a microenvironment which makes them highly avid to gain a proton, which they do:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfon1yU36bkDdUBgoXigQThqpwk4KBzHr41RiSh_ijjkdM8uo7LzkB8aGXZMqGlQv9xwBAfQK5d0ib8oSDryh686bLk4c1W_6edZdyvSsNosWDHT8rZfQGYrtG2CnHX1mBp7u01OFj69TO2qGKQKgtAr62wC5gCWlzptRD0U7Kbhqi4NkfMmt29A/s872/protonation.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="872" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfon1yU36bkDdUBgoXigQThqpwk4KBzHr41RiSh_ijjkdM8uo7LzkB8aGXZMqGlQv9xwBAfQK5d0ib8oSDryh686bLk4c1W_6edZdyvSsNosWDHT8rZfQGYrtG2CnHX1mBp7u01OFj69TO2qGKQKgtAr62wC5gCWlzptRD0U7Kbhqi4NkfMmt29A/w299-h400/protonation.png" width="299" /></a></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Working on the basis of electrostatic coupling between well investigated antiporter-like pumping modules it looks to me very much like the protonation of aspartate D72 provides the "kick" to the messy fourth proton pathway between NuoN and the small membrane subunits NuoA, J and K:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj0ot7UUHNJwYGESNn4MjtcX5qQgTE9r0kXa5K1CCvEW6H2iPBpFKz4xcWr5mzF9jeiuCc4PVP09eQHPvGvddb02oRmPCvLJI-YJ6mqkWRMPyruCxcqg7pLnzCFgWdiooTeRX0uMYN3PdQxNnO8o5ci9W0f34RviCC3ZQ2IMTpv6GPHrQ8yovwyEw/s879/kick.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="879" data-original-width="820" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj0ot7UUHNJwYGESNn4MjtcX5qQgTE9r0kXa5K1CCvEW6H2iPBpFKz4xcWr5mzF9jeiuCc4PVP09eQHPvGvddb02oRmPCvLJI-YJ6mqkWRMPyruCxcqg7pLnzCFgWdiooTeRX0uMYN3PdQxNnO8o5ci9W0f34RviCC3ZQ2IMTpv6GPHrQ8yovwyEw/w374-h400/kick.png" width="374" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Up until now pretty much all of what I have described is what is reported in the paper from their extensive modelling work. Now I'm going to speculate.</div><div><br /></div><div>I don't think these NuoH protons go anywhere towards being transported to the periplasm. I think they go back the way they came. One of the crucial steps after the reduction of CoQ to CoQ2H is the restoration of the protonation of the amino acids which have provided the protons to join with the electrons on CoQ to give a neutral molecule. It's not at all clear where these replacement protons might come from, so I feel free to speculate. In this particular complex I example we are talking about reprotonating aspartate D139 and tyrosine Y87, either side of the CoQ binding pocket. Like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnRc78NHUmnstJJx2LBtNkp5Cz6ET7q0Jie7SzEkfdtl2AH44-wqPzLiZYdnpePx-PByDduc7eanryLIcHFz6y8otrkIaxmQ1fdJO_OVuhXBIc_0sjEU_zgUxvkVNUkPjoaoiKKOWfKqm7srKzVdcgNge0mfCt3HM_Vw6PKYDHGGBl6LAmYvZMUg/s866/restoration.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="866" data-original-width="652" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnRc78NHUmnstJJx2LBtNkp5Cz6ET7q0Jie7SzEkfdtl2AH44-wqPzLiZYdnpePx-PByDduc7eanryLIcHFz6y8otrkIaxmQ1fdJO_OVuhXBIc_0sjEU_zgUxvkVNUkPjoaoiKKOWfKqm7srKzVdcgNge0mfCt3HM_Vw6PKYDHGGBl6LAmYvZMUg/w301-h400/restoration.png" width="301" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>In particular the restoration of protonation of aspartate D139 will allow it to return to a hydrogen bonded to histidine H38 position and allow protein conformation to return to the baseline level overall, leaving the system ready to fire again.</div><div><br /></div><div>This speculation is compatible with a non proton pumping function of the half-channel in NuoH/Nquo8 but a crucial function in transmitting the energy from CoQ reduction to the antiporter modules. It also gives a speculative mechanism for the reprotonation of the amino acids deprotonated in CoQ reduction. I like the idea. It makes sense (which clearly does not mean it is correct!).</div><div><br /></div><div>I would also guess that in an optimised system that only two protons are used to effect the aspartate D72 "kick" and these two protons are the ones which are returned to neutralise the changes around the CoQ binding pocket.</div><div><br /></div><div>I'm now set to try and work out what evolution was doing to set up a pre-adaptation to this rather bizarre system. Fingers crossed.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com11tag:blogger.com,1999:blog-36840063.post-61246503969299445502023-12-20T05:35:00.004+00:002023-12-20T08:24:27.888+00:00Life (38) Water WiresThese people have ideas about the water channels for the fourth proton in modern complex I.<div><br /><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024853/" target="_blank">Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I</a></div><div><br /><div><div>They even made a film about its water channel from the cytosolic proton source to the central horizontal transfer zone, and it's available in the supplementary data to down load:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><iframe allowfullscreen='allowfullscreen' webkitallowfullscreen='webkitallowfullscreen' mozallowfullscreen='mozallowfullscreen' width='320' height='266' src='https://www.blogger.com/video.g?token=AD6v5dxUA2YckbKtDERx8sT9RE4wQdS9yeJb2yQPmuttF9K7B6iFiJO5DugUUJzcyX5a52TLxuf8_Z4yfSw' class='b-hbp-video b-uploaded' frameborder='0'></iframe></div><div><br /></div><div>I had to look very carefully in slo-mo, advancing the frames manually, at some points one by one, to work out what is happening. It's simpler if you edit out the (undoubtedly very important) shuffling of hydrogen bonds to the isoleucine on the right hand side of the image. I've made a simplified gif of the track of the proton from bulk solvent at the top to where it protonates the glutamate at the bottom. This is only a half-channel, quite where the second half-channel is located is unclear but there are several possibilities discussed. Here's the grossly simplified gif with the proton highlighted in blue and the shuffling of hydrogen bonds to the isoleucine cut out:</div><div><br /><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgTmkWUTm9vzqVpbVf385uhWrqk8TNd0sOtkJtgyxW4JrHTCYaXMECHxFKV4-C0CRELEhC9Avh5X28d9sXBsCt5lKDUs8K9TFq2Yjcj5hhl_Agd4nb9aDE5MeyTvplHN-2nEmayaH0RNCSOi6GXMVx3sLE5EKjkG17q-kLFRX7y4V20NqAyKe-QsQ/s928/Hydronium%20proton%20channel.gif" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="928" data-original-width="880" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgTmkWUTm9vzqVpbVf385uhWrqk8TNd0sOtkJtgyxW4JrHTCYaXMECHxFKV4-C0CRELEhC9Avh5X28d9sXBsCt5lKDUs8K9TFq2Yjcj5hhl_Agd4nb9aDE5MeyTvplHN-2nEmayaH0RNCSOi6GXMVx3sLE5EKjkG17q-kLFRX7y4V20NqAyKe-QsQ/w379-h400/Hydronium%20proton%20channel.gif" width="379" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>We can summarise this as a proton from bulk solvent:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi9BHwCLOPb69llelxZ1tJXqoHUKuO7r4WfTsNjZI0LAJ92wDsHbbz7z5NihZ7YSz4g6TJ5TGGYu4lSH1TXVRO1mkkbZimXO_zb0acqbPXoIWEAMejWCg7GTRhKSyEvrKDndFLfK56W447Vqb6Q3jFE5-d-gUn51e-Luf2_EJAFnd67nUYtr4t6Ig/s1129/Ch4%201.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1070" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi9BHwCLOPb69llelxZ1tJXqoHUKuO7r4WfTsNjZI0LAJ92wDsHbbz7z5NihZ7YSz4g6TJ5TGGYu4lSH1TXVRO1mkkbZimXO_zb0acqbPXoIWEAMejWCg7GTRhKSyEvrKDndFLfK56W447Vqb6Q3jFE5-d-gUn51e-Luf2_EJAFnd67nUYtr4t6Ig/w303-h320/Ch4%201.png" width="303" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>"travels" to a "half way" glutamate and protonates it, which reconfigures the proteins to expel the water molecules and so closes the input side of the channel:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhkTJpJ8KzsTrrb2DcwP1lmUZ-iZTh1NASdZ5Mhx_W9I7kNd6QVsGjZZegKZWtM9AKxf1vK4RWhmt0NMM6aJDSk-wKhg0TL0DjrH6QEW3kmu5N-U7z4xIFxxXAP6xsrGNiVCb_NxmVA7O7zzjbD0l2n4Y1MtsNHCCM3dtw3snGQtjBb2MnWiWtWpg/s1129/ch4%202.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1070" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhkTJpJ8KzsTrrb2DcwP1lmUZ-iZTh1NASdZ5Mhx_W9I7kNd6QVsGjZZegKZWtM9AKxf1vK4RWhmt0NMM6aJDSk-wKhg0TL0DjrH6QEW3kmu5N-U7z4xIFxxXAP6xsrGNiVCb_NxmVA7O7zzjbD0l2n4Y1MtsNHCCM3dtw3snGQtjBb2MnWiWtWpg/s320/ch4%202.png" width="303" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Now let's speculate wildly, as you do. Next comes the "kick" from the doohickey in the redox arm and transmitted through NuoH (or Nqo8 in bacteria):</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiFZA-bDX3yb36RVz5crNlylqvMYM6yLv4H0xzyl09HBh336xnH0Pj80MpHTlI5B-viGt0X9HA5lELczJswfaO-rNhq85TnmvUfUshLRyvdpizPwph27VVNbu5ydkCNXPpKLQpHC3zGlA4O0-t0NKpfYN2t-bNor8ibtUmCGShT74yXol6RqsdhVw/s1231/ch4%203.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1231" height="293" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiFZA-bDX3yb36RVz5crNlylqvMYM6yLv4H0xzyl09HBh336xnH0Pj80MpHTlI5B-viGt0X9HA5lELczJswfaO-rNhq85TnmvUfUshLRyvdpizPwph27VVNbu5ydkCNXPpKLQpHC3zGlA4O0-t0NKpfYN2t-bNor8ibtUmCGShT74yXol6RqsdhVw/w320-h293/ch4%203.png" width="320" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>There is a convenient lysine to accept it. Tucked in behind the lysine is another glutamate which can next be protonated:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgdPm-aosutQomtmmriru-euDazZTtoMLy31-qvmi6cxQFeOOtW972X9G6qpOcsfhQK3rmDgH7DwoHg1UpBCvDwkv9_wubkUGIooW5CZQIk4Nc_FJmsn3WiIbPbCREWH3-3sXPX6wuT5IAV8WnYUdPH8-xr0wNKXIfPczx0K1m03-1KWam_lIh2Q/s1231/ch4%204.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1231" height="293" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjgdPm-aosutQomtmmriru-euDazZTtoMLy31-qvmi6cxQFeOOtW972X9G6qpOcsfhQK3rmDgH7DwoHg1UpBCvDwkv9_wubkUGIooW5CZQIk4Nc_FJmsn3WiIbPbCREWH3-3sXPX6wuT5IAV8WnYUdPH8-xr0wNKXIfPczx0K1m03-1KWam_lIh2Q/s320/ch4%204.png" width="320" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>to open the water channel to the periplasm</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh4DleLEXVnYA_Kd2LH8LK7JRIY0vGJFRvt7vnxzmcE2CVQouVW2r7YVpOY7O_QRCeruukvHfMADtxRSR-JOe7GBH3qUhcRO2tsXzDXD2F9okuN0fAdz1fR4m2nXJG8K-iRCcndRnvzSQr4l9RlyUiZpJFO0z57zc7UhK72RP7Eaj3_gty5USLq8g/s1022/ch4%205.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1022" data-original-width="658" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh4DleLEXVnYA_Kd2LH8LK7JRIY0vGJFRvt7vnxzmcE2CVQouVW2r7YVpOY7O_QRCeruukvHfMADtxRSR-JOe7GBH3qUhcRO2tsXzDXD2F9okuN0fAdz1fR4m2nXJG8K-iRCcndRnvzSQr4l9RlyUiZpJFO0z57zc7UhK72RP7Eaj3_gty5USLq8g/w258-h400/ch4%205.png" width="258" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and complete the transfer to the 4th proton:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgmkZJMm5DT0RjwAJ0oIGWoeIX7Bze0H4lZOilGNZDjI1kc9BnKV8pzTJYv0z3Vmdf4j3Oh_WovGIN-HovrwXa6wP1rMknb16vAw32ryMNY6VfiCiAS1jXi3EDYkxFnzZMmK1UJrLALE30BJYLYONDxyYFC0N_RwFzlnX1zZqPy24H9Uc983NYlHw/s1022/ch4%206.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1022" data-original-width="568" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgmkZJMm5DT0RjwAJ0oIGWoeIX7Bze0H4lZOilGNZDjI1kc9BnKV8pzTJYv0z3Vmdf4j3Oh_WovGIN-HovrwXa6wP1rMknb16vAw32ryMNY6VfiCiAS1jXi3EDYkxFnzZMmK1UJrLALE30BJYLYONDxyYFC0N_RwFzlnX1zZqPy24H9Uc983NYlHw/s320/ch4%206.png" width="178" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This fourth channel is messy. Nuos N, M and L are clearly lifted straight from the MRP antiporter as complete units. The above pathway is a hotchpotch of one edge of Nuo N and the small membrane subunits Nuos A, J and K. If you had to guess, these small subunits might be remnants of the Na+ channel of the MRP antiporter but I've not seen this hypothesised anywhere.</div><div><br /></div><div>I'll pause here because the principle of water wires and proton transfer appears to be very generic, the three tidy channels will be functionally very similar to the messy fourth channel described here.</div><div><br /></div><div>Unlike NuoH. That's the next post. It's totally different.</div><div><br /></div><div>Peter</div></div></div></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com4tag:blogger.com,1999:blog-36840063.post-75153425225031607562023-12-12T20:42:00.005+00:002023-12-14T19:19:24.104+00:00Life (37) Just a gif of complex IIf we take the complex I from the <a href="https://www.sciencedirect.com/science/article/pii/S0005272812000394?via%3Dihub" target="_blank">doohickey paper</a><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiSHSCtoMvO4R5E_7YPPVWzwOqk81ckgjAhU0ZGpP8fLzuBCXEekz1RD_zWeGKQvoI_ekxnkeDVuP3go8pHjr9Y7qMRpwXh4BayzXMF8jsZnxFnqecQLmwCFzt-AicuTzGqyD8T8-GhakBKeiOzSHF-xrHPCyXsFKcmi1ye9l7_jdHzfL8rIyfC4w/s758/Comp%20I%20doohickey.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="629" data-original-width="758" height="333" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiSHSCtoMvO4R5E_7YPPVWzwOqk81ckgjAhU0ZGpP8fLzuBCXEekz1RD_zWeGKQvoI_ekxnkeDVuP3go8pHjr9Y7qMRpwXh4BayzXMF8jsZnxFnqecQLmwCFzt-AicuTzGqyD8T8-GhakBKeiOzSHF-xrHPCyXsFKcmi1ye9l7_jdHzfL8rIyfC4w/w400-h333/Comp%20I%20doohickey.png" width="400" /></a></div><br /><div><br /></div><div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and also the mirror image of complex I from the <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547640/" target="_blank">water channels paper</a></div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgcVsT4r5tV9pukmcZvo8B3rzl0bU7suXXT8v8H-i_EfW7pj0RqU0j-d-BsfptSMBIwXPbxokxGGGnkXUDpN8_g0Cx9uonsxXvj7HP8GwXTY0HGxPgPUA6PiImheR3FCFLEld9M9-ao9m0FnEQecTDnVWcu0aELHFXPpcS8s-MmsjCD5vWJP5-6iQ/s854/Comp%20I%20water%20channels.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="656" data-original-width="854" height="308" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgcVsT4r5tV9pukmcZvo8B3rzl0bU7suXXT8v8H-i_EfW7pj0RqU0j-d-BsfptSMBIwXPbxokxGGGnkXUDpN8_g0Cx9uonsxXvj7HP8GwXTY0HGxPgPUA6PiImheR3FCFLEld9M9-ao9m0FnEQecTDnVWcu0aELHFXPpcS8s-MmsjCD5vWJP5-6iQ/w400-h308/Comp%20I%20water%20channels.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and highlight the important bits, we get this</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjKtf1Bwl65ibyz6ex5__5C99ReiJNtzgBVpbMJ2T-2XYdanySmM0WRTe_VCoR7axBnBdgYRowwGyRH6WhOD7NBCO5OpUnJ_VHbEwTAvkEfnYXtIXYWTdUTQKwyNZw_BvLpzbl2bz4rf4xtfnYCwYRH1WxJqkVmvBYnsYAsG27m7nhSaxD3jts5cg/s720/Doohickey%20and%20water%20channels.jpg" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="540" data-original-width="720" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjKtf1Bwl65ibyz6ex5__5C99ReiJNtzgBVpbMJ2T-2XYdanySmM0WRTe_VCoR7axBnBdgYRowwGyRH6WhOD7NBCO5OpUnJ_VHbEwTAvkEfnYXtIXYWTdUTQKwyNZw_BvLpzbl2bz4rf4xtfnYCwYRH1WxJqkVmvBYnsYAsG27m7nhSaxD3jts5cg/w400-h300/Doohickey%20and%20water%20channels.jpg" width="400" /></a></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which can be rearranged to give this</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjHXQ_GqoXiTSm9SRkjTXCqJ094du2e9K7V1B6BybsTcBWaKAT0zRfUUGzD4XWdIFmMEiMB6urNN4LNZ48lE7HPisD7TcyTx-EnrdezDQObyFOkqX5gpxNcL3x1qDkOtTj3MX9WEclSgFCio2Lx26MRd-2vQIs1y4N5jZmYyOBbk6-qwRWqKuZykA/s670/Superimposed.jpg" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="539" data-original-width="670" height="321" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjHXQ_GqoXiTSm9SRkjTXCqJ094du2e9K7V1B6BybsTcBWaKAT0zRfUUGzD4XWdIFmMEiMB6urNN4LNZ48lE7HPisD7TcyTx-EnrdezDQObyFOkqX5gpxNcL3x1qDkOtTj3MX9WEclSgFCio2Lx26MRd-2vQIs1y4N5jZmYyOBbk6-qwRWqKuZykA/w400-h321/Superimposed.jpg" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which animates as a gif to show how the change in the doohickey, induced by electron transfer to CoQ, is associated with the protonation (yellow cross proton) of an amino acid in the NuoH/Nqo8 water channel:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh0xwC2SJdPgx-xR6WmT15AfepYAKhKbDPYjNG251KEZ6p9gmXUHjcGcR8HXuJ1hYleypYaafrXKf5tIfGEOY-x02wWBuUTafkXTtY65ao1dfWtHtPuzPEMTSpxF18Ck6ugbIybJgE46Rr3RPnKAAqHyv2mLlmu4EgV7M_5fYCyShghlqQdiwyBfg/s670/protonation%20gif.gif" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="539" data-original-width="670" height="321" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh0xwC2SJdPgx-xR6WmT15AfepYAKhKbDPYjNG251KEZ6p9gmXUHjcGcR8HXuJ1hYleypYaafrXKf5tIfGEOY-x02wWBuUTafkXTtY65ao1dfWtHtPuzPEMTSpxF18Ck6ugbIybJgE46Rr3RPnKAAqHyv2mLlmu4EgV7M_5fYCyShghlqQdiwyBfg/w400-h321/protonation%20gif.gif" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and then we can add in the orange-crossmarked pumped protons like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgcEdz77as3RNiE0qcA_9jNVe34i8m8kLX8DECKplyUwjBvSMC0AtSujezIQf9uX_h0uSwF6oA3pBt8lwxnjiHvIeHooW3FH-rN_0cbwJ-Szcd_5Hyut5Pfwk-tyCWO-QofIvxIErGSgZc1eD78_VSM4c_oRqPZ1qhkNW-zMGL1IE2OTMwVgsogbw/s670/ezgif.com-gif-maker%20(2).gif" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="539" data-original-width="670" height="321" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgcEdz77as3RNiE0qcA_9jNVe34i8m8kLX8DECKplyUwjBvSMC0AtSujezIQf9uX_h0uSwF6oA3pBt8lwxnjiHvIeHooW3FH-rN_0cbwJ-Szcd_5Hyut5Pfwk-tyCWO-QofIvxIErGSgZc1eD78_VSM4c_oRqPZ1qhkNW-zMGL1IE2OTMwVgsogbw/w400-h321/ezgif.com-gif-maker%20(2).gif" width="400" /></a></div></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This story explains the simple translocation of the three protons through the three antiporter-like subunits but gives us no insight as to how the fourth proton might be translocated.</div><div><div><br /></div><div>And trying to reverse engineer the doohickey-NuoH water channel seems like the best chance of guessing at what happened as an intermediate process between using a geothermal proton gradient and generating a metabolic proton gradient. Each step must be immediately beneficial to early LUCA.</div><div><br /></div><div>Peter</div></div></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com7tag:blogger.com,1999:blog-36840063.post-28936205852190615222023-12-10T15:06:00.004+00:002023-12-10T16:40:05.783+00:00Life (36) Complex I. Protons are large<div>Now it's time to look at this paper:</div>
