Monday, June 08, 2015

ATP Synthase

It's a very interesting time for origin of life speculation. You can look around at the work coming from Lane's group as well as the excellent work by Koonin's group and consider what fits together logically from the various scenarios on offer. None are completely satisfying alone but a mixture of the ideas gives a very pleasant brew. Let's give Koonin some time now:

How do you build an ATP synthase complex? On the surface it looks to be utterly incomprehensible that a lipid embedded, ion gradient driven rotor should turn a stalk to a set of molecular forceps each of which forces a phosphate on to an ADP to give ATP as the rotor turns. It's particularly striking that Nick Lane considers this to be one of the core molecular machines present in LUCA before the separation in to the populations which gave rise to the archaeal and bacterial lineages. Running on a proton gradient. It would need to have been a very early development.

If we buy in to the alkaline hydrothermal vent scenario (Koonin doesn't) for the origin of life we have microscopic "pockets" of metabolism in iron (+/- nickel) sulphide walled chambers at the boundary of alkaline vent fluid with acidic ocean fluid. In the progress towards something akin to life there has to be the development of both protein and RNA. In a vent system, where conditions at the microscopic scale vary from time to time, the ability for a given replicator (RNA based) to spread itself over the maximum number of protocells would provide a survival benefit. The development of any sort of cell membrane is clearly a hindrance to this process, potentially terminal. The formation of simple pores of protein derivation are not inconceivable and would allow the continued spread of successful RNA to a maximum number of protocells. Pores do not have to be complex. RNA spread can be concentration based.

RNA will form double helices. Not with the solidity of those derived from DNA, but double helices never the less. There is a large family of proteins which unwind helices to allow replication, be that of DNA or RNA. Many of them use ATP to facilitate the process. They are very basic machines by evolutionary standards and there is no reason why they were not as available early in evolution as simple membrane pores.

Bear in mind that one of the core products of protometabolism is an acetyl thioester capable, energetically speaking, of producing ATP (was that as long ago as February?).

If we have these basics in mind we are in a position to go to this rather nice speculative opinion piece by Koonin's group. Here is figure 2:

Across the top we have the possibility of an ATP driven RNA helicase sticking itself to a membrane pore and using ATP to power RNA export through a proto membrane bound protocell. The helicase, if it sticks itself to the membrane pore, will extrude an RNA strand out of the protocell. As any helicase passes down an RNA strand, it rotates. If the helicase is fixed to the pore, the RNA will rotate as it passes through the pore. The right hand diagram shows the next development with an RNA strand replaced by a protein strand, not an impossible transformation and still potentially of benefit to a proto replicator.

Working along the middle row from right to left we have a protein translocator using ATP to export a protein using the same rotary machinery inherited from the RNA helicase/translocase. The + and - within the circles represent electrostatic charges which normally hold the pore and helicase stationary relative to each other. The middle diagram has a red protein which is physically stuck in the pore. The rotary component, powered by ATP, pushes/twists against the membrane bound section while the electrostatic charges resist rotation. Something has to give and in the process an ion or more is squeezed outwards as the pore derived section moves against the stator section.

The machine extrudes ions by consuming ATP. Ion gradients have many uses to protocells so the development may well have been advantageous at the time. The machine also works perfectly well in reverse so, given an appropriate ion gradient, it will generate ATP as ions enter the protocell. The ion gradient itself is a whole different ball game. Just for now recall that in those archaea and bacteria which lack cytochromes, the gradient is of Na+ ions, not H+ ions. Another post there.

This is a plausible derivation for the bacterial/mitochondrial F type ATP synthase.

The archaeal V type ATP synthase has very similar ion pump and ATP synthase sections to the F type, but the central stalk is quite different. In fact it doesn't look much like a stalk at all, see the bottom left diagram in the above figure 2. It looks more like a protein used to stabilise the pore and helicase sections against each other. It does the same job as the central stalk of the F type synthase but its origins are clearly different.

The ATP synthase complex was developed twice.

