Wednesday, February 06, 2013

Protons: Where is FeS cluster N-1a?

I picked up this paper from one of Nick Lane's books, can't remember which one but probably PSS. The paper itself is very, very interesting and delves deeply in to redox potentials, especially within the iron-sulphur centres of complex I, to the sort of level which is way beyond my ability to critique. So I'm accepting their findings pretty much as presented, with the usual caveats about mitochondial studies. In particular it worries me that we can study superoxide generation in mitochondria at partial pressures of oxygen around 100mmHg (room air). No mitochondria see this concentration of oxygen in vivo.

The group make a pretty good case that the FeS centre N-1a is the primary source of superoxide generation from complex I when mitochondria are being fed on NADH generating substrates, in particular when there is a large excess of NADH per unit NAD+.

It also appears to be the source of superoxide under succinate driven activation of complex II, probably secondary to reverse electron flow up the FeS clusters secondary to markedly reducing the CoQ couple.

The group didn't look at activated fatty acids but the F:N ratios would suggest to me that palmitic acid and upwards (chain length of fully saturated fats) would behave much as succinate does, by reducing the CoQ couple in a very similar manner. I've argued for some time that superoxide is the physiological switch to turn off glucose metabolism (ie trigger insulin resistance at the individual cell level) when generous levels of saturated fats predominate for oxidation.

This leaves open the simple question of what, exactly, is the N-1a FeS cluster of complex I? What does it do? What does complex I look like anyway? How much iron-sulphur does it contain? I did a quick Google image search for the structure of iron-sulphur chain of complex I and it turned up some beautiful pictures. I rather like this one from the Netherlands:

Look at the string of FeS clusters, shown in red, running through the protein bed. Cluster N-1a is the one right adjacent to FMN, probably set behind it rather than as close as it looks. The one off to the right is N7. Modern complex I almost certainly never originated as a proton pump but its deepest ancestor rather looks to have been a layer of iron pyrites, FeS. Up at the top of the picture, in yellow, is the flavin mono nucleotide which transfers electrons from NADH to the FeS chain. At the bottom, in pink, is ubiquinone of the CoQ pool, ready to transfer electron equivalents to complex III. The CoQ pool is fascinating as having it reduced (in this paper by supplying succinate to complex II) appears to cause reverse flow up the FeS chain as far as (and perhaps beyond) the side branch to N-1a, with superoxide generation as the result. Palmitic acid probably does the same but obviously reduces the CoQ pool using electron-transferring-flavoprotein dehydrogenase (which lacks a catchy name) rather than succinate dehydrogenase (complex II), as we've discussed previously.

Both NADH and CoQ move electron equivalents around on a macroscopic scale by moving physically. The FeS clusters shuttle electrons by quantum tunnelling. This is a very short distance phenomenon. Below are the sorts of distances between the various FeS clusters in complex I. If anyone thinks the arrangement of FeS clusters is in any way a random set up I suspect they have no concept of what 4 billion years means.

We can ignore the N7 cluster as it's not really in the FeS chain at all, being over 20Å out to one side and it is not highly conserved. Cluster N-1a is highly conserved but it too is not really part to the chain of FeS clusters. It also sits out to one side, about 11Å out from FMN. This is a perfectly reasonable tunnelling distance to transfer electrons to and from FMN, but to hand an electron on to the N3 cluster (and so to the rest of the FeS chain) is over 19Å, a relatively low probability electron transfer. So N-1a looks as if it might be functioning as a reflection of the redox state of FMN, which reflects the reduction of the NADH/NAD+ couple. With a highly reduced NADH/NAD+ couple there is a spare electron on N-1a just waiting for an oxygen molecule to convert to superoxide and signal "no more" at the macroscopic (cellular) level... It will be interesting to see how this message is carried from N-1a to wherever it affects cell function but for the time being it's the superoxide generation which I find interesting per se.

I think it is worth pointing out that N-1a is directly sat at the very first step of the first complex of the electron transport chain. Where else would you put the control point? Very neat...

This gives us a nice view of complex I and N-1a linked superoxide generation which sets us up to look at what happens when you destroy complex I by growing mice with the TFAM gene knocked out in their adipocytes... Let's go there soon, if not next.