<div><br /></div>
<div>
<a href="https://www.ncbi.nlm.nih.gov/pubmed/28716925">Symmetry-related proton transfer pathways in respiratory complex I</a>
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<div>
We can start with this image of the membrane part of complex I from <i>Thermus thermophilus</i> which shows the water channels of the antiporter-like pumping units. The NADH reduction site is not shown but is at the right hand end, above the membrane. Nqo8 is the equivalent of mitochondrial NuoH, the universal adaptor between the redox limb and the membrane section of all similar complexes:<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhsnUcJY6e-SZ7OSSaIDStV0-2iCvD_A1q5bJYLe-4Dy8K7tMwmpFCv6L5r0eFWQfnyVjWwEHaQV7oK5LFi0WOGEU1ix_WVk0oEz1YiqefV4h-J5IutXJqh92XMGzSIjyJxeNq5L8M_FIZcCsBugApQbWi6NUDwqwvBwWM7vW_TMjfQThh0V0-jrA/s1115/Water%20channels.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em; text-align: center;"><img border="0" data-original-height="448" data-original-width="1115" height="161" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhsnUcJY6e-SZ7OSSaIDStV0-2iCvD_A1q5bJYLe-4Dy8K7tMwmpFCv6L5r0eFWQfnyVjWwEHaQV7oK5LFi0WOGEU1ix_WVk0oEz1YiqefV4h-J5IutXJqh92XMGzSIjyJxeNq5L8M_FIZcCsBugApQbWi6NUDwqwvBwWM7vW_TMjfQThh0V0-jrA/w400-h161/Water%20channels.png" width="400" /></a><br /><br /><br /><br /><br /><br /><br />Water channels are important
if you want to move protons through a protein. The problem is that protons are
hydrated, at their simplest they can be thought of as the hydronium ion, H<sub>3</sub>O<sup>+</sup>,
but it seems more likely that 4-6 water molecules form a structure around
this giving a large molecule restricted to channels where there are adequate ionised amino acids to form an hydrophilic environment rich in water molecules.</div><div><br /></div><div>The sketch shows the three conventional water pathways through Nqo12, 13 and 14 and a less straight forward probable channel through Nqo8 where the proton exit route is shown as a dashed section of black line rather than the solid line associated with the clear cut water channels in Nqo12, 13 and 14.</div><div><br /></div><div>The channels open and close in response to the protonation of key amino acids. This is the schematic of how this group feels complex I works:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEja6G8lToTXhdNvOVXTWahHdlC7wusxTg6HNgw_JksdIdfte_91FnN0T5dKDLOxy7Ihh_5WivYX8v9U9WrjgHH1MAORrnlJ5scBCvNidzxRIpZELfyvFKsj_HRJB17YNqjW-9Aoh7JMfSyUXTGIqB1IWcZw-klcbADWVs011VUZ-KbGq7O93-sc-Q/s1133/Original%20Fig%206.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1133" height="399" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEja6G8lToTXhdNvOVXTWahHdlC7wusxTg6HNgw_JksdIdfte_91FnN0T5dKDLOxy7Ihh_5WivYX8v9U9WrjgHH1MAORrnlJ5scBCvNidzxRIpZELfyvFKsj_HRJB17YNqjW-9Aoh7JMfSyUXTGIqB1IWcZw-klcbADWVs011VUZ-KbGq7O93-sc-Q/w400-h399/Original%20Fig%206.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>I'll briefly go in to detail of the section marked within the red oval to look at the key mechanism:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhizIxUBst_7KQycltPsktX4r6CBEM995wSsZ3dpTRkuxtk76aPegaY7touCIJX_Hatw51zGMZQD4iSDr4xHwWZEJE-pb67qQ606BGTwCazTYjCD_5506jWz6-_eK3fgJR_NMdIBdxRJIcbQWa6UDQLZ_9MyhOdcmqQNcZiyxNfoG0xZ_ZaNex35w/s1133/red%20circle%20kick.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1133" height="399" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhizIxUBst_7KQycltPsktX4r6CBEM995wSsZ3dpTRkuxtk76aPegaY7touCIJX_Hatw51zGMZQD4iSDr4xHwWZEJE-pb67qQ606BGTwCazTYjCD_5506jWz6-_eK3fgJR_NMdIBdxRJIcbQWa6UDQLZ_9MyhOdcmqQNcZiyxNfoG0xZ_ZaNex35w/w400-h399/red%20circle%20kick.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The pumping function is driven by the protonation of an amino acid in Nqo8 affecting a lysine/glutamic acid pair within Nqo14. While Nqo8 is de-protonated the lysine (K)186 is electrostatically attached to the glutamate (E)112.</div><div><br /></div><div>While Nqo8's amino acid is protonated it attracts the -ve charged glutamic acid and repels the +ve charged lysine.</div><div><br /></div><div>When Nqo8 is de-protonated the lysine and glutamate return to their original electrostatic association. In detail it looks like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7PWfPyO8JC2JPM-ltIpUaXf6i23dzEYq24wexymYcelq9dMB5BMGa7txLnPGjlxsnjHiS-aUtGiwIdNWhIV5fJXNwhAkG2Li3rRbsaDO3XJFWAVWjN1kcHg-JokfpLpyGfn4vtoiAs3VREGnZXu8rMbGX_UhPUaQ2pXDOdPBi_7x9ZlB-nrQv-w/s1366/kicking.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1366" height="330" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7PWfPyO8JC2JPM-ltIpUaXf6i23dzEYq24wexymYcelq9dMB5BMGa7txLnPGjlxsnjHiS-aUtGiwIdNWhIV5fJXNwhAkG2Li3rRbsaDO3XJFWAVWjN1kcHg-JokfpLpyGfn4vtoiAs3VREGnZXu8rMbGX_UhPUaQ2pXDOdPBi_7x9ZlB-nrQv-w/w400-h330/kicking.png" width="400" /></a></div><br /><div style="text-align: left;"><br /></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The process is repeated at the junctions between Nqo14-13 and 13-12. There is a typo in the diagram on the top line, lysines (K)345 and (K)385 are shown as protonated but clearly can't be for the scheme to work.</div><div><br /></div><div>Aside: The junction 13-12 actually has a glutamine bumping up against aspartate/lysine electrostatic bonded couplet. This seems to work but is not as intuitive as the glutamate/lysine pairs. I'll leave it alone as the story is complicated enough as it is. End aside.</div><div><br /></div><div>I've corrected these "blue +" symbols to open circles to make sense of it and have highlighted the repeats with ovals circles again:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgQlCE8uMZKK3FiFUSY2jZHnrui96oXkSELMvtUlg5BC_on18JgmDv2wXfs0XYEnsDXHUuIQTuu0CGd0-S1mjvsQs20yNZoZeSvIMZoN1jk5G9KuU25Or0tQYKsthUw6qznzPPsHrponXiYSwXBWezQKV-_ne0uLFwMiVj_sL3XawBqUmOQbDGdcg/s1133/repeat%20circles.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1133" height="399" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgQlCE8uMZKK3FiFUSY2jZHnrui96oXkSELMvtUlg5BC_on18JgmDv2wXfs0XYEnsDXHUuIQTuu0CGd0-S1mjvsQs20yNZoZeSvIMZoN1jk5G9KuU25Or0tQYKsthUw6qznzPPsHrponXiYSwXBWezQKV-_ne0uLFwMiVj_sL3XawBqUmOQbDGdcg/w400-h399/repeat%20circles.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /></div><div><br /></div><div>Having got that sorted let's fade Nqo12 and 13 out and look at pumping in Nqo14. In the resting state the cytosol side water channels are open and this allows protonation of the central lysine residue:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgHimrSfkUkZ0_EzCiKIeAtZKB80EpH0_ToV6M32sIq6nFcGkOFaFGtUcOTm8RFqeRfeOT8CQpGofkUicK1TsdlDMkEbGlq2QLY7Xpb0HtaUykot5VbvMYUq9OJErW4OOwSPvfOjUURWaSG3-gbgyRCKruudamuD2UERsj1m_DCJBRmxlXp8ATlYg/s1260/Nqo14%201.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="593" data-original-width="1260" height="189" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgHimrSfkUkZ0_EzCiKIeAtZKB80EpH0_ToV6M32sIq6nFcGkOFaFGtUcOTm8RFqeRfeOT8CQpGofkUicK1TsdlDMkEbGlq2QLY7Xpb0HtaUykot5VbvMYUq9OJErW4OOwSPvfOjUURWaSG3-gbgyRCKruudamuD2UERsj1m_DCJBRmxlXp8ATlYg/w400-h189/Nqo14%201.png" width="400" /></a></div><br /><div><br /><br /><br /><br /><br /><br /><br />Nqo8's amino acid then protonates</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgTGSFoLdfypBphJHVvm4uub-qtNwzV36DdXvS7-a_oklWa3RS3FHLyR53J0LyWrcbVnvsyULvXhy104qk19Wxqru5QWRlsztwGM83gPTiMhzyk00Z-tiAjQcu2R5PO6FhWEyqUM9J1pb_jRCTd1LZgLu1KbcEqzKqa8TCECy9qEkJ6qz_R8Xj8IQ/s1260/Nqo14%202.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="593" data-original-width="1260" height="189" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgTGSFoLdfypBphJHVvm4uub-qtNwzV36DdXvS7-a_oklWa3RS3FHLyR53J0LyWrcbVnvsyULvXhy104qk19Wxqru5QWRlsztwGM83gPTiMhzyk00Z-tiAjQcu2R5PO6FhWEyqUM9J1pb_jRCTd1LZgLu1KbcEqzKqa8TCECy9qEkJ6qz_R8Xj8IQ/w400-h189/Nqo14%202.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and separates the adjacent lysine/glutamate pair which pushes the orange-crossed proton from its lysine to another lysine adjacent to Nqo13. It closes the cytosolic water channels in the same action</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh1GFA7sJ8uuvVfK-HfM6zqmaAPxslWJtE5NadDxilbFM-c7Z20Zc8LGdz62HN-0aSEW7cG6WI4EuknShJjfiH0RilmdcEuDaaVZbph_WsD9lw7QmDPXCTRrZwUDmgBfOBBXjJ9fY15O_ysHypQ-ON37J1TEVC8v85mk4LFdwPJaZJsoCu1zpJn5A/s1287/Nqo14%203.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="652" data-original-width="1287" height="203" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh1GFA7sJ8uuvVfK-HfM6zqmaAPxslWJtE5NadDxilbFM-c7Z20Zc8LGdz62HN-0aSEW7cG6WI4EuknShJjfiH0RilmdcEuDaaVZbph_WsD9lw7QmDPXCTRrZwUDmgBfOBBXjJ9fY15O_ysHypQ-ON37J1TEVC8v85mk4LFdwPJaZJsoCu1zpJn5A/w400-h203/Nqo14%203.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This displaced orange-crossed proton binds to Nqo13's lysine/glutamine pair and so propagates the effect along the central water channel within the membrane arm</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg5tJs9kyTZKfXRCpLRTt_LHy3TvXfPvom664eHOx9O7AMvWnQy8doljGhcxSLSXDPE5XNAv8v_nPWgBAYH3HVeeLfrVJWer5nxft1nfnAnc3l1juFQi4MD_7jywrwbXD2yFevKV27pBN_wVF1BO4SJCfspRB_4VgRtawUOjdiqAIYYQiBt1Pim2w/s1285/Nqo14%204.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="652" data-original-width="1285" height="203" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg5tJs9kyTZKfXRCpLRTt_LHy3TvXfPvom664eHOx9O7AMvWnQy8doljGhcxSLSXDPE5XNAv8v_nPWgBAYH3HVeeLfrVJWer5nxft1nfnAnc3l1juFQi4MD_7jywrwbXD2yFevKV27pBN_wVF1BO4SJCfspRB_4VgRtawUOjdiqAIYYQiBt1Pim2w/w400-h203/Nqo14%204.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This movement also opens the periplasmic water channels</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjE8fY5B1FuGyo1n9wTlaYGDg82gWNCY8MkZyusDlWb8p7e-fD68naqj6501sSVfi35bbg-Rvw-tsMEVLRv2-BBjVpBhyphenhyphenPewGJjgRUjXCGVnPHZULEEgbpEdxLgpPcLosbcNzZExFnN3sbBh0g3Vir2nt-kEMcqhRFlhUuGSatGT9O6ILzd7ay0uw/s1285/Nqo14%205.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="625" data-original-width="1285" height="195" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjE8fY5B1FuGyo1n9wTlaYGDg82gWNCY8MkZyusDlWb8p7e-fD68naqj6501sSVfi35bbg-Rvw-tsMEVLRv2-BBjVpBhyphenhyphenPewGJjgRUjXCGVnPHZULEEgbpEdxLgpPcLosbcNzZExFnN3sbBh0g3Vir2nt-kEMcqhRFlhUuGSatGT9O6ILzd7ay0uw/w400-h195/Nqo14%205.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>allowing protons to leave the cell. This allows the lysine/glutamine pairs to return to their starting locations as the yellow-crossed proton exits Nqo8</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhk5zJawJgpkVZi7-AuIhXSbs_GW7kE0ge4217PO8qglHoRVv2sJCvTj60I2NrqLEM7CRZXwK4Pj3-bxlUl-hv7nTJOEv8WND_4C2fOyQVw-yDNoWpSWLEadyvNGFYghJNAVaFbT8BsLgaAm5-kK0KCwYEvekVlPKYSb_-biK6NYhvvVkMdxQ0gGg/s1285/Nqo14%207.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="593" data-original-width="1285" height="185" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhk5zJawJgpkVZi7-AuIhXSbs_GW7kE0ge4217PO8qglHoRVv2sJCvTj60I2NrqLEM7CRZXwK4Pj3-bxlUl-hv7nTJOEv8WND_4C2fOyQVw-yDNoWpSWLEadyvNGFYghJNAVaFbT8BsLgaAm5-kK0KCwYEvekVlPKYSb_-biK6NYhvvVkMdxQ0gGg/w400-h185/Nqo14%207.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br />As the protons exit, the associated conformational changes open the cytosolic water channels and the cycle is ready to repeat</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjrhmgR4eZm9NiIDaEYf0Hn3Pys-YKYPcOgqbk8JQWyHLgvEIBlvIQ4hIyaxhfuE9N1ZkQBDMq3t15F6N0mTtqIUpZ-GZAEPZL0p5hzd4LX435hwZpL96oPqX8MjVUSFYgaogelUSHETAim16QGUsRDaWuPIRALvtFLJYT5LdWzgeefAvS0eh_9fA/s1443/Nqo14%208.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="679" data-original-width="1443" height="189" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjrhmgR4eZm9NiIDaEYf0Hn3Pys-YKYPcOgqbk8JQWyHLgvEIBlvIQ4hIyaxhfuE9N1ZkQBDMq3t15F6N0mTtqIUpZ-GZAEPZL0p5hzd4LX435hwZpL96oPqX8MjVUSFYgaogelUSHETAim16QGUsRDaWuPIRALvtFLJYT5LdWzgeefAvS0eh_9fA/w400-h189/Nqo14%208.png" width="400" /></a></div><br /><div><br /><br /><br /><br /><br /><br /><br /><br />This is all very nice and neat, typos excepted, with four protons pumped per cycle. But there is a fly in the ointment. As the authors comment</div><div><br /></div><div>"Despite a significant N-side entry channel, we observe no clear exit pathways to the P side of the membrane within Nqo8."</div><div><br /></div><div>There is no route out of Nqo8, exactly as there is no route out for protons in NuoH of human mitochondria or the equivalent in any of the other membrane bound hydrogenases of strict anaerobes.</div><div><br /></div><div>Which gives us insightful constraints on where the system might have come from.</div><div><br /></div><div>Peter</div>
Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com8tag:blogger.com,1999:blog-36840063.post-61799579552641722202023-11-28T05:59:00.000+00:002023-11-28T05:59:20.485+00:00Life (35) NiFe pumping<p>Just a quick run down of a possible mechanism of the membrane bound NiFe hydrogenases taken from the scheme for complex I function as suggested:<br /><br />"A similar scheme can be used for the mechanism of NiFe-hydrogenases, where charge variations in the NiFe site drive conformational changes resulting in proton translocation."<br /><br />quoted from</p><p><a href="#"></a><a href="https://pubmed.ncbi.nlm.nih.gov/22386882/" target="_blank">The coupling mechanism of respiratory complex I — A structural and evolutionary perspective</a></p>which was the basis of the <a href="https://high-fat-nutrition.blogspot.com/2023/11/life-34-complex-i-pumping.html" target="_blank">last</a> post. The modern NiFe system is pretty much the same as the CoQ system but it needs a simple summary here to allow us to try and run it in reverse, more in the LUCA style, and to try and to try and see what it might have been doing before it converted to a pump.<div><br /></div><div>Here we are, ready to start. These electrons have come from ferredoxin rather than NADH:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgD6SRjCtgAGlFYecz0tKM2OtlnZlfc_HkO4zzMIU9BM4KpXWP_qMh2DAunI1zhPPHssbVV3iOdG7sNI_r6AEoenX5Wk58eLV_bJMaDzRNU372D_Lhr-386ioYgDxhkgs9axSaUhba2_BTjdNSleeHv8LKcRzYrAYuDl6fhHbxKJoDZWW0c9Jostg/s943/NiFe1.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="943" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgD6SRjCtgAGlFYecz0tKM2OtlnZlfc_HkO4zzMIU9BM4KpXWP_qMh2DAunI1zhPPHssbVV3iOdG7sNI_r6AEoenX5Wk58eLV_bJMaDzRNU372D_Lhr-386ioYgDxhkgs9axSaUhba2_BTjdNSleeHv8LKcRzYrAYuDl6fhHbxKJoDZWW0c9Jostg/w375-h400/NiFe1.png" width="375" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />The electrons transfer from the FeS clusters to the NiFe cluster and the repulsive effect shown in the purple arrows disappears:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-2wbJd7h9zGaLZji-5t_m2OOn3lTGgbiRGwCqhofg7hecB8NFhy0CLmqG6COjNA0yNFVDtqPlaQtZuEUxwgCaxMM_LGHpD1PHni6MLvwko-2ZbIHnID677veqOXaPh98a_92HPF_wQfP03ITL18e6Fjh1C84XvmrxfYev0sheyhqWaimQbS71LQ/s943/NiFe2.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="943" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-2wbJd7h9zGaLZji-5t_m2OOn3lTGgbiRGwCqhofg7hecB8NFhy0CLmqG6COjNA0yNFVDtqPlaQtZuEUxwgCaxMM_LGHpD1PHni6MLvwko-2ZbIHnID677veqOXaPh98a_92HPF_wQfP03ITL18e6Fjh1C84XvmrxfYev0sheyhqWaimQbS71LQ/w375-h400/NiFe2.png" width="375" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and is replaced by the large purple arrows signifying marked attraction:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjMD8vlY_3bsL3XrKL-IEWKXkmRx0wMUXrwItqmyCb0SNKmn4PBOcQMpgaqYNBumC4yT5Eb_4ivfjzN_oHf6RROutF5iBhf5YSWLlnZjv265l1M6lZwrCsqX2-mdOOde60zlGd0sNhw2x9MRFdi-xc48192SWC2XqmqW4asRzwcP5Srb_poUDs55w/s947/NiFe3.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="947" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjMD8vlY_3bsL3XrKL-IEWKXkmRx0wMUXrwItqmyCb0SNKmn4PBOcQMpgaqYNBumC4yT5Eb_4ivfjzN_oHf6RROutF5iBhf5YSWLlnZjv265l1M6lZwrCsqX2-mdOOde60zlGd0sNhw2x9MRFdi-xc48192SWC2XqmqW4asRzwcP5Srb_poUDs55w/w373-h400/NiFe3.png" width="373" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and subsequent conformation change and ion pumping:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOO4elhvEBGoLsDmrX8Dck7ko-2-rwrOA0yd2zRz-ZU4PrFBzL9s92i3iX5rqKMWd3zsTd20yHfsCB4wAeTP5yO8LTn8-MZdi_EfaI8HWMPTfuSAdBsI2T9XNY6EAdSP5roaU4teKwGibEqoXObjHgffq4f8Ux9sl583sRwh3lLpkWwv4iTqQm5g/s883/NiFe4.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="852" data-original-width="883" height="386" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOO4elhvEBGoLsDmrX8Dck7ko-2-rwrOA0yd2zRz-ZU4PrFBzL9s92i3iX5rqKMWd3zsTd20yHfsCB4wAeTP5yO8LTn8-MZdi_EfaI8HWMPTfuSAdBsI2T9XNY6EAdSP5roaU4teKwGibEqoXObjHgffq4f8Ux9sl583sRwh3lLpkWwv4iTqQm5g/w400-h386/NiFe4.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Next the two electrons have to be transfered to two protons to yield the molecular hydrogen which is the final product, comparable to the two hydrogens which exit on CoQ2H. This has mostly been studied in the soluble NiFe hydrogenases. The mechanism will be conserved and consensus suggests that it will involve an hydride ion, H<sup>-</sup>, attached to the Ni atom.</div><div><br /></div><div>So we can start like this</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgP5NuT2ucv1EPYHGHxWt_KO0uKfnsJ41hTDBWRjMLVIoiuLoKpzBP7IiCho1nWLtykK_mgFEgurUsUmYM55zD8-R1dRZvU2HkepVfQcbwxRYEOiq7exA1YQvKKuHaUeeBdruxawaHfjl8pkSlsTWXZjo-JvpsK847rxf0d4b1d8OQehJHNWP-6xw/s883/NiFe5.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="852" data-original-width="883" height="386" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgP5NuT2ucv1EPYHGHxWt_KO0uKfnsJ41hTDBWRjMLVIoiuLoKpzBP7IiCho1nWLtykK_mgFEgurUsUmYM55zD8-R1dRZvU2HkepVfQcbwxRYEOiq7exA1YQvKKuHaUeeBdruxawaHfjl8pkSlsTWXZjo-JvpsK847rxf0d4b1d8OQehJHNWP-6xw/w400-h386/NiFe5.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which will rearrange to this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg-bEkN6tFRot2D4C4J5tXFkR9stO1wVgBf3A6xJlpfoXLjkGH1nGsAcZU8vQaT2VEGFKmHusQUlXzfEzvLAkFVoKFsoez_EeVOmDzhqErICcPHpEeD8q94srMh4XNVefRnQyc1LR9ok98O7o7vwQ1VGguIIDw0Ryb0kiJx0XY4A3Z_vcsfy7PSRA/s883/NiFe6.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="852" data-original-width="883" height="386" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg-bEkN6tFRot2D4C4J5tXFkR9stO1wVgBf3A6xJlpfoXLjkGH1nGsAcZU8vQaT2VEGFKmHusQUlXzfEzvLAkFVoKFsoez_EeVOmDzhqErICcPHpEeD8q94srMh4XNVefRnQyc1LR9ok98O7o7vwQ1VGguIIDw0Ryb0kiJx0XY4A3Z_vcsfy7PSRA/w400-h386/NiFe6.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>giving us the hydride intermediate before exchange of the electron and acquisition of a proton from the amino acid Y:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgV3LKBM_PukYaXK6T2PRWT9w2q6DFeqrypuiFVL4jwMqeAGJsKVKb3VyHJ8S24uqwyzrLHOyzh5Mz_-UI_PP9joIK2TVxNC2MLpSj771yTQu9Blkd0RLb1HJFHAjDSk7l1U3afyKbUHnShpvkTF7G69lZZwctL1lFSHi8JN4Gzoxan_Oj0D2IhkQ/s883/NiFe7.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="852" data-original-width="883" height="386" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgV3LKBM_PukYaXK6T2PRWT9w2q6DFeqrypuiFVL4jwMqeAGJsKVKb3VyHJ8S24uqwyzrLHOyzh5Mz_-UI_PP9joIK2TVxNC2MLpSj771yTQu9Blkd0RLb1HJFHAjDSk7l1U3afyKbUHnShpvkTF7G69lZZwctL1lFSHi8JN4Gzoxan_Oj0D2IhkQ/w400-h386/NiFe7.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>to give us molecular hydrogen</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj00EIPrWL7QhW1_OeICqNQUvl3W9mazrXrs7AvZJZ1jwElYUXkatj6_t_6yVxCpMbY8q_d32bAKqlHyRF0MjBwBwW_rNxyiyWwuqTwLRx8lF7clbZlnRHlsRqR3rovFkCInkPBg1sEvFtWkt9bN27y64eqvSihTv_oVvl6Zzk2PS3yh_8CeTr3LA/s993/NiFe8.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="852" data-original-width="993" height="344" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj00EIPrWL7QhW1_OeICqNQUvl3W9mazrXrs7AvZJZ1jwElYUXkatj6_t_6yVxCpMbY8q_d32bAKqlHyRF0MjBwBwW_rNxyiyWwuqTwLRx8lF7clbZlnRHlsRqR3rovFkCInkPBg1sEvFtWkt9bN27y64eqvSihTv_oVvl6Zzk2PS3yh_8CeTr3LA/w400-h344/NiFe8.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>To get us back to the start position we need, as per complex I/CoQ2H function, to supply of a pair of electrons and a pair of protons:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgQnlINmy6i40Hh53Uk09402X2o1ATkpAjzLYRNX5Lfs-qMfOs8JjxZWB8Wp9G4MKK_Id0qR1gvmjihS3YsXsAV_aE1BDK2dcKMaAd5RNzPv9PmzWUB9oHULlR0BoRqJpLt1WOJH9KxrGLa0NWwXe6F6BcuYAe4pRLGCh_EIEG4hXtOYBPvND0yNQ/s1075/NiFe9.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="935" data-original-width="1075" height="348" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgQnlINmy6i40Hh53Uk09402X2o1ATkpAjzLYRNX5Lfs-qMfOs8JjxZWB8Wp9G4MKK_Id0qR1gvmjihS3YsXsAV_aE1BDK2dcKMaAd5RNzPv9PmzWUB9oHULlR0BoRqJpLt1WOJH9KxrGLa0NWwXe6F6BcuYAe4pRLGCh_EIEG4hXtOYBPvND0yNQ/w400-h348/NiFe9.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and we're good to go again:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhFmDSzQiRDvOBQSevYMe1o3vS1cKuOhbD7xNttAjc1WCG17sNoEZZjAOIvfeyCyaofi3Q-aFpeVQ0H4-T7IXyfNHJIFQEwNBAzzhcqaExquadiqD1l58REgQZ5HlAwH97UH6S-oi_jbG7BqXfmN0AGxqDDX1RI7QyAgwPnOrNSGYBWwP5tHOYP7g/s943/NiFe10.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="943" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhFmDSzQiRDvOBQSevYMe1o3vS1cKuOhbD7xNttAjc1WCG17sNoEZZjAOIvfeyCyaofi3Q-aFpeVQ0H4-T7IXyfNHJIFQEwNBAzzhcqaExquadiqD1l58REgQZ5HlAwH97UH6S-oi_jbG7BqXfmN0AGxqDDX1RI7QyAgwPnOrNSGYBWwP5tHOYP7g/w375-h400/NiFe10.png" width="375" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>That will do for to today. Obviously there is perhaps less interest in how anaerobic hydrogen evolving bacteria and archaea pump either protons or Na<sup>+</sup> ions at the most frugal limits of bio-energetics when compared to complex I. So detailed work on the reverse process, where hydrogen powers a proton or Na<sup>+</sup> gradient, is thin on the ground but this is where we need to look to see how a pH driven system for carbon fixation/ferredoxin generation might be converted to a system to preserve the energy derived from hydrogen as a proton/Na<sup>+</sup> gradient which is a pre requisite to leaving the vent system as an independent organism.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com2tag:blogger.com,1999:blog-36840063.post-62067638617005881622023-11-26T06:03:00.002+00:002023-11-26T19:54:49.876+00:00Life (34) Complex I pumping<div>Before trying to work backwards from complex I via NiFe hydrogenases to consider how such a system might have evolved I've spent a couple of weeks on Fig 6 of this paper:<br /><br /><a href="#"></a><a href="https://pubmed.ncbi.nlm.nih.gov/22386882/" target="_blank">The coupling mechanism of respiratory complex I — A structural and evolutionary perspective</a><a href="#"></a><br /><br />For orientation this is section A from Fig 1 in the same paper:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhP2iKTtkQkaMcq9rUHK-ZjWCn_6Y5wqPR6FVJgCw-KpDiUtE6oUJC55TApraQ-ok2AsbUTvqLAcPx7jJVEXgYBlWDGVnbXtYXQEUgewgutxt2LmOhwXrzWkmVX2qtpjhM9hhrZliGrJaUy9C7DGdiDGyZ_kDpEaWndfHi4Stj-YJkG7WLJol2pmw/s1500/C%20I%20pump.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1500" height="301" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhP2iKTtkQkaMcq9rUHK-ZjWCn_6Y5wqPR6FVJgCw-KpDiUtE6oUJC55TApraQ-ok2AsbUTvqLAcPx7jJVEXgYBlWDGVnbXtYXQEUgewgutxt2LmOhwXrzWkmVX2qtpjhM9hhrZliGrJaUy9C7DGdiDGyZ_kDpEaWndfHi4Stj-YJkG7WLJol2pmw/w400-h301/C%20I%20pump.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br /><br /><br />and most of the rest of the post is going to be about the area outlined by the red oval.</div><div><br /></div><div>Fig 4 is an overlay of this area taken from complex I with the same area from the NiFe hydrogenase of the sulphate reducing bacterium<span style="box-sizing: inherit;"><i> Desulfovibrio gigas</i></span><i><span style="color: #222222; font-family: Harding, Palatino, serif;"><span style="background-color: white; box-sizing: inherit; font-size: 18px;">.</span></span></i> Both of these complexes show marked conservation of their structures in this area, as do all similar complexes:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjG2_S4V2-PPo7tkAdD-fUr_ykuYxR1bu2he2u2QUw-ft45YZfhsU9AG_3RNOBqdZpwxi1NyB1-sISjtijw2N5veawi2VuCaBgvmXTmw4mHXD-uRa3jYdFrB_QNMCpLcUXKyIHNh6o9WWSQL9KH2w5LU1-fAFz-xLUAa9KTKAFYTFsFAPmpGhsJ1Q/s1129/Overlay%20Ci%20and%20NiFe%20hydrogenase.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="937" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjG2_S4V2-PPo7tkAdD-fUr_ykuYxR1bu2he2u2QUw-ft45YZfhsU9AG_3RNOBqdZpwxi1NyB1-sISjtijw2N5veawi2VuCaBgvmXTmw4mHXD-uRa3jYdFrB_QNMCpLcUXKyIHNh6o9WWSQL9KH2w5LU1-fAFz-xLUAa9KTKAFYTFsFAPmpGhsJ1Q/w333-h400/Overlay%20Ci%20and%20NiFe%20hydrogenase.png" width="333" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and if I duplicate the image I can add in the electron pathways like this:</div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhZd1_LDveg45dkNSD89HIiFttXiSpgaDrP34gr6W3ORRITFcUAyOiLbH4cg7dRpGgFeCZY69FScFZ4t_4AZkEDAN8ZQHyPQHU_0i3RbKA3fMh54Vvjur1vyRXJnmD8xNt9doswpIXOc0t90_FaSMgHSl3skfabKmvcZJVSfRZdfYI4OLRSm-lE6Q/s1418/Comp%20I%20two%20pathways.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><br /></a><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhZd1_LDveg45dkNSD89HIiFttXiSpgaDrP34gr6W3ORRITFcUAyOiLbH4cg7dRpGgFeCZY69FScFZ4t_4AZkEDAN8ZQHyPQHU_0i3RbKA3fMh54Vvjur1vyRXJnmD8xNt9doswpIXOc0t90_FaSMgHSl3skfabKmvcZJVSfRZdfYI4OLRSm-lE6Q/s1418/Comp%20I%20two%20pathways.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="904" data-original-width="1418" height="255" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhZd1_LDveg45dkNSD89HIiFttXiSpgaDrP34gr6W3ORRITFcUAyOiLbH4cg7dRpGgFeCZY69FScFZ4t_4AZkEDAN8ZQHyPQHU_0i3RbKA3fMh54Vvjur1vyRXJnmD8xNt9doswpIXOc0t90_FaSMgHSl3skfabKmvcZJVSfRZdfYI4OLRSm-lE6Q/w400-h255/Comp%20I%20two%20pathways.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>In this next image I've put in both pathways and a funny little white, green and blue doo-hickey which converts the energy of the reaction in to a conformational change in the protein responsible for driving ion translocation. The doo-hickey is what Fig 6 is going to look at in detail. I chose the colour scheme here to match that in Fig 6:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJac66W0rXLqIBpierFJKkYfMBNN4EbsJN2X1NNmohZHLQybE2aGBYrldpOAniYLj0pE4hpERo8wDd7ctMV0popQdYYQ3GxoPhzut_6rG_rzwENWmRP48XehFrNzu7LuhSO5W2gwHOXz0tMyqyiE2EnnMzGCr_JUUxW9H8RJH9n_Qpwecco8M3TQ/s1129/Pump%20doo%20hickey.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="972" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjJac66W0rXLqIBpierFJKkYfMBNN4EbsJN2X1NNmohZHLQybE2aGBYrldpOAniYLj0pE4hpERo8wDd7ctMV0popQdYYQ3GxoPhzut_6rG_rzwENWmRP48XehFrNzu7LuhSO5W2gwHOXz0tMyqyiE2EnnMzGCr_JUUxW9H8RJH9n_Qpwecco8M3TQ/w345-h400/Pump%20doo%20hickey.png" width="345" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This is the un-altered original Fig 6. I'll explain it step by step in the following section:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEihJFlDPCqRkHP4r1Wq0ZL080iQGcQeh3bwBB4XkS9sZsBGhM91xWuPuU6DV14ijQBwcLsPY7fB7R6m203jWqRg8itwoGq8cMYRsGRNE8oymQibwnm_Dm_bc4NyJuWWcZwyGzNnX43W8Wr-Qd3TpcI7lHGcW3Tt39dcUAVbhjDVAFK0wUEScLO_mA/s1502/pump%20fig%206%20original.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="852" data-original-width="1502" height="228" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEihJFlDPCqRkHP4r1Wq0ZL080iQGcQeh3bwBB4XkS9sZsBGhM91xWuPuU6DV14ijQBwcLsPY7fB7R6m203jWqRg8itwoGq8cMYRsGRNE8oymQibwnm_Dm_bc4NyJuWWcZwyGzNnX43W8Wr-Qd3TpcI7lHGcW3Tt39dcUAVbhjDVAFK0wUEScLO_mA/w400-h228/pump%20fig%206%20original.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>In my modified images a solid red circle is a full electron negative charge. Open circles in black and white represent charge distributions of the magnitude used for hydrogen bonding, either +ve or -ve as appropriate. A solid red cicle with a +ve is a proton.</div><div><br /></div><div>Part 1 can be thought of as the open configuration. Electrons have arrived down the FeS chain from NADH to the terminal clusters N2 and (probably) N6a. Their negative charge induces a change in the yellow protein which, in combination with hydrogen "repulsion" between key amino acids opens the binding site to allow CoQ to enter. The original image had just two small + signs, within black circles, to designate mildly positively charged amino acid residues on the "top" end of the green protein. The same mild positive charge is also present on amino acids X and Y of the blue protein but were missing from the original image so I've added them in. These mildly positive areas are doing the opposite of hydrogen bonding, what I called hydrogen repulsion. The proteins are held apart, that's what the purplish thick arrows signify:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhI71bKDv0Nz0wMirgiIV1krcoez36oZA8JeC0tTHTcRdCTMNa3LmMpsTepiYEWgUv8K_V6TbMUD_HcCVpPVZT6zrrBcd6MVd0rMCv9qiWr936K5B9HjmSa76hWWuTJ1kNjazwNT4OWOliT7ggJaUlWsOgJyDlI9aJcNH7EgVql8pNgPA1ipH8xBQ/s943/pump%201.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="943" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhI71bKDv0Nz0wMirgiIV1krcoez36oZA8JeC0tTHTcRdCTMNa3LmMpsTepiYEWgUv8K_V6TbMUD_HcCVpPVZT6zrrBcd6MVd0rMCv9qiWr936K5B9HjmSa76hWWuTJ1kNjazwNT4OWOliT7ggJaUlWsOgJyDlI9aJcNH7EgVql8pNgPA1ipH8xBQ/w375-h400/pump%201.png" width="375" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Now oxidised CoQ docks with its binding site. Note that the pair of keto oxygens on CoQ are mildly -ve charged so form normal attractive hydrogen bonds to the mildly +ve amino acid residues X and Y on the blue protein. I've put in the partial charges eliciting the normal hydrogen bonding which are absent from the original image using hydrogen bonding -ves within small open circles:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhCkwFHJ9tVEvUg2HhB1-PzCaI-GiqVqPXoOrpPEfptMHfyTk-Dn4HQwCPJoOpnyKU_32d7mPs9nMZ7dYRA4gznZAVgqJrjeMFCnePJHtUe2w9DOSyXPVmwAGC_yjPI0PEJDqLfJFqFALz-Apw_IQJeDsEVemriDwe1OcxbJmxFMIxyA0pvEPYTBw/s937/part%202%20pump.