It looks very much as if the pore/helicase combination was ubiquitous in LUCA but the system of locking them together is different in the archaeal vs bacterial lineages. That seems very profound to me when thinking about LUCA.

The scenario does not have the ATP synthase as primordial. The system evolved as an ATP consuming machine, not an ATP generating machine. It has no hallmarks of the core power producing system in LUCA. Where did the ATP come from to power the engine which was eventually to become the dynamo?

I commented in my post on the reduction of CO2 to CO that the acetyl thioester produced was quite capable of substrate level phosphorylation. It is able to produce molecules in the energetic range of ATP and undoubtedly did so, because we are here today we are and running on ATP.

ATP is undoubtedly what worked best for the RNA helicase as it appears to have carried this preference forwards to the protein translocase and hence the ion pump which reversed role to become a dynamo. If the helicase had been powered by acetyl phosphate rather than ATP I can see no reason why metabolism might not be based around such a molecule. We use ATP because that's what worked best in the motor which, once it reversed to become a dynamo, unleashed a ubiquitous supply of energetic substrate based on an ion gradient.

Just to recapitulate: An proton gradient is essential to power protometabolism because without it you cannot reduce CO2 to CO to develop acetyl thioester and its assorted phosphorylated derivatives. This substrate level phosphorylation is utterly proton gradient driven but does not require proton translocation per se, although proton translocation may have been used to produce a localised environment which reduced ferredoixin. A source of direct ATP synthesis was required to power an immediately useful translocase which allows development of an ion pumping motor which could subsequently be reversed to provide an ATP generating dynamo as the membrane energetics changed.

The change in membrane energetics was probably related to a biologically generated Na+ gradient in addition to the geochemical H+ gradient. If the sodium ion gradient was supported by anti porting on a geothermal H+ gradient, it was both free and nearly unlimited in availability. So we have to think about antiporters.



Jack Kruse said...

Peter you really need to read Pollack latest book and his research. Lane needs to as well. Here is what the alkaline vents give us.........An exclusion zone in water. Why? Alkaline pH's build the exclusion zone in water. When water is charge separated it becomes a battery capable of doing work without ANY CELL. Water's key to the formation of life: All three kingdoms of life make energy via chemiosmosis.........what does it require? A membrane impermeable to protons. What does an EZ exclude.......? Protons. This is Pollack 101. You only need the smallest hydrophilic protein to gain this don't a cell contrary to what evolutionary biologists think right now today. How do you get a small protein at a vent? Thermophoresis. Nick got that correct. Once you link Pollack, then read the other UK scientists, Jim Al Khalili's latest book. It extends Pollack and Lane and really shows you what Lane is missing. Nick is brilliant but his tunnel vision is killing his ideas.

Jack Kruse said...

Protein turnover is a synonym for elevated ubiquitin marking. Recall, from Lane's mouth himself, that eukaryotes spend 80% of their total energy budget on protein synthesis. High ubiquitin rates, lower the redox potential by lowering the net negative charge in a cell faster than any other process in biology. Which brings us directly to the eukaryotic cell membrane. What separated it from prokaryotes? The presence of DHA. For 600 million years it has not been replaced one time while everything else in eukaryotes has changed dramatically as Lane as amply shown us. Consider the mitochondrial consequences of these action now. Free radical leak is designed to work with DHA in eukaryotic cell membranes to optimize respiration while simultaneously eliminating dysfunctional mitochondria. KEY POINT: The mitochondria that leak the MOST free radicals will leak the most light because of a lack of DHA in the surrounding membranes. The more a mitochondria leaks, the more copies it is capable of making. This is why I made the prediction in Ubiquitination 6 that those who chronically use ketosis without DHA would likely have no superoxide pulses from their mitochondria. This would give you senescent mitochondria with no hope of recycling them leading to organ failure or oncogenesis. As a result, people would have to live with their altered stretched out respiratory proteins. This causes them to live the back half of their life with defective mitochondria. Defective mitochondria on a long term basis, regardless of dietary choices, lead to oncogenesis.