Purposelessness said...

Hi Peter!

It's a little bit like christmas whenever you post.

I feel like I should go back to the start of the series and reread the basics of reverse electron transfer... But I'll post a few papers and think out loudly before that:

Complex II can produce superoxide when complex I and III are inhibited and succinate is low:
"However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool."

Acute hypoxia seems to make complex III generate superoxide:

No idea what that means, haven't read the fulltexts yet, just looks interesting. I also have some reading to do where the superoxide ends up, I have read a few abstracts that make it sound like there are differences.

Anonymous said...

I got all motivated and copied/pasted the whole proton series into a word doc today - plus a few notable comments. Came to around 40 pages in 11 font Arial.

That's about as far as my motivation stretched today, what with life and kids getting in the way.

Bought Nick Lane's PSS book too. S'pose I should read that first?

As you were.

Purposelessness said...

So I have just reread the adipocyte TFAM knockout paper, and I noticed that there are earlier studies too. For example, knocking out TFAM in muscle tissue has similar effects:

Makro said...

I guess the proper term is "whoopsie"?

Galina L. said...

At the moment the Mediterranean Diet which is "Naturally high in monounsaturated and polyunsaturated fats, particularly as a replacement for saturated fats " is the most politically correct one.

Puddleg said...

So I presume that succinate isn't going into Complex II directly to be digested in some quantum fashion, but is instead feeding it via the TCA cycle.
In which case it ought to be be via TCA-generated FADH2, which is the proximal fate of succinate in the TCA cycle.
If so this supports my concept that the inputs to the TCA cycle exert strong control over the outputs.
"Cycle" might even be a misnomer in some cases; it sometimes looks to me like segments are performing independently, as if it's not always a question of acetyl-CoA playing ring-a-ring-a-rosy ad infinitum.
And then my mind boggles and I need to lie down in a darkened room.

Peter said...


Yes, succinate never appears alone except in isolated mitochondrial experiments. The TCA usually provides 3 NADHs at the same time of course... Pushing electrons up the complex I FeS chain seems much better at generating superoxide if there is plenty of NADH/NAD+ at the top end...

Purposelessness, yes on several fronts. Try Pubmeding "pseudohypoxia" too. A bit contentious but...

Chip Spittter, there are aspects of interest in all three of Nick Lane's books but a huge amount of his current thinking is on his website, you get there by googling Nick Lane. Lots of stuff. Some classic quotes I'd love to get to one day. He's not too impressed with Rimonabant as might relate to glaucoma, nice.


Anonymous said...

Thanks for the heads up re Lane. I've visited his website but obviously didn't look around properly.

Peter said...

George, forgot (mea culpa!), of course there is valine and the proprionate from end beta oxidation of odd chain fatty acids, which do feed straight in to complex II and bypass all of that NADH generation between acetyl CoA and succinate...


Peter said...


LMAO on that one. Forensic statistics to unearth the buried crimes of cardiologists-past.


karl said...

@ Marco
Interesting - that is an observational paper ( I like real experiments ) - but the results are not unexpected to me.

How many people will die due to Argo-business based medical advise?

Makro said...

@Karl "Interesting - that is an observational paper ( I like real experiments ) - but the results are not unexpected to me. "

It´s actually an RCT.

Here is the design description of the study:

"a single blinded, parallel group, randomized controlled trial conducted in 1966-73; and an updated meta-analysis including these previously missing data."

Full text:

Luckily, the re-meta-analysis only gets p=0.06 for demonstrating harm, so keep chugging down that seed oil - It´s heart healthy(TM)!

Makro said...

The paper is so full of hilarity that... well, let´s just get some factoids out there:

- The number of trials on the topic is small (3 pure N6 trials).

- Out of the three trials, two got desk-drawered for more than a decade. (SDHS for about 40 years and MN Coronary for just 16 years)

- Note that mixed N3/N6 interventions seem to help you. Pure N6 kills you dead.

- Be sure to read table 5 carefully. "Mortality outcomes according to longitudinal changes in dietary fatty acid intake".

Those saturated artery-clogging fats sure are some nasty stuff!

Makro said...

Oh, but I should add that the study was successful in one regard: They lowered cholesterol by 13%!