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="937" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhCkwFHJ9tVEvUg2HhB1-PzCaI-GiqVqPXoOrpPEfptMHfyTk-Dn4HQwCPJoOpnyKU_32d7mPs9nMZ7dYRA4gznZAVgqJrjeMFCnePJHtUe2w9DOSyXPVmwAGC_yjPI0PEJDqLfJFqFALz-Apw_IQJeDsEVemriDwe1OcxbJmxFMIxyA0pvEPYTBw/w378-h400/part%202%20pump.png" width="378" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The two electrons transfer from their FeS clusters to CoQ to give a strongly negatively charged oxygen atom on either side of CoQ, call it CoQ<sup>2-</sup>. I've used the same red circles to accentuate the small negative signs used in the original diagram:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJO37d2O2O1gGD2vFJTREqexzmdW01VsBRvkbaFgEKWkU8dROj2cflueSUJehKOen1sN5H8fml8gzeDnt4oaBXZj_-pqFrAB1CcUaWpGcPLGgy-cbGFL2LVCXlVbT4PnzmtKh4O1QXUt1bk4XM6CsDcNsX0Muay_0KRfqLmRxq7o56_aWYwNXyeQ/s937/pump%20fig%203.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="937" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJO37d2O2O1gGD2vFJTREqexzmdW01VsBRvkbaFgEKWkU8dROj2cflueSUJehKOen1sN5H8fml8gzeDnt4oaBXZj_-pqFrAB1CcUaWpGcPLGgy-cbGFL2LVCXlVbT4PnzmtKh4O1QXUt1bk4XM6CsDcNsX0Muay_0KRfqLmRxq7o56_aWYwNXyeQ/w378-h400/pump%20fig%203.png" width="378" /></a></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>These strongly negatively charged oxygen atoms exert a pull (dark purple arrows pointing together) which induces the conformational change in to the location of the green protein which causes the actual pumping. This is shown in section 4 where the green protein has moved "upwards" and pumping has happened. In the same process the exit of the electrons from the FeS clusters allows the yellow protein back in to its starting conformation:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh6D5VaXliQZ-d05kDS_dgtq17wJ_Fh424Pt0ul0T0MxmZ9IuOT7GiH8iBBdFqX-jLl-LG0ctPUFtEA9fzFUSLqvelXppxLiQYNUXo1fxPtkuVIjAV10QLE79qJwuw2YmLKLbjYdA-Benyt0-uYTXek8qYMsSSByayYkH-X2L3nR-6YzhnkoCmsCg/s883/pump%20fig%204.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="827" data-original-width="883" height="375" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh6D5VaXliQZ-d05kDS_dgtq17wJ_Fh424Pt0ul0T0MxmZ9IuOT7GiH8iBBdFqX-jLl-LG0ctPUFtEA9fzFUSLqvelXppxLiQYNUXo1fxPtkuVIjAV10QLE79qJwuw2YmLKLbjYdA-Benyt0-uYTXek8qYMsSSByayYkH-X2L3nR-6YzhnkoCmsCg/w400-h375/pump%20fig%204.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The next change is subtle. All that has happened is that CoQ<sup>2-</sup> has taken the protons from the amino acids X and Y to form neutral CoQ2H, leaving the amino acids with the full negative charges. The covalent bonds rearrange slightly and the red -ve circle charges move a tiny distance on to the blue protein:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiS8DLrQvNMOVHDYF3HXJ37XonAtYeqHraMUfGCMpLklInQKiNFoj5P3CtOKZVt3guNy27623cKzIzhP7ZamVLQ75PaqnZTEtpr1xr62it-qt2YdRqksC4LqHsikDEnoftv6B4lFncjE9HDRFs4PTz5CX-7QzC-MNVt8qwZe7vrp_xkVdcE8AhAZA/s883/pump%205.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="816" data-original-width="883" height="370" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiS8DLrQvNMOVHDYF3HXJ37XonAtYeqHraMUfGCMpLklInQKiNFoj5P3CtOKZVt3guNy27623cKzIzhP7ZamVLQ75PaqnZTEtpr1xr62it-qt2YdRqksC4LqHsikDEnoftv6B4lFncjE9HDRFs4PTz5CX-7QzC-MNVt8qwZe7vrp_xkVdcE8AhAZA/w400-h370/pump%205.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>At this point CoQ2H is electrically neutral but still held in place by hyrdogen bonding to the now negatively charged amino acids X and Y, as are the two mildly positively charged amino acids (black +ves in white circles) on the green protein.</div><div><br /></div><div>Two things have to happen to return the complex to the section 1 configuration. Protons must enter the active site to allow the amino acids X and Y to lose their net negative charges and electrons must be replaced in the FeS clusters N2 and N6a to allow the yellow protein to assume the "open" configuration as CoQH2 exits:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipUQ3AMkFcx-roZT5CF9kY64U4BxkYtQwWcoXYxuILowDuhnDfbP4z-V3Pn73Yt97SSTFuToNXyROFNHbrWkPFJpg3VtQW-cNrOxPSNYi5zyHR58oP4zEeNRC2x9QcnMcQZhACIO7uTO9bhaoG-MHxDMsaxh4O3w9Z-lTokBmupAx9TG1wMp5tcg/s1066/2H+.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="920" data-original-width="1066" height="345" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEipUQ3AMkFcx-roZT5CF9kY64U4BxkYtQwWcoXYxuILowDuhnDfbP4z-V3Pn73Yt97SSTFuToNXyROFNHbrWkPFJpg3VtQW-cNrOxPSNYi5zyHR58oP4zEeNRC2x9QcnMcQZhACIO7uTO9bhaoG-MHxDMsaxh4O3w9Z-lTokBmupAx9TG1wMp5tcg/w400-h345/2H+.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Once these two events have occurred then we are back in to the situation in section 1 and the process can repeat:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhI71bKDv0Nz0wMirgiIV1krcoez36oZA8JeC0tTHTcRdCTMNa3LmMpsTepiYEWgUv8K_V6TbMUD_HcCVpPVZT6zrrBcd6MVd0rMCv9qiWr936K5B9HjmSa76hWWuTJ1kNjazwNT4OWOliT7ggJaUlWsOgJyDlI9aJcNH7EgVql8pNgPA1ipH8xBQ/s943/pump%201.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="943" data-original-width="883" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhI71bKDv0Nz0wMirgiIV1krcoez36oZA8JeC0tTHTcRdCTMNa3LmMpsTepiYEWgUv8K_V6TbMUD_HcCVpPVZT6zrrBcd6MVd0rMCv9qiWr936K5B9HjmSa76hWWuTJ1kNjazwNT4OWOliT7ggJaUlWsOgJyDlI9aJcNH7EgVql8pNgPA1ipH8xBQ/w375-h400/pump%201.png" width="375" /></a></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The system is reversible and, given an adequate membrane potential, conformational changes can result in electron translocation in reverse up to the flavin unit where they can be donated to NAD+ to give NADH. Or to oxygen to give superoxide.</div><div><br /></div><div>I think it might be worth taking a pause here before looking at the possible mechanism involved in the NiFe hydrogenase mechanism of action. As the authors comment:<br /><br /></div><div>"A similar scheme can be used for the mechanism of NiFe-hydrogenases, where charge variations in the NiFe site drive conformational changes resulting in proton translocation."</div><div><br /></div><div>Clearly there is nothing ancestral about the CoQ system but it's mechanism of action has to be derived from that of the NiFe hydrogenases, which do appear to carry the signature of the ancestral pumping mechanism.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com3tag:blogger.com,1999:blog-36840063.post-85013387423515716872023-11-12T17:48:00.003+00:002023-11-12T17:58:12.856+00:00Life (33) Transient hunger<div>Just a brief repeat that the origin of metabolism is this reaction at</div><div>pH 6:</div><div><br /> CO₂ + 2e- + E<span style="font-size: x-small;">(in)</span> -> CO + O<sup>2-</sup><br /><br />where the electrons and activating energy E<span style="font-size: x-small;">(in)</span> come from molecular hydrogen in solution at pH 10.</div><div><br /></div><div>The next step is:<br /><br />CO + O<sup>2-</sup> + 2H<sup>+</sup> -> HCOOH + E<span style="font-size: x-small;">(out)</span><br /><br />where E<span style="font-size: small;">(out)</span> is greater than E<span style="font-size: small;">(in)</span> , making the reaction exothermic and not reversible without the input of energy. Because there is no system for resupplying this energy to drive the reaction in reverse, you cannot convert the hydrocarbons back to molecular hydrogen and CO₂. Once the proto-metabolite hydrocarbons are formed they are stable and so able to accumulate as the building blocks of life. It's a one way process.<div><br /></div><div>Biomolecules accumulate. They are energetically stable.</div><div><br /></div><div>So if we look at these conditions:</div><div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzo6pWzojG4nxItzFycsEA8QIqJah1B68ZhMFfiDrvIJAb0sAFGWL8i4KGPoiHvXtGvWTWpof591kqcFzmx2Dyqz2R1bBZ-eAyIK3zNJGzcIJJivjyLZt9cW9Sak0W5QgZkuQO8lQby1gAbhvLdPKQ2p47ECA0REdYqg-N0HEI657H__oZANvjtg/s1454/FeS%20fluids.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1043" data-original-width="1454" height="288" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzo6pWzojG4nxItzFycsEA8QIqJah1B68ZhMFfiDrvIJAb0sAFGWL8i4KGPoiHvXtGvWTWpof591kqcFzmx2Dyqz2R1bBZ-eAyIK3zNJGzcIJJivjyLZt9cW9Sak0W5QgZkuQO8lQby1gAbhvLdPKQ2p47ECA0REdYqg-N0HEI657H__oZANvjtg/w400-h288/FeS%20fluids.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br /><br /><br /><br /><br /><br /><br /><br /></div><div><br /></div><div>there is no need for localised stability, the wiggly line of the interface can move left or right at random. So long as there is an interface, biomolecules will form, be stable and so accumulate.</div></div><div><br /></div><div>That's step one.</div><div><br /></div><div>Next comes the development of another use for the energy provided by hydrogen at an alkaline pH.</div><div><br /></div><div>A very, very early product of evolution was the development of the ferredoxin (Fd) molecule, an FeS cluster bound to one of the most ancient but tightly conserved very small proteins on earth. It allows the capture of both the electrons and the activation energy from our primordial hydrogen source in to a storable form, rather than it being used immediately to fix CO₂. Again the electrons and energy come from hydrogen at pH 10, as per hydrocarbon formation.</div><div><br /></div><div><div>Ferredoxin + 2e- + E<span style="font-size: x-small;">(in)</span> -> Ferredoxin<sup>2-</sup></div><div><br /></div><div>If we take out all intermediate steps we can summarise the generation of Fd<sup>2-</sup> near the origin of life like this</div><div><br /></div><div>H<sub>2</sub> + Fd -> Fd<sup>2-</sup> + 2H<sup>+</sup></div><div><br /></div><div>Fd<sup>2- </sup>(reduced ferredoxin) can supply electrons and energy to drive metabolic processes throughout the protocell, well away from the site where pH gradient recapitulation is driving hydrogen oxidation. I think ferredoxin came well before ATP arrived but it did, and still does do, the same job.</div></div><div><br /></div><div><br /></div><div><br /></div><div>This cannot happen in the core primordial situation. High energy ferredoxin in the presence of a catalyst would react with random protons immediately to regenerate molecular hydrogen and heat. Stability of Fd<sup>2-</sup> requires a cell membrane to protect it from the catalytic primordial FeS and NiFeS surface.</div><div><br /></div><div>This happens automatically when organics accumulate as a coating to the acidic area of the protocell surface. So now an amino acid derived "tube" is needed to get the pH 6 fluid in to the cell and the NiFeS and FeS clusters need to be embedded in some sort of amino acid derived structure to hold them in the correct pH zones. Like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIfEdUQaDN27D9tUYJXNaapEVMlgNUnZSxBKHuFObE_8UbukUFlmMFDaoLaHvCAm64aWcyD4rx8xew6-MhKE6X-YzQ3rXGgGOx80euWPYJYXCTgIQyaFQRtvrzG600XlS3EQ6YbWMNEOc7a34_UDbDOAvcZV1fpmJ0GKSJImjgAHJd_Kod07w_AA/s1379/structures%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1379" height="328" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiIfEdUQaDN27D9tUYJXNaapEVMlgNUnZSxBKHuFObE_8UbukUFlmMFDaoLaHvCAm64aWcyD4rx8xew6-MhKE6X-YzQ3rXGgGOx80euWPYJYXCTgIQyaFQRtvrzG600XlS3EQ6YbWMNEOc7a34_UDbDOAvcZV1fpmJ0GKSJImjgAHJd_Kod07w_AA/w400-h328/structures%20MBH.png" width="400" /></a></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><br /><div><br /></div><div><br /></div><div><br /></div><div>which is the basis of this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEigG93xrQuh2NZ1tTh3yILUkbVigYsnIq39aB2JgJ2KjDgyMPVGqBRKJ2fFOigp09e84qxNq8u2PIOIj8gLfI8H186E2nbLqfks8kAS6RL7BBxo6Q5bGQpLpejhXTMWd5e26uc1QgbAbHtFSCz6JfaN9pfO-bVV13lV2kZstnPOUIsw7vA5jdpGRA/s1493/AA%20based%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEigG93xrQuh2NZ1tTh3yILUkbVigYsnIq39aB2JgJ2KjDgyMPVGqBRKJ2fFOigp09e84qxNq8u2PIOIj8gLfI8H186E2nbLqfks8kAS6RL7BBxo6Q5bGQpLpejhXTMWd5e26uc1QgbAbHtFSCz6JfaN9pfO-bVV13lV2kZstnPOUIsw7vA5jdpGRA/w400-h304/AA%20based%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which we can declutter to this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEii-RQG66U0f9Jw0TWhzqgUeQKZgkbMJP8egfZYcI18BdlyObrrA7AdE0fNaoKYlgEK60iDQ0rV-y6xNcLqbFOadV4eDRf99ZMN8QLqovGBLOPUCr9mtTpvLaiYNBlGWHt0Iz0_CZydtcbe5DE036xLlI3Sne26fbwNpZcntaqp7CYVnkEKLqrBLg/s1493/decluttered%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEii-RQG66U0f9Jw0TWhzqgUeQKZgkbMJP8egfZYcI18BdlyObrrA7AdE0fNaoKYlgEK60iDQ0rV-y6xNcLqbFOadV4eDRf99ZMN8QLqovGBLOPUCr9mtTpvLaiYNBlGWHt0Iz0_CZydtcbe5DE036xLlI3Sne26fbwNpZcntaqp7CYVnkEKLqrBLg/w400-h304/decluttered%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>We are now in a position to speculate about what might happen in a protocell which experiences fluctuations in the supply of hydrogen rich alkaline vent fluid. Loss of vent fluid allows the pH of the protocell to drop towards the acidic pH 6 of the ocean, shown as the red alkaline fluid in the above doodle turning to a more blue acidic fluid, as here below:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEghBRPmm4L-X0eMzn1h71bW6LU2yatWYXUZh7nxFclclD7pX7mWvUD9MRoZy4UnDDtjJuP34NsdguLn3SGqJxCtla2I-TVzw2QYu5ttjP-eLaflcY-ot7x6GPrmq5VddVfMF8YUoBiy75ow5LOBy2er5uUnFQ576xgnTRrPAXIqUEsBzAfddZA8VA/s1493/pH%20fall%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEghBRPmm4L-X0eMzn1h71bW6LU2yatWYXUZh7nxFclclD7pX7mWvUD9MRoZy4UnDDtjJuP34NsdguLn3SGqJxCtla2I-TVzw2QYu5ttjP-eLaflcY-ot7x6GPrmq5VddVfMF8YUoBiy75ow5LOBy2er5uUnFQ576xgnTRrPAXIqUEsBzAfddZA8VA/w400-h304/pH%20fall%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Recall that organic molecules are safe from degradation under these circumstances but that ferredoxin is not. So it is very easy to degrade Fd<sup>2-</sup> to molecular hydrogen, losing some of its stored energy as heat energy. Note the reversal of the arrows in the above diagram as this happens.</div><div><br /></div><div>If this is a short term fluctuation there is the potential to preserve on-going protocell function in two ways. One is to stop the flow of electrons derived from Fd<sup>2-</sup> jumping from the embedded FeS cluster to the embedded NiFeS cluster. All that is needed is either a conformational change to the NiFeS structure to move it physically away from the FeS cluster or to change its local environment to make it an unattractive target for electrons. Changing the shape of a protein in response to protonation is a very simple concept and I've illustrated in this next doodle where the acidified NiFeS support structure has moved the NiFeS cluster away from the FeS cluster due to such a shape change.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgg68HspRL8GG9bjx1tBKBmY-sqs3H16HOVLE1I_jtP-1AFx-vkA2GfQRHxHIa_YQQWw6qsWBplZKtWDdYMqiULc_1Wh97f3rkTYLrGpcsFWhpfAfcGTxHyom_WSVOGDqepDC2ySYKKnvi0UHugajrbqtdEIlHPan8u1-_OYf__QP77tnh7etm9fw/s1493/moved%20NiFeS%20at%20pH%206%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgg68HspRL8GG9bjx1tBKBmY-sqs3H16HOVLE1I_jtP-1AFx-vkA2GfQRHxHIa_YQQWw6qsWBplZKtWDdYMqiULc_1Wh97f3rkTYLrGpcsFWhpfAfcGTxHyom_WSVOGDqepDC2ySYKKnvi0UHugajrbqtdEIlHPan8u1-_OYf__QP77tnh7etm9fw/w400-h304/moved%20NiFeS%20at%20pH%206%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This stops the wasteful generation of molecular hydrogen from Fd<sup>2-</sup>.</div><div><br /></div><div>The second technique is to limit the ingress of protons from the acidic ocean fluid.