Tuesday, November 28, 2023

Life (35) NiFe pumping

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:

"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."

quoted from

The coupling mechanism of respiratory complex I — A structural and evolutionary perspective

which was the basis of the last 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.

Here we are, ready to start. These electrons have come from ferredoxin rather than NADH:

The electrons transfer from the FeS clusters to the NiFe cluster and the repulsive effect shown in the purple arrows disappears:

and is replaced by the large purple arrows signifying marked attraction:

and subsequent conformation change and ion pumping:

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-, attached to the Ni atom.

So we can start like this

which will rearrange to this:

giving us the hydride intermediate before exchange of the electron and acquisition of a proton from the amino acid Y:

to give us molecular hydrogen

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:

and we're good to go again:

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+ 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+ 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+ gradient which is a pre requisite to leaving the vent system as an independent organism.


Sunday, November 26, 2023

Life (34) Complex I pumping

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:

The coupling mechanism of respiratory complex I — A structural and evolutionary perspective

For orientation this is section A from Fig 1 in the same paper:

and most of the rest of the post is going to be about the area outlined by the red oval.

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 Desulfovibrio gigas. Both of these complexes show marked conservation of their structures in this area, as do all similar complexes:

and if I duplicate the image I can add in the electron pathways like this:

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:

This is the un-altered original Fig 6. I'll explain it step by step in the following section:

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.

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:

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:

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 CoQ2-. I've used the same red circles to accentuate the small negative signs used in the original diagram:

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:

The next change is subtle. All that has happened is that CoQ2- 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:

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.

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:

Once these two events have occurred then we are back in to the situation in section 1 and the process can repeat:

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.

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:

"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."

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.


Sunday, November 12, 2023

Life (33) Transient hunger

Just a brief repeat that the origin of metabolism is this reaction at
pH 6:

CO₂ + 2e- + E(in) -> CO + O2-

where the electrons and activating energy E(in) come from molecular hydrogen in solution at pH 10.

The next step is:

CO + O2- + 2H+ -> HCOOH + E(out)

where E(out) is greater than E(in) , 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.

Biomolecules accumulate. They are energetically stable.

So if we look at these conditions:

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.

That's step one.

Next comes the development of another use for the energy provided by hydrogen at an alkaline pH.

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.

Ferredoxin + 2e- + E(in) -> Ferredoxin2-

If we take out all intermediate steps we can summarise the generation of Fd2- near the origin of life like this

H2 + Fd -> Fd2- + 2H+

Fd2- (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.

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 Fd2- requires a cell membrane to protect it from the catalytic primordial FeS and NiFeS surface.

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:

which is the basis of this:

which we can declutter to this:

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:

Recall that organic molecules are safe from degradation under these circumstances but that ferredoxin is not. So it is very easy to degrade Fd2- 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.

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 Fd2- 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.

This stops the wasteful generation of molecular hydrogen from Fd2-.

The second technique is to limit the ingress of protons from the acidic ocean fluid.

We currently have a situation where electrons are still free to travel from Fd2- to the embedded FeS cluster, producing a net negative charge:

All that is now needed is a positively charged area of amino acids in the transmembrane tube structure, like this:

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:

This provides a temporary "pro-survival" state for the protocell using the remaining Fd2- 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 Fd2- pool. This opens the transmembrane channel and allows normal cell function to return:

and we're back where we started from.

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:

Symmetry-related proton transfer pathways in respiratory complex I

especially figures 2A and 6

and here:

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.


Tuesday, October 31, 2023

Life (32) On the correct side of the fence

Preamble. On page 143 of Transformer (I'm assuming everyone has a copy of Transformer) Nick Lane writes:

"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."

Which takes me back to my thoughts on pH differential driving prebiotic chemistry here:

Life (22) FeNi hydrogenase

which contains a serious logical flaw regarding the location of the site for pre-biotic carbon dioxide reduction:

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.

So I've had to rethink the whole basic process with some ideas triggered by various lines in Transformer. Here we go.

The reaction which summarises the origin of life is

CO2 + H2 -> HCOOH

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".

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:

CO2 + H2 -> CO + H2O

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.

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.

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.

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:

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.

The two FeS clusters at the interface are at markedly differing pH conditions.

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.

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.

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:

The process is laid out in Transformer, organics can be formed on the alkaline side of an FeS permeable barrier.

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.

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

Structure of an Ancient Respiratory System

which I doodled on 2019 like this:

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

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:

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.

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.

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.


Sunday, October 29, 2023

Dalton Graham

I'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.

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...

Anyhoo, Tucker sent me the link to this discussion he and Brian Kerley had with Dalton Graham.

As in
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:

Ep. 10: Dalton Graham: How to Induce Fatty Liver


Monday, October 23, 2023


There was a line by karl in a  comment 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:

"My hunch is the ROS signal is more primitive than insulin itself?"

In response:

Hi karl,

Superoxide is crucial to bacterial (and archaeal) growth, division and death.

Mitochondria retain this signalling system based on a bacterial ~200mV membrane potential.

The cell surface membrane (archaeal derived) has relinquished its 200mV potential to mitochondria.

Cell surface signalling still uses superoxide but this is now NOX controlled.

It provides the same signal as mitochondrial superoxide but has differing cues and locations.

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.

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.

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.

These assorted signalling systems cross react across all eukaryotes because the underlying ROS signal is fundamental.

Mitochondrial (bacterial style) ROS from RET in ETC -> mitochondrial division (biogenesis) = Good™.

Cell surface (eukaryote invented) NOX ROS -> cellular division -> tissue growth (+ cancer) = potentially Bad™.

Aside: Bad™ stolen from @KetoCarnivore on X/Twitter. End aside.

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.

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.

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.


Tuesday, October 17, 2023

Insulin mimesis without NOX enzymes (6) MCT versus linoleic acid

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.

Let's recap the Jim Johnson's lab findings about the Surwit diet effects described here.

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:

No one should be surprised that they were also hyperglycaemic and hyperinsulinaemic, ie insulin resistant:

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:

which I can simplify down with some very crude curve fitting by eye in Powerpoint to give this:


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:

An isocaloric moderately high-fat diet extends lifespan in male rats and Drosophila

Oh, and their data presentation is not great.

Here we have the weights. Colour scheme is different to the first study

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.

They were hyperinsulinamic

and they were hyperglycamic.

and obviously they too were insulin resistant.

But this time the longevity curves are reversed and the fat rats die younger than the slim control fed rats, by about 100 days:

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.

This normal physiological response is mediated by reverse electron transfer through complex I acting to inhibit insulin signalling at the insulin receptor/substrate level.

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.

Generating ROS via RET in the ETC is pro-survival and pro-longevity. See here, discussion on another day.

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.

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.

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.

The problems really present when ROS are being generated on a background of an intake of (in this human observational study) 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.

That's what I think is happening.

I think I've said enough for one post. I'll have to write a separate post about RET and Superfly,

the fly featured in the paper nominated for the best graphical abstract of all time, ever. By me anyway.

And about ROS and mitochondrial biogenesis.