So I guess in one sense the trial was a "success".

Like I said, hilarious stuff.

Anonymous said...

Some control participants began substituting polyunsaturated margarine for butter—leading to substantial, but comparatively modest, dietary changes in the same direction as the intervention group. These changes may have attenuated the observed differences in mortality between the groups, leading to an underestimation of the adverse effects of the intervention.

Anonymous said...

My favouite bit of hand-wringing:

Reconciling results from the SDHS with the traditional diet-heart hypothesis
Increasing dietary n-6 LA in place of SFA lowers serum total cholesterol, primarily by reducing low density lipoprotein with little or no effect on high density lipoprotein.61 The traditional diet-heart hypothesis predicts that these favorable, diet induced changes in blood lipids will diminish deposition of cholesterol in the arterial wall, slow progression of atherosclerosis, reduce clinical cardiovascular risk, and eventually improve survival.18 65 As expected, increasing n-6 LA from safflower oil in the SDHS significantly reduced total cholesterol; however, these reductions were not associated with mortality outcomes (results not shown). Moreover, the increased risk of death in the intervention group presented fairly rapidly and persisted throughout the trial. These observations, combined with recent progress in the field of fatty acid metabolism, point to a mechanism of cardiovascular disease pathogenesis independent of our traditional understanding of cholesterol lowering.

Good to know however that these troublesome findings in no way change public health policy. AHA put out a press release telling us to keep chugging corn oil and limit artery-clogging saturated fat:

Puddleg said...

The other thing you get from the reactions proximal to succinyl-CoA entry is GTP, at least in hepatic mitochondria, and GTP can either be used to produce ATP or be used as is for gluconeogenesis and protein synthesis.

"It also has the role of a source of energy or an activator of substrates in metabolic reactions, like that of ATP, but more specific. It is used as a source of energy for protein synthesis and gluconeogenesis."

Another example of inputs controlling outputs?

A cell that could use segments of the TCA independently at times for specific biosynthetic purposes would have a real advantage over cells committed to a strictly balanced cycling.

Sour fruit is full of TCA cycle intermediates in different ratios. Citric acid, malic acid, and no doubt others. I wonder how these come to be there. Do fruit cells have leaky TCAs with cataplerosis in all directions, drowning in substrates?

Makro said...

Sidereal: Sadly, I don´t have the energy to exactly translate the delicious cardiologist hand-wringing that Swedish trade rag Dagens Medicin ran.

Still, his rationalizations for not having to pay any heed were (i kid thee not):

- There were many smokers in the study (IT WAS CONDUCTED 40 YEARS AGO). Side note: I checked. Smoking was slightly more prevalent in the control group...

- 40 years ago, there was a much lower prevalence of diabetics among heart patients. (GEE I WONDER WHY!)

- Today, we have GLORIOUS STATINS!

- The control group ate fewer calories (I checked, non-significant difference).

At least he didn´t claim that they needed to drop cholesterol even more than 13%! Always something.

karl said...

@ Makro

First - it is a meta-study - junk science flag one. ( in real science, each study has to stand on it's own - combining other studies leads to cherry picking and worse )

They can call it a RCT, but using diet surveys has been discredited - then going back and asking them if they followed the instructions makes it an observational study again anyway.

This is a meta-study of junk science ( yet I would tend to agree with the conclusion - it is extremely important to base what we know on real science.

Compare with something like this:

karl said...

A couple of interesting papers:

Thyroid related:

Journal of Clinical Investigation

A CAD treatment based on modulating inflammation :

Puddleg said...

A meta-analysis needn't be junk science at all. In this case, it's a way of saying "here's even more evidence that extra omega 6 PUFA is bad, are you convinced yet?"
It's actually a unique paper that combines the revisited RCT, the meta-analysis, and a review of the mechanisms. It was partly funded by an Alcoholism research body, and as far as Alcoholism is concerned linoleic acid stood convicted a long time ago.
Whatever it is, it is about a bunch of stuff that happened to some Australians, a lot of whom seem to have died prematurely as a result.
I predict that the right journalist can make a best-selling book by talking to the people involved who are still alive and reading the further records that are out there.
Looking up the references I see a few articles published at the time, and there must have been newspaper stories.
It was probably covered by Reader's Digest at the time, and the bestseller that will hopefully be written about it now will make a popular Reader's Digest Condensed Book.
The paper moots omega 6 restriction as the more likely strategy to prevent heart disease.