</div><div><br /></div><div>We currently have a situation where electrons are still free to travel from Fd<sup>2-</sup> to the embedded FeS cluster, producing a net negative charge:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgknWBFYwv7BDhZ5AOiBiDlmU53xlMhRVSitHM-deI8wp4mQ4_vqlH0WMMQozHz8GAdz4jpHiRZFMeD9KjspPHDaG5mCM_TvoeMRpBs66DhiiuVR9zWnKmVLLvOu8JtnsgAb_vaQRNUWTRUrgJUlZT9TjZStNHpuIDMEdE0aqUTxlFMJjAauk7TSw/s1493/FeS%20-ve%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgknWBFYwv7BDhZ5AOiBiDlmU53xlMhRVSitHM-deI8wp4mQ4_vqlH0WMMQozHz8GAdz4jpHiRZFMeD9KjspPHDaG5mCM_TvoeMRpBs66DhiiuVR9zWnKmVLLvOu8JtnsgAb_vaQRNUWTRUrgJUlZT9TjZStNHpuIDMEdE0aqUTxlFMJjAauk7TSw/w400-h304/FeS%20-ve%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>All that is now needed is a positively charged area of amino acids in the transmembrane tube structure, like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEilUUuUXgjC7Elcbqw6Z-odXvadSyfy8bAD-mG0LFGL7Q2T7QHwMfjYFwJPl_T_GJyeP2iPJsudzZagTT-Po16QkF9r2kHiKIMTcRzHMytcsWwECXLrQDDNfPt4Xj4E6Tw8Zuz1MQ_YSk2hgrpKC2fBmjoypX9Bnl-OPtP_cCYhhRLDSFUMRMYovA/s1493/electrostatics%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEilUUuUXgjC7Elcbqw6Z-odXvadSyfy8bAD-mG0LFGL7Q2T7QHwMfjYFwJPl_T_GJyeP2iPJsudzZagTT-Po16QkF9r2kHiKIMTcRzHMytcsWwECXLrQDDNfPt4Xj4E6Tw8Zuz1MQ_YSk2hgrpKC2fBmjoypX9Bnl-OPtP_cCYhhRLDSFUMRMYovA/w400-h304/electrostatics%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and we now have the potential to alter the shape of the transmembrane tube to stop the ingress of protons from the oceanic fluid. All that is needed is for a positively charged localised area of the transmembrane tube to move towards the now negatively charged FeS cluster and produce a conformational change. This can limit acidic fluid ingress and preserve whatever alkaline conditions are still present within the protocell. Note the reinstatement/preservation of some degree of red alkaline pH-ness in to the intracellular fluid zone and lack of blue acidic pH-ness in the protocell area of the catalytic FeS cluster:</div><div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3D2MD-pe9nXx8UAhObVOXmPuv94aShBerVNEUT_X_TZcQNg1M4JKUR3fFB2RT46fNi8j1jFBpOpB3pGFCWXTrkEGk5xQFOnjzWkFAWP5G5Wzwcb2O3vlJVAJJYCG_hMj9YFA6e910mR96GwjDb_jDu8q_g1BA4LxOWvfN1oOgNt666h4o2B6dOQ/s1493/Blocked%20tube%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj3D2MD-pe9nXx8UAhObVOXmPuv94aShBerVNEUT_X_TZcQNg1M4JKUR3fFB2RT46fNi8j1jFBpOpB3pGFCWXTrkEGk5xQFOnjzWkFAWP5G5Wzwcb2O3vlJVAJJYCG_hMj9YFA6e910mR96GwjDb_jDu8q_g1BA4LxOWvfN1oOgNt666h4o2B6dOQ/w400-h304/Blocked%20tube%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div></div></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This provides a temporary "pro-survival" state for the protocell using the remaining Fd<sup>2-</sup> pool while awaiting the prompt (hopefully) return of vent fluid. Return of vent fluid's alkaline conditions removes protonation of the area around the NiFeS cluster, returning it to close proximity of the FeS cluster and establishing electron flow from the now resupplied hydrogen to the now depleted Fd<sup>2-</sup> pool. This opens the transmembrane channel and allows normal cell function to return:</div><div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEii-RQG66U0f9Jw0TWhzqgUeQKZgkbMJP8egfZYcI18BdlyObrrA7AdE0fNaoKYlgEK60iDQ0rV-y6xNcLqbFOadV4eDRf99ZMN8QLqovGBLOPUCr9mtTpvLaiYNBlGWHt0Iz0_CZydtcbe5DE036xLlI3Sne26fbwNpZcntaqp7CYVnkEKLqrBLg/s1493/decluttered%20MBH.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1133" data-original-width="1493" height="304" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEii-RQG66U0f9Jw0TWhzqgUeQKZgkbMJP8egfZYcI18BdlyObrrA7AdE0fNaoKYlgEK60iDQ0rV-y6xNcLqbFOadV4eDRf99ZMN8QLqovGBLOPUCr9mtTpvLaiYNBlGWHt0Iz0_CZydtcbe5DE036xLlI3Sne26fbwNpZcntaqp7CYVnkEKLqrBLg/w400-h304/decluttered%20MBH.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and we're back where we started from.</div><div><br /></div><div>These thoughts came from a combination of Nick Lane's comment about the protonation of Ech (also read MBH, complex I or about 6 other membrane pumps from the same family) in the region of a transmembrane channel coupled with some degree of reverse engineering of complex I mechanism in these two papers, which are another post but if anyone wants to read ahead they're here:<br /><br /><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547640/" target="_blank">Symmetry-related proton transfer pathways in respiratory complex I</a></div><div><br /></div><div>especially figures 2A and 6</div><div><br /></div><div>and here:</div><div><br /><a href="https://www.sciencedirect.com/science/article/pii/S0005272812000394?via%3Dihub" target="_blank">The coupling mechanism of respiratory complex I — A structural and evolutionary perspective</a><br /><br />also figure 6 and the associated legend.</div><div><br /></div><div>It's all about what is reversible and what is not. The above doodles describe a pre-adaptation to a situation where reversal will lead to proton pumping. They're not describing a pump per se but they provide the basis for a pump should that become advantageous.</div><div><br /></div><div>Peter<br /></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com12tag:blogger.com,1999:blog-36840063.post-8926381119123608602023-10-31T06:51:00.000+00:002023-10-31T06:51:00.631+00:00Life (32) On the correct side of the fence<div>Preamble. On page 143 of <a href="https://nick-lane.net/books/transformer-the-deep-chemistry-of-life-and-death/" target="_blank">Transformer</a> (I'm assuming everyone has a copy of Transformer) Nick Lane writes:</div><div><br /></div><div>"The deepest requirement for the proton-motive force might therefore be CO2 fixation. The prime example is the "energy converting hydrogenase" or Ech. This membrane protein has four iron-nickle-sulphur clusters, which transfer electrons from H2 to ferredoxin. Two of the clusters sit right next to a proton channel in the membrane, and their properties depend on proton binding, which is to say, the local pH. So when Ech binds protons, it can accept electrons from H2 (in the jargon, it is more easily reduced). And when the protons detach, Ech becomes more reactive, and can now force its electrons on to ferredoxin, which in turn pushes them on to CO2. Then the incoming protons bind Ech again, and the cycle repeats itself."</div><div><br /></div><div>Which takes me back to my thoughts on pH differential driving prebiotic chemistry here:</div><br /><a href="https://high-fat-nutrition.blogspot.com/2019/02/life-22-feni-hydrogenase.html" target="_blank">Life (22) FeNi hydrogenase</a><br /><br />which contains a serious logical flaw regarding the location of the site for pre-biotic carbon dioxide reduction:<br /><br /><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhQdt4BmETfsEl0rVVxPvUbjCpdRAzE0LYuWmKD0q4AvzjGMdzMXycuZdVlKMJ-R87cTUrt6WjLqOEC-_K3KWdKX0uFZTqzhS_cPkuXXYOhQjVX5bAAu6z85-zAdXHq80V2AnaxKg/s1600/Slide05.png" style="clear: left; margin-bottom: 1em; margin-right: 1em; text-align: center;"><img border="0" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhQdt4BmETfsEl0rVVxPvUbjCpdRAzE0LYuWmKD0q4AvzjGMdzMXycuZdVlKMJ-R87cTUrt6WjLqOEC-_K3KWdKX0uFZTqzhS_cPkuXXYOhQjVX5bAAu6z85-zAdXHq80V2AnaxKg/s400/Slide05.png" width="400" /></a><div><br /></div><div>In this diagram organic synthesis is happening on the acidic (pH 6), oceanic side of the FeS barrier. From where it would simply wash away in to the rest of the ocean. Metabolism actually happens on the alkaline, vent side in a constrained cell-like structure with walls made up of iron sulphide mineral.<div><br /></div><div>So I've had to rethink the whole basic process with some ideas triggered by various lines in Transformer. Here we go.<br /><br /><div><br /></div><div>The reaction which summarises the origin of life is<div><br /></div><div>CO2 + H2 -> HCOOH</div><div><br /></div><div>After formic acid formation the generation of the core building blocks of metabolism is, energetically, all down hill. I think it was Nick Lane who described this as a "free lunch you're paid to eat".</div><div><br /></div><div>Fascinatingly, you could mix hydrogen with carbon dioxide in a jar and, even if you watched it for 4 billion years, nothing would happen. The problem is that the initial step of the conversion, which is this:</div><div><br /></div><div>CO2 + H2 -> CO + H2O</div><div><br /></div><div>is not a free lunch. It requires energy, it is a lack of activation energy which stops the reaction occurring spontaneously. Note it is not remotely as simple as written, the water on the right hand end just there to balance the equation, see below.</div><div><br /></div><div>It *will* occur spontaneously if the CO2 is held at pH 6 and the H2 is held at pH10, in the presence of an iron-sulphur catalyst. An iron/nickel sulphur catalyst works even better but simple FeS seems to do the job.</div><div><br /></div><div>So this is a crystal of iron sulphide, part of the wall of a tubule structure in a "white non-smoker" alkaline hydrothermal vent. Imagine it's a few billion atoms thick and many billions of atoms long.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiKNxFufqiH7H8j7fs4bnezHhpsPQMLPa5UVC5QOVseEbdjiHbhbFiE1bBGXJCVV2PkBBoI9Riivfy91_O8oynD6a0V4LcOOh1BTlpgssoLqmuKqS2-S3cWlEAgdQje9Vsx-eg9YtOr1pFNvqyOQfy0k3ONaFmIgJNpkmkD4oCTFGkFh9NKwUCFfQ/s1454/FeS%20matrix.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="568" data-original-width="1454" height="156" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiKNxFufqiH7H8j7fs4bnezHhpsPQMLPa5UVC5QOVseEbdjiHbhbFiE1bBGXJCVV2PkBBoI9Riivfy91_O8oynD6a0V4LcOOh1BTlpgssoLqmuKqS2-S3cWlEAgdQje9Vsx-eg9YtOr1pFNvqyOQfy0k3ONaFmIgJNpkmkD4oCTFGkFh9NKwUCFfQ/w400-h156/FeS%20matrix.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>In some areas of the tubule wall vent fluid is trying to pass outwards and ocean fluid is trying to pass inwards. I've depicted an interface between two flow areas as a wavy line:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzo6pWzojG4nxItzFycsEA8QIqJah1B68ZhMFfiDrvIJAb0sAFGWL8i4KGPoiHvXtGvWTWpof591kqcFzmx2Dyqz2R1bBZ-eAyIK3zNJGzcIJJivjyLZt9cW9Sak0W5QgZkuQO8lQby1gAbhvLdPKQ2p47ECA0REdYqg-N0HEI657H__oZANvjtg/s1454/FeS%20fluids.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1043" data-original-width="1454" height="288" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjzo6pWzojG4nxItzFycsEA8QIqJah1B68ZhMFfiDrvIJAb0sAFGWL8i4KGPoiHvXtGvWTWpof591kqcFzmx2Dyqz2R1bBZ-eAyIK3zNJGzcIJJivjyLZt9cW9Sak0W5QgZkuQO8lQby1gAbhvLdPKQ2p47ECA0REdYqg-N0HEI657H__oZANvjtg/w400-h288/FeS%20fluids.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br /><br /><br /><br /><br /><br /><br /><br /></div><div><br /></div><div>To make life simpler I've now ignored the billions of other FeS layers to just show the layer adjacent to the inner side of the vent tubule. I've put in the pHs of the fluids too.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOMAkWhKY9Q5RA8zIbwMWqb_TvkyWabADqrheGFjhKwFKdxxG5lFRAEHE7GoC1DC468K6zgO7WSzZwp4Ls7KTAdrI3czpoV38r5HMF9gQG5uQ1gDSlLTwbvtqyEHjWzDNOQqnUqan1ZcXdmyNQQWF2vbHWdTn2h7X23thq3lZ0-nQ1gx8nKTDJXg/s1431/monolayer%20FeS.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1016" data-original-width="1431" height="284" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOMAkWhKY9Q5RA8zIbwMWqb_TvkyWabADqrheGFjhKwFKdxxG5lFRAEHE7GoC1DC468K6zgO7WSzZwp4Ls7KTAdrI3czpoV38r5HMF9gQG5uQ1gDSlLTwbvtqyEHjWzDNOQqnUqan1ZcXdmyNQQWF2vbHWdTn2h7X23thq3lZ0-nQ1gx8nKTDJXg/w400-h284/monolayer%20FeS.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The two FeS clusters at the interface are at markedly differing pH conditions.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj8e1cIqu2IW2oFI64kkN4KCcrBip6OGw3vHFmO4qM29IqQuVkr2LyPMhfqlYrwIW-goBQmDJpw_1oNUHP5IKooIdee9aVEhE29ooE3I9CdZNQG18-c7mV3yKmf36Q3GXqkdOZvqnDISy_6MOIdX73gDsdK2NkTxcYT0v9Mnst2tB25Pa_qOkw8Aw/s1431/pH%20FeSs.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1031" data-original-width="1431" height="289" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj8e1cIqu2IW2oFI64kkN4KCcrBip6OGw3vHFmO4qM29IqQuVkr2LyPMhfqlYrwIW-goBQmDJpw_1oNUHP5IKooIdee9aVEhE29ooE3I9CdZNQG18-c7mV3yKmf36Q3GXqkdOZvqnDISy_6MOIdX73gDsdK2NkTxcYT0v9Mnst2tB25Pa_qOkw8Aw/w400-h289/pH%20FeSs.png" width="400" /></a></div><br /><div><br /></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The red part of the intrinsically catalytic FeS surface can split a molecule of vent derived H2 in to two protons and two electrons. The protons react with hydroxyl ions in the alkaline vent fluid while the electrons hop "down hill" to the acidic FeS part of the surface in blue.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJuhBUhrnmhoj3ptCBZGY4QuS6IAEpTF_s-tDMECEP6YWUhxKcJ37mn_e-mUxPSIk5tpAdwMF3v60CfZ9IZTk5-FxFTV0lfOVS7kafW4dEwxgX4hBYpK3Wa6eICy65F6ZUK6PanZmUdKul_iz1g2oC6n31CuBniPNFXc5V8bKdTocd9GRZRTo1oA/s1431/charged%20surface.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1031" data-original-width="1431" height="289" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhJuhBUhrnmhoj3ptCBZGY4QuS6IAEpTF_s-tDMECEP6YWUhxKcJ37mn_e-mUxPSIk5tpAdwMF3v60CfZ9IZTk5-FxFTV0lfOVS7kafW4dEwxgX4hBYpK3Wa6eICy65F6ZUK6PanZmUdKul_iz1g2oC6n31CuBniPNFXc5V8bKdTocd9GRZRTo1oA/w400-h289/charged%20surface.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This, ephemerally, provides a negatively charged FeS surface. The electrons are clearly destined to eventually react with protons from the acidic fluid but Nick Lane explains in great detail how that process can be indirect, via CO2 interacting with the charged surface, to allow the proton and electron to recombine as an hydrogen, but this time as part of a hydrocarbon rather than as hydrogen gas. He's not particularly forthcoming on the origin of the charged surface, hence my above doodles to suggest how it might plausibly develop.</div><div><br /></div><div>Lane goes through the conversion of CO2 to acetate at a charged surface in a step by step guide. The title of the section is "Magic surfaces" and it runs from p133 to 140 of Transformer. I've yet to find a better description. This is a wholely abiotic process. My limited summary of the "difficult" initial step is like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgG2gFwd7-uZoZyCxLo-S8FINDGPEk0FC7ZKq9fkZ5CHpxCqYA5GpKfl715aGBBuY_PzpH4TrIXwJ8y0WphyEcpz9FjaUtWY_NREfijWY40xbPRBzwC7OpT0-BqBM2rPO3nt-XCumpkm-sQ33CrbC3v1VHJRIVbS4bnNm2FDsIK7U_21sapwLq4iw/s1431/summary.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1031" data-original-width="1431" height="289" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgG2gFwd7-uZoZyCxLo-S8FINDGPEk0FC7ZKq9fkZ5CHpxCqYA5GpKfl715aGBBuY_PzpH4TrIXwJ8y0WphyEcpz9FjaUtWY_NREfijWY40xbPRBzwC7OpT0-BqBM2rPO3nt-XCumpkm-sQ33CrbC3v1VHJRIVbS4bnNm2FDsIK7U_21sapwLq4iw/w400-h289/summary.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The process is laid out in Transformer, organics can be formed on the alkaline side of an FeS permeable barrier.</div><div><br /></div><div>Nick Lane addresses the formation of this set up on page 146. He has shown that protons cross FeS barriers very easily and hydroxyl ions do so only slowly, facilitating the situation I've outline above. Nice. Electrons move over the surface of the FeS barrier briefly before combining with CO2, protons travel through the FeS barrier to provide the driving pH differential.</div><div><br /></div><div>I've taken these concepts and overlain them on a cartoon of the power module from the membrane bound hydrogenase (MBH) of pyrococcus furiosus (I have a better cartoon for MBH than I do for Ech, they share a functionally homologous power unit so this part is interchangeable). This is Fig4 A from</div><div><br /></div><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003862/" target="_blank">Structure of an Ancient Respiratory System</a></div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgAnaUnnb0RFk9Fo8B1-5t1GcJnayX2FpIz-IsD951Wo2tyx7RU4xMQVF-9znvytN7BaEtHPIaz5hXUkH-RdIfJXwt0tsupyh2w-ja3d48hiIb7G2SwksDRuF99VKUinsgw3Z1Y0LaeuQsLQEAatav7R5FIjsP7s353_U_3k3CQ7uyVCMdvzm_1cg/s799/A%20from%20fig4.