When mitochondria leak MORE free radicals the signal is to CORRECT the respiratory protein deficit by increasing the capacity. Capacity means more new mitochondria are made. Recall that mitochondria are clones made from your maternal line. Mitochondrial DNA codes only for the proteins on respiratory changes. This DNA copies itself way faster than nuclear DNA by design. When respiratory proteins increase their atomic size they lose the ability to tunnel electrons and protons.

When the respiratory proteins increase the distance between the 45 subunits that make up cytochrome 1, there is a 10 fold loss in quantum tunneling of electrons. That causes massive losses in energy and really lowers ATP production. When ATP drops proteins cannot fold or unfold properly to bind water to make an EZ. This is where Ling and Pollack brilliance decouple from Mitchell's flub. ATP is important not to drive biochemical or enzymatic flux. Ling showed that ATP could not possibly provide the only battery for life because The lower the EZ the lower the net negative charge is in a cell. Capacity can also relate to the amount of respiratory proteins capable of being made from our 13 mitochondrial genes. The more mitochondria a tissue has the better it tends to function bio-energenically. The more mitochondria a cell has, generally the CLOSER mitochondria come to approaching the nucleus distance wise to nucleus to coadapted better. This affects the "sea of water" in your cells around your proteins. In the mitochondria a loss of water around it allows the inner mitochondrial membrane to become more leaky to protons. Why? A loss of the EZ causes proton permeability which destroys chemiosmosis and all tunneling. This has massive down stream effects on epigenetic expression.

Tan Yew Wei said...
This comment has been removed by the author.
Yew-wei Tan said...

To echo Jack's point a little, I do think that Ling's paper on the Nanoproplasm could shed some light (literally) on the ATP Synthase issue --


In which Ling mentions a bunch of studies showing ATP formation without an ionic gradient, following up with:

> The emphasis is shifted from ionic or "osmotic" gradients, central to the membrane-pump theory, to ion adsorption on proteins, central to the Al hypothesis"


> How does the mitochondrion, without the aid of a biochemist, carry out ,two-step manipulations equivalent to laboratory procedures?
> The first step involves exposing the ATPase to Mg2+ (and sometimes K+ ) and Pi, thus producing the "phosphoenzyme."
> The second step involves removing the Mg2 + or K+ already present and replacing it with a different cation, either Ca2+ or Na+ as the case may be.
> Additionally, today we must ask how adsorption of Ca2+ or Na+ creates the phosphate transfer from the ATPase to ADP to synthesize ATP?

The Nanoprotoplasm paper also brings up some interesting points, which I summarised here -- , but can be boiled down to the fact that while ATP is the best Electron Withdrawing Cardinal Adsorbent, other ions could also take it's place.

This could have been the case with earlier versions of the the ATPase, which would "bootstrap" the system with the energy needed to evolve more complex mechanisms of making ATP.

Finally, red light LLLT has been shown to increase ATP synthesis -- . The most plausible mechanism for this is tied to generation of EZ water at sites of ATP production, and it is the charge gradients generated by said EZ water that power the ATPase.

NY said...

Two quotes from a religious scripture:

1. "How dare you deny me. In the beginning heavens and earths were one mass and we tore them apart."

2. "We have created life out of water."

August said...

We need testable hypotheses. Especially to go all in on the alt-theories.

Peter said...


"We discuss the construction and testing of the reactor, describing the precipitation of thin-walled, inorganic structures containing nickel-doped mackinawite, a catalytic Fe(Ni)S mineral, under prebiotic ocean conditions. These simulated vent structures appear to generate low yields of simple organics. Synthetic microporous matrices can concentrate organics by thermophoresis over several orders of magnitude under continuous open-flow vent conditions"

I need to see light shone on water reducing CO2 to formate using H2 as the electron source to take Pollack seriously. It should be easy.


Jack Kruse said...

Peter you dont need a cell to generate a thin membrane to exclude protons......all you need is water and a hydrophilic protein. A sea vent gives that and Pollack has already proven that with experiments. Nick believes one needs a cell for dont. From their autocatalysis takes over ala Stewart Kaufman. As Yew said above, Ling's idea's about the aTPase begin to make some sense. Ruminate on this.