Puddleg said...

@ Carl, colchicine has similar anti-inflammatory effects to niacinamide, which is less toxic and elevates HDL.

This is the only (maybe) relevant paper I could find in a short search.

Nicotinamide is a potent phosphodiesterase inhibitor and suppresses neutrophil chemotaxis and mast cell histamine release.

Makro said...


I mostly agree with George's reply, but here are my points on the topic:

- It´s not primarily a meta study, but a discrete study with an added revised meta study as a bonus.

- I agree that meta studies are always "suspect", due to both the potential for selective inclusion and the mixing of different trials with different methods and populations, but they have some use for getting an overview of the different trials conducted in a field.

Some study design comments:

(a) - I wouldn´t call a regular rolling food log given to trial participants a "diet survey".

(b) - The participants were actually provided with the N-6 rich margarine and oils to be used in the trial.

(c) - Going by blood tests the N6 group successfully dropped their blood cholesterol relative to the control group during the trial from 281.3 to 243.9 (between-group p<0.001)

So, unless the food logs were faked, the provided oils and margarines thrown away, and large drop in blood cholesterol faked, I´d say compliance was pretty good in this trial.

- The "ideal" trial design that you link to (I.e. closed metabolic ward, etc.) also illustrates the practical limits of such a design (n=22, t=22 weeks).

Before we can increase funding for nutritional research by a couple of orders of magnitude, that´s not something that you´ll be able to use in practice to get at any long-term effects.

Makro said...

Addendum: Also, good luck getting experiments on prisoners, patients of mental institutions etc. past an IRB these days...

Stipetic said...

The Sydney Diet Heart Study was definitely a RCT. No doubt about that. Ramsden's forensic work to address heart risk does not negate this. No controversy there. Marko makes a great point(s) about the oil (and margarine) being provided to the intervention group. And, as pointed out by Sidereal, in the discussion Ramsden goes out of his way to present the omega-6s in the best possible light possible. The actual harm is likely much worse than presented.

Ramden's re-evaluation of his previous meta-analysis based on these new findings should not be seen as anything controversial. It is still a meta-analysis, but he's had the foresight of trying to separate the effects of omega-3s from the omega-6s (which is why he went back to re-evaluate the SDHS in the first place), unlike the jokers at Harvard--William Willet anyone?--who lump all polyunsaturates into one big slimy gelatinous mess and tell you to slurp down your corn oil based on the positive results. Retards.

As far as meta-analysis go (and I despise them), Ramden's is damn good and well justified.

Anonymous said...

I don't own a dog so this may not be something at all new - but an advert on TV the other day made me laugh and feel quite depressed at the same time.

20% less fat! And quite unpalatable by the looks of it.

Meanwhile, across the ditch, it seems subsisting on coke reduces your life expectancy quite a bit.

karl said...

I guess I've taken some flack for being wary of a paper that's conclusion I think is correct... perhaps it is a better paper than I thought.

Interesting bit about mouse papers:

Here is the paper:

From the paper, it looks like mice are particularly bad models for human inflammation. They really need to use some other animal for CAD research.

Puddleg said...

Karl's perfect study would be valuable and might generate new ideas
BUT skeptics would say it was just one time, one place, one kind of people, what about the other side of the world?
Whereas meta-abalysis for all its flaws will EVENTUALLY show overall trends. No one MA is perfect, but keep up-dating them, and we have that stumbling towards knowledge that seems to be progress in this field.
Ancel Keys never did a meta-analysis so far as I know. But the thing is to keep doing them as results keep coming in, reinterpreting as insights into mechanisms like omega-3/6 arise, etc.
Anthony Colpo has a good rant up crowing about this study. As well he might.

Puddleg said...

Karl, why not use pigs?
Surely the closest thing, and we can eat them when we're done.

Mouse has different factors driving Treg-Th17 differentiation from human.
That will influence inflammatory response to diet.

Unknown said...

Out of all the proton series post this one is the closest to what I am working on. The physics of transition metals is where your switch reside Peter. Keep it up.