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="799" data-original-width="690" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgAnaUnnb0RFk9Fo8B1-5t1GcJnayX2FpIz-IsD951Wo2tyx7RU4xMQVF-9znvytN7BaEtHPIaz5hXUkH-RdIfJXwt0tsupyh2w-ja3d48hiIb7G2SwksDRuF99VKUinsgw3Z1Y0LaeuQsLQEAatav7R5FIjsP7s353_U_3k3CQ7uyVCMdvzm_1cg/w345-h400/A%20from%20fig4.png" width="345" /></a></div><br /><div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which I doodled on 2019 like this:</div><div><br /><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0RPEw5OEofGsZVkH6OPun6_MPb7leSYJbprsVe8ESIxVuW_GprJoQ3Jjl4olNwgZRIpb7bn1jM3Hint4y7wbPr9NURiYSejQKUHofB0XtTKfXbWR6dLYB5bWGz0D65BrimOx-wg/s1600/FeNi+overlay.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1079" data-original-width="1335" height="322" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg0RPEw5OEofGsZVkH6OPun6_MPb7leSYJbprsVe8ESIxVuW_GprJoQ3Jjl4olNwgZRIpb7bn1jM3Hint4y7wbPr9NURiYSejQKUHofB0XtTKfXbWR6dLYB5bWGz0D65BrimOx-wg/s400/FeNi+overlay.png" width="400" /></a></div><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /></div><div><br /></div><div><br /></div><div><br /></div><div>and which I can now overlay the origin of organic synthesis scenario from my above doodles (rearranged slightly and new colour scheme) to give this</div></div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhygq_noC6G1EvGBmhEeXhO0aemwVIt2KdKQK1eBZ1bgv9Vm-ktWD5P7EzJNiE4HIAKmIK_mBqGQ8rZ_mwyyYRzFeHBS5jBt-vKRJgtcbbD3LL1t2x1SFw7CmlOjT03mrhhFny7gAcFLI8EBSUdxAdM1EHX3HVaHlmazrN3agTDqDfBLfHChXZkhA/s1414/rearranged.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1414" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhygq_noC6G1EvGBmhEeXhO0aemwVIt2KdKQK1eBZ1bgv9Vm-ktWD5P7EzJNiE4HIAKmIK_mBqGQ8rZ_mwyyYRzFeHBS5jBt-vKRJgtcbbD3LL1t2x1SFw7CmlOjT03mrhhFny7gAcFLI8EBSUdxAdM1EHX3HVaHlmazrN3agTDqDfBLfHChXZkhA/w400-h320/rearranged.png" width="400" /></a></div><br /><div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>If we blank out the enzyme we can compare the two electron/proton pathways one above the other. Oh, and I've put in the Ni atom which pyrococcus uses in its hydrogenase like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgvgJ7GKI0YuLMtAKAxkSgi7Eat_FQUcPr8iSHx-C3AyqY5SH0wPfhlL8XeH2J7djCrv46_MKIjrAsneIieicsfpA6l5c7AjZ6TMPV0FLFFmmBXgZETZJF2o_LYshIrO8jCn71I2h4jYVLAhMWVkY0Hbj6L3_t0wobrGzKtrGuOESOcm55QNqr9bQ/s1414/comparison.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1216" data-original-width="1414" height="344" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgvgJ7GKI0YuLMtAKAxkSgi7Eat_FQUcPr8iSHx-C3AyqY5SH0wPfhlL8XeH2J7djCrv46_MKIjrAsneIieicsfpA6l5c7AjZ6TMPV0FLFFmmBXgZETZJF2o_LYshIrO8jCn71I2h4jYVLAhMWVkY0Hbj6L3_t0wobrGzKtrGuOESOcm55QNqr9bQ/w400-h344/comparison.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The enzyme just needs to recapitulate the pH differential at the active site to allow the reaction to proceed. Quite how you get from this pH driven abiotic process to the same process embedded in a massive and complex enzyme system is not important here, what matters is that the basic core is clearly preserved. Getting the system to run in reverse and coupling it to an antiporter as a pump is another story.</div><div><br /></div><div>Just in case I haven't mentioned it, the reason that the power units of Ech and MBH are so important is that they are directly homologous to the power unit of complex I and the multiple other related membrane pumps. Which means that the system, clearly derived from the inorganic process at the origin of metabolism, is pretty well ubiquitous.</div><div><br /></div><div>The next step is to examine how a abiotic system using hydrogen as fuel can function as a proton pump which generates hydrogen as waste and what it might do with any spare electrons. That can wait for another day.</div><div><br /></div><div>Peter</div></div></div></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com1tag:blogger.com,1999:blog-36840063.post-69572077158793760762023-10-29T06:36:00.002+00:002023-10-29T06:39:55.710+00:00Dalton GrahamI'm in a bit of a quandary about blogging. I've had to rethink some of my thoughts on the origin of life and the function of the membrane bound hydrogenases which kicked it all off and how they might have lead to superoxide being used as the core growth signal on Earth.<br /><br />I've also got a number of ideas I want to get written down about the differences between hydrogenated coconut oil driven obesity and linoleic acid driven obesity. Plus I'm about to run out of free time in the very near future so argh...<div><br /></div><div>Anyhoo, Tucker sent me the link to this discussion he and Brian Kerley had with Dalton Graham.</div><div><br /></div><div>As in</div><div><br /><a href="https://pubmed.ncbi.nlm.nih.gov/37267350/" target="_blank">Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet</a><br /><br />discussed in a slightly tangential way here <br /><br /><a href="https://high-fat-nutrition.blogspot.com/2023/10/insulin-mimesis-without-nox-enzymes-5.html" target="_blank">Insulin mimesis without NOX enzymes (5) MCT plus linoleic acid</a><br /><br /></div><div>Which gives me about four more things I'd like to blog about but might not get time for. Especially listen to what chow does to liver fat content in the long term and about unpublishable but highly enlightening observations. Oh, and a shared view of peer review. Oh and what it's like to be junior linoleio-phobe in a conventional lab. Oh, and... Never mind, just give it a listen:</div><div><br /></div><a href="https://tuckergoodrich.substack.com/p/ep-10-dalton-graham-how-to-induce" target="_blank">Ep. 10: Dalton Graham: How to Induce Fatty Liver</a><div><br /></div><div class="separator" style="clear: both; text-align: center;"><iframe allowfullscreen="" class="BLOG_video_class" height="266" src="https://www.youtube.com/embed/jvyZx4QTYNk" width="320" youtube-src-id="jvyZx4QTYNk"></iframe></div><div class="separator" style="clear: both; text-align: center;"><span style="text-align: left;"><br /></span></div>PeterPeterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com11tag:blogger.com,1999:blog-36840063.post-33284150669725932932023-10-23T10:14:00.001+00:002023-10-23T10:15:03.370+00:00Credo<div>There was a line by karl in a <a href="https://high-fat-nutrition.blogspot.com/2023/10/insulin-mimesis-without-nox-enzymes-6.html?showComment=1697842221298#c3664604257281939434" target="_blank">comment</a> to my last post which triggered a summary of my current thinking about life and signals. A lot of this covers ideas I've had for a long time but not blogged about and, as I can never be sure a post will write itself, here is a thought dump to lay it out without any detail or references. As karl said:</div><div><br /></div><div>"My hunch is the ROS signal is more primitive than insulin itself?"<br /><br />In response:</div><div><br />Hi karl,</div><br />Superoxide is crucial to bacterial (and archaeal) growth, division and death.<br /><br />Mitochondria retain this signalling system based on a bacterial ~200mV membrane potential.<br /><br />The cell surface membrane (archaeal derived) has relinquished its 200mV potential to mitochondria.<br /><br />Cell surface signalling still uses superoxide but this is now NOX controlled.<br /><br />It provides the same signal as mitochondrial superoxide but has differing cues and locations.<br /><br />Larger multicellular organisms use a pancreas to condense/combine these ROS signals in to a redox signal carried by a pair of -S-S- double bonds (ox stress marker, think of as a glutathione G-S-S-G mimetic) between two short peptides to encode and transmit an assessment of whole body redox status from the circulation, via the circulation.<br /><br />Individual cells respond/resist this signal by making superoxide in response to it then modulate it using their own locally derived NOX/RET superoxide signals.<br /><br />There is no need for it to be the insulin/insulin receptor combination that carries this signal, any -S-S- di-peptide and an appropriate receptor will do. Most metazoans use insulin/insulin receptor but plants, protozoa and yeasts made different choices but they all do the same job.<br /><br />These assorted signalling systems cross react across all eukaryotes because the underlying ROS signal is fundamental.<br /><br />Mitochondrial (bacterial style) ROS from RET in ETC -> mitochondrial division (biogenesis) = Good™.<br /><br /><div>Cell surface (eukaryote invented) NOX ROS -> cellular division -> tissue growth (+ cancer) = potentially Bad™.</div><div><br /></div><div>Aside: Bad™ stolen from @KetoCarnivore on X/Twitter. End aside.</div><div class="css-1dbjc4n r-1awozwy r-z2wwpe r-6koalj r-1q142lx" style="-webkit-box-align: center; -webkit-box-direction: normal; -webkit-box-orient: vertical; align-items: center; background-color: white; border-radius: 4px; border: 0px solid black; box-sizing: border-box; display: flex; flex-basis: auto; flex-direction: column; flex-shrink: 0; font-size: 15px; margin: 0px; min-height: 0px; min-width: 0px; padding: 0px; position: relative; z-index: 0;"></div><div><br />That LUCA, even before her division in to bacteria/archaea and while living in an anoxic hydrothermal vent, had a globin ancestor to bind O2, an SOD ancestor (based on Fe) to convert superoxide to H2O2 and a catalase to detoxify H2O2 suggests to me that O2 was available, rare/precious and used as a signal (by accepting an electron -> superoxide) of "membrane" potential (ie opportunity to grow, ie superoxide signal) which had to be processed and terminated at a time even before cell membranes were genetically specified.</div><div><br />Obviously catalase derived O2 would be re-stored by the globin for re-use. LUCA (in a deep anoxic ocean) would have derived a meagre supply of precious O2 from the radiolysis of water, a process still used by deep earth-crust bacteria living at a mile below the nearest O2 supply at the earth's surface, even today.<br /><br />This is my most simplistic view. Without it I can't understand mice becoming obese eating an "high fat" diet. With it I’m spared the deeper intricacies of intermediary metabolism, a complex system if ever there was one.<br /><br />Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com7tag:blogger.com,1999:blog-36840063.post-17930308099285531222023-10-17T17:23:00.001+00:002023-10-17T17:28:20.270+00:00Insulin mimesis without NOX enzymes (6) MCT versus linoleic acid<div>I have a preference for median lifespan when thinking about the potential effect of an intervention on a population because it is looking at the population as a whole, rather than a few (quirky?) isolated individuals who make up the maximum longevity crowd. Or should I say the maximum longevity chosen few? I guess it confirms my biases too. Which I like.</div><div><br /></div><div>Let's recap the Jim Johnson's lab findings about the Surwit diet effects described here.</div><div><br /></div><div><a href="https://www.cell.com/cell-reports/pdfExtended/S2211-1247(17)30862-8" target="_blank">Reduced Circulating Insulin Enhances InsulinSensitivity in Old Mice and Extends Lifespan</a></div><div><br /></div><div>I have removed the lowest insulin gene group data and just included those with a normal insulin phenotype. A number metabolic snapshots were taken at around the 80 weeks of age mark. Surwit fed animals were heavier on the scales and fatter by DEXA compared to chow fed:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh8WyDxSgKOLyXrPJAFSuOjcVU9P8NWgfrwYhHJjgyOy6BoTaTVmR57doZtApCVXj3OGmGdgaVoB75TJfMvwBUbwhuc9QhvN995lRdBvoxV9vvzNhPUjAPfyKtVigZj891lxrK28sDfGiAnb-93C2TfnrPqWXNGvipVvytrZrpP9lQlNjkYJ_Dqiw/s987/Johnsons%20weights%20and%20fat.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="835" data-original-width="987" height="339" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh8WyDxSgKOLyXrPJAFSuOjcVU9P8NWgfrwYhHJjgyOy6BoTaTVmR57doZtApCVXj3OGmGdgaVoB75TJfMvwBUbwhuc9QhvN995lRdBvoxV9vvzNhPUjAPfyKtVigZj891lxrK28sDfGiAnb-93C2TfnrPqWXNGvipVvytrZrpP9lQlNjkYJ_Dqiw/w400-h339/Johnsons%20weights%20and%20fat.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>No one should be surprised that they were also hyperglycaemic and hyperinsulinaemic, ie insulin resistant:</div></div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgHz5qySDQv8JFINhaimGZEZvz21IEOpToqBfAA7Em-QqOzwRjFNDk0BOJmU1WKjgw5wN2Vws035w35D6SoEltJI9-ygyAN7wHd3XnHeMg5GBY4n3eUneKGVJRtXuHUYsimX_aAOejWd_UwjIuMGVRijENpsI2xzAW5ZOYOhVCrnKL_XeekdTnFvQ/s1127/JJ%20ins%20gluc.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="783" data-original-width="1127" height="278" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgHz5qySDQv8JFINhaimGZEZvz21IEOpToqBfAA7Em-QqOzwRjFNDk0BOJmU1WKjgw5wN2Vws035w35D6SoEltJI9-ygyAN7wHd3XnHeMg5GBY4n3eUneKGVJRtXuHUYsimX_aAOejWd_UwjIuMGVRijENpsI2xzAW5ZOYOhVCrnKL_XeekdTnFvQ/w400-h278/JJ%20ins%20gluc.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>What should be extremely surprising is that these Surwit fed mice while being obese, hyperinsulinaemic, hyperglycaemic and insulin resistant, lived over 100 days longer than their slim chow fed relatives. The data are in here:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh97w6SZB1GZBkI255d7YEMmkXoA2wLws2EgJA-L46cZm8323D1nq5c7nkHAxN_IKDbwZXRVBWgF7-WW2WSBEHXQJk-0Ci4hqrhNyxnnaOKz7TqjuFNB4_xvEIKhTQaqsWCS1VoNVsn_D9EoAIcRtK9V0hcvrRi5o3ub4ZgjRakwi9-pkJWiLvnXw/s1129/JJ%20original.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1000" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh97w6SZB1GZBkI255d7YEMmkXoA2wLws2EgJA-L46cZm8323D1nq5c7nkHAxN_IKDbwZXRVBWgF7-WW2WSBEHXQJk-0Ci4hqrhNyxnnaOKz7TqjuFNB4_xvEIKhTQaqsWCS1VoNVsn_D9EoAIcRtK9V0hcvrRi5o3ub4ZgjRakwi9-pkJWiLvnXw/w354-h400/JJ%20original.png" width="354" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>which I can simplify down with some very crude curve fitting by eye in Powerpoint to give this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEikGleXfVWyzH0u8QL84jhFJxyfZM0BFrW8BX13y5hJBui594ObJUJcb6pK6wA_Vwd5ZcohAGQE5xMmSWawzgsNovjexJNzQsX5OibELWonFZLKB_5D2BC2aIDbKx_qcE1XnCO77ogGoqreyy_9zqMC1E-1EbH2AosyChTOP9-btJUvq5Bwqx9GBw/s1129/JJ%20simp.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1052" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEikGleXfVWyzH0u8QL84jhFJxyfZM0BFrW8BX13y5hJBui594ObJUJcb6pK6wA_Vwd5ZcohAGQE5xMmSWawzgsNovjexJNzQsX5OibELWonFZLKB_5D2BC2aIDbKx_qcE1XnCO77ogGoqreyy_9zqMC1E-1EbH2AosyChTOP9-btJUvq5Bwqx9GBw/w373-h400/JJ%20simp.png" width="373" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Interesting.</div><div><br /></div><div>Now let's look at some rats fed an obesogenic diet based on lard. There are some good data in here although it does not give us the linoleic acid content of the lard:<br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/33440166/" target="_blank">An isocaloric moderately high-fat diet extends lifespan in male rats and Drosophila</a></div><div><br /></div><div>Oh, and their data presentation is not great.</div><div><br /></div><div>Here we have the weights. Colour scheme is different to the first study</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgpTTuq7y4YP4qGDwZ6WCmS8N-dcHrv5e5gfmivphwBhNp5aqGZkHi5HpY6UsbqrmUoCKjZFT8vkRASGl4CfBPKJbryVlKF8uqk-mxqU2XdVHuiJPyYS_mlcldcEHy7gv6gIWsfW3TaC_dUtsj4z5cIiY6ijK5cnlhs_HmVOV3jMhROkgWXcUx28Q/s1504/Wights%20lard.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1000" data-original-width="1504" height="266" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgpTTuq7y4YP4qGDwZ6WCmS8N-dcHrv5e5gfmivphwBhNp5aqGZkHi5HpY6UsbqrmUoCKjZFT8vkRASGl4CfBPKJbryVlKF8uqk-mxqU2XdVHuiJPyYS_mlcldcEHy7gv6gIWsfW3TaC_dUtsj4z5cIiY6ijK5cnlhs_HmVOV3jMhROkgWXcUx28Q/w400-h266/Wights%20lard.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><a href="https://pubmed.ncbi.nlm.nih.gov/33440166/" target="_blank"><span style="color: black;"><br /></span></a><br /><br /><br /><br /><br /><br /><br /><br /><br />You can't tell if the weight loss toward the end was from the surviving rats eating less or that the fattest rats died earliest. Probably a bit of both. But the lard fed rats were fat.</div><div><br /></div><div>They were hyperinsulinamic</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjmHfYrp9jftQahqF8ud6XFEwlzA_b3vpZJPWiPeBG-WmrvfVBctHr0R5BtRu-991CIH8z7xjH_CP4lrJgHRJa2Hk2WJvRwj1Ew2ETGRAIqToEKNSf_FgS3ElhfawtnOqcJVdx_xCKTxeyNYNzFS-fYFTwsxnAWkdlVxCDH2R136BG6QJxEUNmaYw/s1293/lard%20graphs%20simp.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1293" height="349" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjmHfYrp9jftQahqF8ud6XFEwlzA_b3vpZJPWiPeBG-WmrvfVBctHr0R5BtRu-991CIH8z7xjH_CP4lrJgHRJa2Hk2WJvRwj1Ew2ETGRAIqToEKNSf_FgS3ElhfawtnOqcJVdx_xCKTxeyNYNzFS-fYFTwsxnAWkdlVxCDH2R136BG6QJxEUNmaYw/w400-h349/lard%20graphs%20simp.png" width="400" /></a></div><br /><div><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />and they were hyperglycamic.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiDa_dFyYDqq5phYz89nkzXChOv_3rha3jZhezapZRcWeERcXuxva9-OOHqoy6JnDG-1Es7j58pWwYSOMMQrWALKxEssWq62sTgOH-88NWEsguC5eQr-H2Ew0f9cavQ-S7ffd8LKgImPwRwr-CpIInLPNlLAbioHng7q7-KbTFWSU-95t9EPVz4Pw/s1325/glucose%20lard.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="966" data-original-width="1325" height="291" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiDa_dFyYDqq5phYz89nkzXChOv_3rha3jZhezapZRcWeERcXuxva9-OOHqoy6JnDG-1Es7j58pWwYSOMMQrWALKxEssWq62sTgOH-88NWEsguC5eQr-H2Ew0f9cavQ-S7ffd8LKgImPwRwr-CpIInLPNlLAbioHng7q7-KbTFWSU-95t9EPVz4Pw/w400-h291/glucose%20lard.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /><br /><br />and obviously they too were insulin resistant.</div><div><br /></div><div>But this time the longevity curves are reversed and the fat rats die younger than the slim control fed rats, by about 100 days:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiXNmraVWZ9MMn5HQ2_P6FxrrFq1P5dOVNg-SbN0Gs6kQpcIAglFIGcbhLpbPG0lbRjP6xQQh7tgTPZU985bHGAJhDPkqf7J5EBT5HKlCtM0FuVT9W2AjAJn_IPUV9HfjUEpWChLr00gutaQP57ZNLW30iPMMzyZGfbJVV8-dBMsKQSzjvw0FJylw/s1441/median%20lard.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1129" data-original-width="1441" height="314" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiXNmraVWZ9MMn5HQ2_P6FxrrFq1P5dOVNg-SbN0Gs6kQpcIAglFIGcbhLpbPG0lbRjP6xQQh7tgTPZU985bHGAJhDPkqf7J5EBT5HKlCtM0FuVT9W2AjAJn_IPUV9HfjUEpWChLr00gutaQP57ZNLW30iPMMzyZGfbJVV8-dBMsKQSzjvw0FJylw/w400-h314/median%20lard.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>So we have the mice in Jim Johnson's lab and the rats in the lab in Harbin, China. Both have comparable levels of obesity and insulin resistance. Both are oxidising FFAs when insulin should be suppressing FFA availability in peripheral tissues. In both cases excess energy is being supplied from fatty acids so there is an absolutely normal physiological reduction/rejection of some of the calories which are being taken up by cells using insulin facilitation.</div><div><br /></div><div>This normal physiological response is mediated by reverse electron transfer through complex I acting to inhibit insulin signalling at the insulin receptor/substrate level.</div><div><br /></div><div>In animals made obese using fully hydrogenated coconut oil in a Surwit diet the mitochondria are normal and mitochondrial/cytoplasmic membranes lipids are low in linoleic acid as you would expect from 1-2% LA in the diet. So high-physiological ROS from oxidising fatty acids will inhibit the insulin cascade, as they must, but in the process will only encounter "physiological" levels of linoleic acid and only generate "physiological" levels of 4-HNE, 13-HODE, 9-HODE etc. These lipoxides, like superoxide, are normal signalling molecules. A low level of generation is normal and generally beneficial. Probably essential.</div><div><br /></div><div>Generating ROS via RET in the ETC is pro-survival and pro-longevity. See <a href="https://pubmed.ncbi.nlm.nih.gov/27076081/" target="_blank">here</a>, discussion on another day.</div><div><br /></div><div>The obesity induced by increased dietary linoleic acid is different. Here the adipocytes are large because they fail to limit their insulin cascade adequately. Under these conditions adipose tissues will store an excess of lipid from all sources without insulin being elevated, indeed in the earliest stages insulin signalling will have been enhanced and IR subnormal. The core initiating problem being that linoleic acid is present in levels which generate too small an ROS signal, so fail to limit caloric ingress and storage. Also there is good evidence that linoleic acid is preferentially oxidiseg compared to saturated fats.</div><div><br /></div><div>However excess basal lipolysis secondary to adipocyte size will release all species of FFAs, arguably with some favourites, the problem now is that adipocytes *ignore* insulin. It doesn't matter how insulin sensitised you might have been via LA in order to become obese. If basal lipolysis is up, it's up. And insulin no longer matters. The exact mix of FFAs delivered to the periphery (especially muscle) becomes less important. All cells oxidising any type of fat generate more ROS than cells oxidising glucose.</div><div><br /></div><div>One of the secondary problems with high linoleic acid diets is that, separate from their obesogenic effects in mitochondria, the LA molecules are also present at increased levels in tissue lipids.</div><div><br /></div><div>The problems really present when ROS are being generated on a background of an intake of (<a href="https://pubmed.ncbi.nlm.nih.gov/16446749/" target="_blank">in this human observational study</a>) of 17% of calories as linoleic acid. Apart from making you obese this provides an environment where ROS which *should* meet a "normal" level of LA (ie derived from 2% in the diet) are actually encountering an environment derived from 17% LA in the diet. Generating *physiological* levels of 4-HNE etc is out of the question when all the ROS can "see" is freely available double bonds to interact with. Lipids are converted to lipoxides in supraphysiological quantities, cellular damage ensues and this chain of redox damage manifests as what we describe as pathology.</div><div><br /></div><div>That's what I think is happening.</div><div><br /></div><div>I think I've said enough for one post. I'll have to write a separate post about <a href="https://www.cell.com/cell-metabolism/fulltext/S1550-4131(16)30112-7?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1550413116301127%3Fshowall%3Dtrue" target="_blank">RET and Superfly</a>,</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiKzkaPRvpHMI_75KMz5SPJ-UptZq7wkc65cIpORSWZMWg5nZ2eCw1m5CNm1b-9Z1Ui2H9ouhinEZbavkDatbXdsdK5Ks0fgfT9U3odibsJ5c5muWwFKpu2fRSgLrE7sNj31gLU8AaT2z7v1gmlFJeRGllbyR7jd0AGab5CE2pxUv7FgUzPQM7Rqw/s675/NDI1%20fly.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="675" data-original-width="616" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiKzkaPRvpHMI_75KMz5SPJ-UptZq7wkc65cIpORSWZMWg5nZ2eCw1m5CNm1b-9Z1Ui2H9ouhinEZbavkDatbXdsdK5Ks0fgfT9U3odibsJ5c5muWwFKpu2fRSgLrE7sNj31gLU8AaT2z7v1gmlFJeRGllbyR7jd0AGab5CE2pxUv7FgUzPQM7Rqw/w365-h400/NDI1%20fly.png" width="365" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>the fly featured in the paper nominated for the best graphical abstract of all time, ever. By me anyway.</div><div><br /></div><div>And about ROS and <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358708/" target="_blank">mitochondrial biogenesis</a>.</div><div><br /></div><div>Peter<br /></div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com28tag:blogger.com,1999:blog-36840063.post-55649087447430023342023-10-15T16:34:00.007+00:002023-10-15T16:35:36.115+00:00Insulin mimesis without NOX enzymes (5) MCT plus linoleic acid<p>You can become obese by having your adipocytes fail to limit insulin signalling. That is what linoleic acid plus an insulogenic diet does, pure Protons.</p><div>You can also become obese by making your liver alone insulin resistant in combination with an insulogenic diet. The Surwit diet approach.<div><br /></div><div>These concepts put us in to a position where we can examine this study, HT to Tucker:<br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/37267350/" target="_blank">Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet</a></div><div><br /></div><div>In particular we can look at these curves:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfUYmvEHC6TMzW2AlFfm0ApwUtcr_oHuCjWpDBuYZPocEI-Fuyq0S0VMgbM87Kxr9ftpYpYk_0BqTjPcynYJRuqvLbDCbFUV6TSg8hZetW3YtdzPOfAmk5jn8U90aKxhcN3g6KKQaVypGKPMGFbQsDUNd5gqQNjP3pOZiA06mB3p6JGcQ2P1YM-w/s960/weight%20gains.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="484" data-original-width="960" height="201" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjfUYmvEHC6TMzW2AlFfm0ApwUtcr_oHuCjWpDBuYZPocEI-Fuyq0S0VMgbM87Kxr9ftpYpYk_0BqTjPcynYJRuqvLbDCbFUV6TSg8hZetW3YtdzPOfAmk5jn8U90aKxhcN3g6KKQaVypGKPMGFbQsDUNd5gqQNjP3pOZiA06mB3p6JGcQ2P1YM-w/w400-h201/weight%20gains.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br />Obviously blue circles are the chow and red triangles show the 8% linoleic acid, high overall fat diet. The latter is as obesogenic as you might expect. The surprising curve is the beige 1% LA line. This diet is clearly less obesogenic than the high LA diet but is not exactly slimming when compared to the chow line. And it supplies only 1% of calories from LA.</div><div><br /></div><div>So do *all* high fat diets make you (and rodents) obese?</div><div><br /></div><div>The macro of the diets are described in Table 1. I've cut off the vitamin E parts as they are a different subject altogether and might be worth a post some day.</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGifdZzBgQq3zzZypd4NA4dtQKRgkCgbaQi5lnHLQ1LLEqIyWXx-xiFwTBUuYuvywJwu12bx1R1EEnMwh6ovityZInH7kxit1ObFQqry2rCz8BEzXxww2PjRi_1_WjMe-FiJ26-aMyOGRiDbVMHBW-kJKsroevsP2yth9Ebf_OHc_D5sEPl7O4uw/s837/macros.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="231" data-original-width="837" height="110" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGifdZzBgQq3zzZypd4NA4dtQKRgkCgbaQi5lnHLQ1LLEqIyWXx-xiFwTBUuYuvywJwu12bx1R1EEnMwh6ovityZInH7kxit1ObFQqry2rCz8BEzXxww2PjRi_1_WjMe-FiJ26-aMyOGRiDbVMHBW-kJKsroevsP2yth9Ebf_OHc_D5sEPl7O4uw/w400-h110/macros.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>If you come at this from the point of view that saturated fats are slimming and LA is obesogenic then the low LA diet causing a significant level of obesity is a paradox.</div><div><br /></div><div>In the legend to Table 1 there is a link to supplementary information which I followed, trying to find whether fructose was included in the diets (the paper doesn't actually say) but I did find out that the bulk of the fat in both the 35% fat diets is, as you may have guessed, fully hydrogenated coconut oil.</div><div><br /></div><div>The 1% low LA diet is a classical Surwit derivative (possibly without the sucrose, I don't think it matters) and is inducing hepatic insulin resistance via ETC derived high levels of ROS to allow penetration of glucose/insulin to peripheral sites. The adipocytes see too much insulin for too much time and get fat.</div><div><br /></div><div>The 8% high LA diet has exactly the same problem from its MCT components as the 1% LA diet but this time it's compounded by peripheral adipocyte exposure to high levels of LA causing pathological insulin sensitivity, delivered directly via chylomicrons from the gut, bypassing the liver. They get high insulin exposure secondary to MCTs acting in the liver to allow increased glucose/insulin to the systemic circulation PLUS a pathological inability to resist insulin's fattening signal in the periphery via LA, ie adipocyte Protons effect on top of localised hepatic insulin resistance. Double wammy, double weight gain.</div></div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com8tag:blogger.com,1999:blog-36840063.post-43468979098028174912023-10-14T12:33:00.001+00:002023-10-20T20:16:42.296+00:00Insulin mimesis without NOX enzymes (4) MCT<div>Before I get to talk about this (highly recommended) paper:</div><div><br /></div><div><a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0286726" target="_blank">Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet</a></div><div><br /></div><div>which came up in a discussion between Tucker and Brad a few months ago, I have to clarify as well as I can how fully hydrogenated coconut oil, one of the most saturated fat sources on the planet, is obesogenic, even under low linoleic acid conditions. The post is already part written but I want to get the MCT mechanism down here as clearly as possible using one of my long term doodles.</div><div><br /></div><div>Apologies if it's getting repetitive, this is really an executive summary. Here we go.</div><div><br /></div><div>One basic concept of the Protons hypothesis is that saturated fats shut down insulin signalling when fatty acids are freely available and glucose is scarce. Classically this is under fasting conditions where allowing glucose in to muscles etc would rapidly deplete the body of glucose stores which are helpful to maintain brain function under fasting conditions. It applies whole-body. Like this:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg3Q0wIqmUvKcr2Xiw_fj3Vu49_3me5R5qe3NBR5_ssDK8uRzYRzMW4DWSkCvb8pu8siDc4FPjJMpN9XICQ-US0YbAt5wxDDJ2TDXkN5LHTk26DYEQjrVPgQdTs5NrLltCJQAK7Sxs8cl5nwe0vRB1uIz1CJDyNV_ZLJMujsutat1Iprxad6_OzcA/s914/palmitate.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="664" data-original-width="914" height="290" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg3Q0wIqmUvKcr2Xiw_fj3Vu49_3me5R5qe3NBR5_ssDK8uRzYRzMW4DWSkCvb8pu8siDc4FPjJMpN9XICQ-US0YbAt5wxDDJ2TDXkN5LHTk26DYEQjrVPgQdTs5NrLltCJQAK7Sxs8cl5nwe0vRB1uIz1CJDyNV_ZLJMujsutat1Iprxad6_OzcA/w400-h290/palmitate.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Under high fatty acid availability the ROS derived from reverse electron transfer through complex I of the mitochondrial electron transport chain inhibit insulin signalling at the insulin receptor/receptor substrate level, ie at the cell surface. Simple and obviously adaptive. This is also the system which fails when linoleic acid in bulk is introduced to the diet.</div><div><br /></div><div>The next concept is that metabolic substrates which do not cause insulin secretion from beta cells will generate their own insulin-mimicking ROS signal using NADPH oxidases, also at the cell surface of nutrient recipient cells. At low levels these ROS inhibit the "insulin signalling suppression system" and allow the insulin signalling to take off, in the absence of insulin per se:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj741xwlzbrSQlKc0hC18SGqihhQ3OToy0lvP8DeU_uYtUvGBv2xt5Df-9mUra8oTLzMAF5WgagZsWZKqiQ_SiCwQ9DmKq5uD59CDNve6yBzlPhbc4zIakrUyqJl6mtYTpkTKNnNhp8m4gHK6fTxt1tDx1GRDGcRmyqMOgjOnxlalI5FL2m_v3snw/s1504/low%20dose%20NOX%20activators.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1070" data-original-width="1504" height="285" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj741xwlzbrSQlKc0hC18SGqihhQ3OToy0lvP8DeU_uYtUvGBv2xt5Df-9mUra8oTLzMAF5WgagZsWZKqiQ_SiCwQ9DmKq5uD59CDNve6yBzlPhbc4zIakrUyqJl6mtYTpkTKNnNhp8m4gHK6fTxt1tDx1GRDGcRmyqMOgjOnxlalI5FL2m_v3snw/w400-h285/low%20dose%20NOX%20activators.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Mostly this happens in the liver because that's where most of the fructose/ethanol/acetate ends up. High doses are a separate story.</div><div><br /></div><div>Aside: An high concentration of these substrates, ie two cans of cola giving >17mmol/l fructose in the systemic circulation or doing ethanol shots, will generate severe inhibition of the insulin cascade at the insulin receptor/insulin receptor substrate level whole-body, via NOX hyperactivation. And cellular damage from ROS spilling everywhere will result, ie Badness. Back to low level discussions. End aside.</div><div><br /></div><div>The situation with MCTs is different from both of the above. They too are delivered primarily to the liver, not to the systemic circulation, and taken up rapidly by the mitochondria. All undergo beta oxidation to acetyl-CoA but only some of this acetyl-CoA will enter the Krebs cycle to provide FADH2 and NADH to the electron transport chain. The rest will be diverted to ketone bodies without providing any input to the ETC, and so without generating the inhibitory ROS you would expect from a saturated fat. I consider this is what happens, almost only in hepatocytes:<br /><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjeLOglxdxgfb6ZKryUsfmIQtjvB0N_rvGkGuoDeApwrkDR68uL-OH2EuS18cWsbg5WkFwRaqYJpiJEPtRsOEMgoSVsd26VnTL9y7pJFSRtMjvKo8y-uaBGi-c88hK4f8ACYlYL9CituyTrt49YE8jh-qRX7L9bXTI_qs_cMuVFJJCZuhnz-_LIEA/s1504/MCT%20activating%2050.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1100" data-original-width="1504" height="293" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjeLOglxdxgfb6ZKryUsfmIQtjvB0N_rvGkGuoDeApwrkDR68uL-OH2EuS18cWsbg5WkFwRaqYJpiJEPtRsOEMgoSVsd26VnTL9y7pJFSRtMjvKo8y-uaBGi-c88hK4f8ACYlYL9CituyTrt49YE8jh-qRX7L9bXTI_qs_cMuVFJJCZuhnz-_LIEA/w400-h293/MCT%20activating%2050.png" width="400" /></a></div><br /><div><br /><br /><br /><br /><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Low levels of MCTs (in this doodle I've suggested 50micromol/l, this is just for a thought experiment, not data based) arrive at the liver and have absolutely nothing to do with NADPH oxidase enzymes while generating low levels of ROS via mitochondria. These "low" levels are high enough to disable the "insulin signalling inhibitory system" and allow the insulin cascade to be activated but not high enough to directly inhibit the insulin receptor/substrate complex. This activation is insulin "mimesis" without substrate going anywhere near NOXs 1,2, 4 etc. But only in hepatocytes because that's where MCTs are diverted to.</div><div><br /></div><div>The result is to sequester glucose in to hepatocytes so reduce penetration of glucose past the liver and in to the systemic circulation. The pancreas sees reduced glucose, so secretes less insulin. Lipolysis increases, you might lose weight.</div><div><br /></div><div>Under Surwit-like diet conditions with coconut oil at 35-60% of calories things are different. Here large amounts of MCTs are delivered to the liver, I've suggested 600micromol/l but who knows what the portal vein concentrations really are? These MCTs still "waste" acetyl-CoA to produce ketones but the increased bulk supply means more acetyl-CoA can enter the Krebs cycle/ETC. Enough to generate ROS at levels which will inhibit activation of the insulin cascade at the insulin receptor/substrate level:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEibX5BPv-lqnzLjThNgUefIZ2BMCtpEsBMFE94Bubl1uPr9scJk7SD1YWR_7sQh5IIhHYd-eVC6ufah7rHcuiMw9ynISPiNzf_p5353deEJlwD8C_6mY3kSvAqgdykcs08_X3xdyTC71ACJ91QUtUV082f4ZlyYPc0BHziwEKuDGRLnOR8EsyG81A/s1504/High%20MCT%20liver.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="1112" data-original-width="1504" height="296" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEibX5BPv-lqnzLjThNgUefIZ2BMCtpEsBMFE94Bubl1uPr9scJk7SD1YWR_7sQh5IIhHYd-eVC6ufah7rHcuiMw9ynISPiNzf_p5353deEJlwD8C_6mY3kSvAqgdykcs08_X3xdyTC71ACJ91QUtUV082f4ZlyYPc0BHziwEKuDGRLnOR8EsyG81A/w400-h296/High%20MCT%20liver.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>Under conditions of suppressed hepatocyte insulin signalling dietary derived glucose will penetrate past the liver and in to the systemic circulation. This will require the pancreas to crank out more insulin. And so inhibit lipolysis. As in the obesogenic Surwit diet.</div><div><br /></div><div>That is all for now.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com4tag:blogger.com,1999:blog-36840063.post-41163476155371753842023-10-12T04:57:00.001+00:002023-10-12T14:22:12.008+00:00Insulin mimesis and NOX enzymes (3) MCTSo now it's time to see how it is possible to understand the obesogenic effects of fully hydrogenated coconut oil in Surwit diets and also the recognised potential for MCT induced weight loss effects. Oooh, another paradox!<div><br /></div><div>I've been skirting around this subject for years and I still do not have nice solid answers but this is getting closer.</div><div><br /></div><div>It's worth thinking, initially, about fructose as a weight loss agent.<br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/11812757/" target="_blank">Inclusion of low amounts of fructose with an intraduodenal glucose load markedly reduces postprandial hyperglycemia and hyperinsulinemia in the conscious dog</a><br /><br />Going back to this canine model of intraduodenal glucose infusion +/- 5% fructose we have these curves:</div><div><br /></div><div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjACLMnQlQviSn7gBuJZO3h5Nxmu3iYVT-FCjxAL8jGNfm4QnjFEGAZ0ABO1DysTElKEYsH1laKnGkarmCwUpXP_OINOhWfot33LDI4rpLwmjtb3bRivzFNJKnoZdGqT14rJ1Rjn9q6Bq2-1XF6MhrktOvxqc1jTI-8ycLMFPrCD1dqgiOWz9A/s1104/GFG%20insulin.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="891" data-original-width="1104" height="323" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjACLMnQlQviSn7gBuJZO3h5Nxmu3iYVT-FCjxAL8jGNfm4QnjFEGAZ0ABO1DysTElKEYsH1laKnGkarmCwUpXP_OINOhWfot33LDI4rpLwmjtb3bRivzFNJKnoZdGqT14rJ1Rjn9q6Bq2-1XF6MhrktOvxqc1jTI-8ycLMFPrCD1dqgiOWz9A/w400-h323/GFG%20insulin.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The fructose supplemented line (open circles) shows a markedly reduced insulin excursion, a direct result of the reduced penetration of glucose past the liver. Fructose, acting solely at the liver, is imitating insulin via ROS from an NADPH oxidase. Glucose gets stored:</div></div><div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjYqtSbCDNVBXKdJhqDkZL3udIGxB6qfMmDY0wajQYJYr1fH0I8uxD8gl5QF7Tj4KglKY2X_VDSEfynSkvCLiNOXZoLk3Mf4Au1M9U_lSLINoWe8S2peN9OS44ergABVaxHXUezs_sgaUqH29mG-zddMYU6H0JKT7EJDcMHPtigGGy2uJRCxHA/s1152/glucose%20fructose%20glucose.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="843" data-original-width="1152" height="293" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjYqtSbCDNVBXKdJhqDkZL3udIGxB6qfMmDY0wajQYJYr1fH0I8uxD8gl5QF7Tj4KglKY2X_VDSEfynSkvCLiNOXZoLk3Mf4Au1M9U_lSLINoWe8S2peN9OS44ergABVaxHXUezs_sgaUqH29mG-zddMYU6H0JKT7EJDcMHPtigGGy2uJRCxHA/w400-h293/glucose%20fructose%20glucose.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>The pancreas sees a markedly reduced glucose level, so responds with a markedly reduced insulin secretion (200pmol/l down to 100pmol/l).</div><div><br /></div><div>If you were an adipocyte in the periphery you are not going to see the fructose acting as insulin via ROS to sequester glucose as hepatic glycogen. This all happens in the liver. All an adipocyte sees is 100pmol/l of insulin instead of 200pmol/l.</div><div><br /></div><div>What is that going to do to your rate of lipolysis?</div><div><br /></div><div>I would expect it to be higher in the fructose supplemented state.</div><div><br /></div><div>Aside. There is s small rise in lactate with the fructose infusion but I doubt it would offset an halving of the insulin level. End aside.</div></div><div><br /></div><div>Does this cause weight loss? I don't know that anyone has asked that question in this form for humans using fructose. </div><div><br /></div><div>Acetate, which I consider to induce a similar signalling response to fructose, certainly does. Drinking vinegar for weight loss appears to work. The effect is not huge but appears real and is mechanistically logical:<br /><br /><a href="https://pubmed.ncbi.nlm.nih.gov/19661687/" target="_blank">Vinegar intake reduces body weight, body fat mass, and serum triglyceride levels in obese Japanese subjects</a></div><div><br /></div><div>So maybe fructose is a weight loss drug, in modest amounts, on a background of a starch/linoleic acid diet.</div><div><br /></div><div>So if we get back to medium chain triglycerides for weight loss, here they used either coconut oil or MCT oil (both worked):</div><div><br /><a href="https://pubmed.ncbi.nlm.nih.gov/36386906/" target="_blank">Coconut oil and medium-chain fatty acids attenuate high-fat diet-induced obesity in mice through increased thermogenesis by activating brown adipose tissue</a><br /><br /></div><div>You can get a marked decrease of weight gain in a (mouse) model of lard-driven obesity by <i>small</i> amounts of MCTs.<br /></div><div><br /></div><div>Like fructose and to some extent acetate, low doses of MCTs are predominantly diverted directly to the liver via the portal vein from the gut. If they generate modest doses of ROS, ie at insulin mimetic levels, in the liver alone, they will allow the diversion of glucose in to hepatic storage and so reduce penetration of glucose to the systemic circulation and that will reduce the need for insulin secretion. Peripheral adipocytes will see less insulin and so store less, or release more, fatty acids.</div><div><br /></div><div>To me that makes sense. Increasing hepatic insulin-like signalling derived from very modest MCT ROS generation protects the peripheral adipocytes from glucose/insulin exposure.</div><div><br /></div><div>Exposure to higher levels of MCTs, ie Surwit-like diets, is undoubtedly obesogenic.</div><div><br /></div><div>As we increase the proportion of MCTs in the diet this gut-to-liver channel increases delivery giving increased storage of glucose as hepatic glycogen (no problem) and an attempt at hepatic storage of MCT fatty acids. But MCTs aren't stored, they are rapidly oxidised to give ketones plus mitochondrially derived ROS. At high enough exposure there will be enough ROS to finally resist insulin signalling within the hepatocytes.</div><div><br /></div><div>Hepatic insulin resistance allows more insulin to penetrate past the liver to the systemic circulation and so to reach peripheral adipocytes. It's not essential for MCTs per se to reach those adipocytes in any quantity, though if they do so at insulin mimetic levels they will compound the problem.</div><div><br /></div><div>Back in March this year I <a href="https://high-fat-nutrition.blogspot.com/2023/03/how-can-insulin-resistance-cause-weight.html" target="_blank">pointed out how</a> mixed coconut MCTs at "physiological" concentration are experimentally confirmed generators of ROS at a level which will phosphorylate Akt in isolated neurons, insulin mimesis. I was also clear that "supra-physiological" exposure to octanoate inhibited correct development of adipocytes, ie caused insulin resistance. These effects applied to hepatocytes, which are the primary target of dietary MCTs, would be quite enough to explain the Surwit effects <i>and</i> weight loss effects, depending on dose.</div><div><br /></div><div>Summary: Low dose MCTs are insulin-mimetic and primarily delivered to the liver only. They protect peripheral adipocytes from insulin exposure and allow weight loss/limit weight gain.</div><div><br /></div><div>High dose MCTs provide insulin resistance levels of ROS in hepatocytes and facilitate insulin's penetration to peripheral adipocytes. Any low levels of MCTs reaching peripheral adipocytes will provide low levels of ROS to augment fat storage by insulin per se.</div><div><br /></div><div>It is perfectly possible to generate obesity with highly saturated MCT based diets. Even at 2% linoleic acid.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com12tag:blogger.com,1999:blog-36840063.post-42333672884754914802023-10-11T06:51:00.001+00:002023-10-12T04:21:07.576+00:00Insulin mimesis and NOX enzymes (2) Lactate<div>I wasn't going to torture lactate in the usual manner but Alan Couzens on Twitter suggested the two essentials for weight loss were to oxidise lipids and to minimise lactate. I was vaguely aware that lactate is a potent inhibitor of lipolysis and that rapidly rising lactate with exercise is going to tank your fatty acid availability. So I assumed lactate is an insulin mimetic and this is a quick hit searching Pubmed for lactate and lipolysis. GPR81 and HCAR1 are different names for the same receptor:</div><br /><a href="https://pubmed.ncbi.nlm.nih.gov/19047060/" target="_blank">Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81</a><div><br /></div><div>which obviously suggests that this might be happening:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2LF2doZECG6-aXZFtKxfQ_B-jrkbPN6QMDwFE5qTB5w5mEGv8xukYz1ETKM7rbqpEd5CaA15_CguweNQM1_vvMTe6a7ctvizuk2Vd5jRh_aXIj3I8eQh0C5inakyKOvCviCTy2lZzK5aNobll6su2LLyp3SCb9jj4B6SnjJONjzo6LAYgekqoGg/s1504/lactate%20basic.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="941" data-original-width="1504" height="250" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg2LF2doZECG6-aXZFtKxfQ_B-jrkbPN6QMDwFE5qTB5w5mEGv8xukYz1ETKM7rbqpEd5CaA15_CguweNQM1_vvMTe6a7ctvizuk2Vd5jRh_aXIj3I8eQh0C5inakyKOvCviCTy2lZzK5aNobll6su2LLyp3SCb9jj4B6SnjJONjzo6LAYgekqoGg/w400-h250/lactate%20basic.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>However there was nothing here to relate this to NADPH oxidases, so I added some more search terms and got this paper:</div><div><br /></div><div><a href="https://pubmed.ncbi.nlm.nih.gov/37688977/" target="_blank">Lactate-upregulated NADPH-dependent NOX4 expression via HCAR1/PI3K pathway contributes to ROS-induced osteoarthritis chondrocyte damage</a></div><div><br /></div><div>which is more interesting.</div><div><br /></div><div>Recall that acetate primes NOX4 in neutrophils to be ready to <a href="https://pubmed.ncbi.nlm.nih.gov/34330996/" target="_blank">protect you from severe sepsis</a>? By phosphorylating it.</div><div><br /></div><div>Lactate does something similar and allows me to add this part to the doodle:</div><div><br /></div><div class="separator" style="clear: both; text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiG89nPgtFrvuSqJ2MAuL1lf53w5JbC4ZUbAZKWM-JERSedoJShmtyM1UxvN9Pm12c7cT5-hQayZNvWcOAup5lkzwF2PBHBh39B2NRDqB5hmDBhupgkfyUoU5KWfTnVQNd1esfhs9G2OKMVkML7tcaxvGM4JmJM7FwZr8-DfF4RfU2TYuY1hLtSig/s1504/lactate%20feedback.png" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" data-original-height="945" data-original-width="1504" height="251" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiG89nPgtFrvuSqJ2MAuL1lf53w5JbC4ZUbAZKWM-JERSedoJShmtyM1UxvN9Pm12c7cT5-hQayZNvWcOAup5lkzwF2PBHBh39B2NRDqB5hmDBhupgkfyUoU5KWfTnVQNd1esfhs9G2OKMVkML7tcaxvGM4JmJM7FwZr8-DfF4RfU2TYuY1hLtSig/w400-h251/lactate%20feedback.png" width="400" /></a></div><br /><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div><br /></div><div>This, in chondrocytes, is a positive feedback loop. When they want to to reject insulin signalling, they really will generate toxic levels of ROS, which will ruin your knees. I wonder if this positive feedback is a generic trait too. Okay, I'll leave lactate alone soon.</div><div><br /></div><div>Interestingly, in people undergoing total knee replacement surgery serum lactate can be as high as 13mmol/l at rest. Even in "normal" controls one individual had a lactate of 9mmol/l at rest. One feature of metabolic syndrome seems to be that you walk around with the lactate levels of a young athlete under exercise while sitting still, even without a cup of sugary tea by your hand...</div><div><br /></div><div>Fascinating.</div><div><br /></div><div>Peter</div>Peterhttp://www.blogger.com/profile/14527788116058656094noreply@blogger.com3