Yew-wei Tan said...

I don't have the full text for 'Photochemical reduction of carbon dioxide to formate mediated by ruthenium bipyridine complexes as homogeneous catalysts' --!divAbstract

But the title is pretty self-explanatory.

Here's another indirectly related paper 'Photochemical generation of carbon monoxide and hydrogen by reduction of carbon dioxide and water under visible light irradiation' --


> Visible light irradiation of solutions of Ru(2,2′-bipyridine)32+, cobalt(II) chloride, and carbon dioxide in acetonitrile/water/triethylamine generates simultaneously carbon monoxide and hydrogen.
> The reaction involves photoinduced reduction of CO2 and H2O, triethylamine serving as electron donor in the Ru(2,2′-bipyridine)32+/Co2+ system.

Ru(2,2′-bipyridine)32+ looks like this -- , which of course is very similar looking to the Reaction centres of Chlorophyll (which uses Mg2+ in the middle of 4 Nitrogens)

triethylamine and acetonitrile are simple Carbon and Nitrogen compounds

The key factor in this case is likely the light absorption spectrum of Nitrogen containing rings (either 5- or 6-membered). The Metals serve to "coordinate" multiple of such Nitrogen rings, which increases the chances of light-mediated electron transfer to CO2.

Adding more free compounds seems to lose the coordination effect. Quote:

> Addition of free bipyridine strongly decreases CO generation but increases H2 production

This suggests that the free bipyridine absorbs incoming light, but doesn't "channel this energy to CO2", and reduces efficiency of CO2 reduction.


Of course looking at the reactiosn of Formate Dehydrogenase, we again see the interplay between the Nitrogen-containing rings of NAD and/or the "coordinated' 4 Nitrogens bordering the central Iron ions in the ferricytochrome

NAD-dependent reaction
Formate + NAD+ ↔ CO2 + NADH + H+

Cytochrome-dependent reaction
Formate + 2 ferricytochrome b1 ↔ CO2 + 2 ferrocytochrome b1 + 2 H+

Very similar sort of chemical reactions when compared to the Ru(2,2′-bipyridine)32+, cobalt(II) chloride system, and I wouldn't be surprised if the presence of Iron and Nitrogen rings alone in the IR light (and possibly green light) of the vents would have allowed all these compounds to exist at various states of along the continuum of equilibrium.

Peter said...

Photochemical generation of carbon monoxide and hydrogen by reduction of carbon dioxide and water under visible light irradiation.

This is a lovely paper by two very bright people who have gone on to major careers in evolutionary thinking and photochemistry. Fascinating to read what they are researching today, as well as the original paper.

So we have a system where an organic chromophore captures a photon of visible light and transfers the energy to a Rb Co redox couple which allows the generation of the redox potential to reduce CO2 to CO using H2. Or water to H2 in the absence of CO2. This is a fascinating, fully man made redox system. Abiotic excepting the human brains which thought it up.

Great stuff.

Now, the question was, how does light acting on water allow H2 to reduce CO2 to CO. The context was structured water and the development of a charge separation across the border of an exclusion zone for H+.How does this proton gradient drive such a reaction? This, unless I am very mistaken, is where structured water exclusion zone is relevant to the origin of life.

The paper, undoubtedly, does not illuminate an answer to the problem. Citing it does not help support your contention that light acting on water allows the reduction of CO2 to CO in the presence of H2.

Perhaps this is not your contention? It looks as if it is when viewed from the outside.


Peter said...

Actually, the H2 was fully being generated, not being used as a reductant. My mistake. We have a redox couple which can split water. Back to exclusion zones....


Jack Kruse said...

Peter, EZ's are markedly effected by mechanical sources of energy. There are huge mechanical disturbances in the sea and pressure alone dramatically increases the EZ in water by itself. This is why life began there and this predates all autocatalysis in my opinion. The ATPase manifests because of the massive charges that charge separated water can create.
We use this to our benefit in surgery in the CNS too, in humans. FYI.

Yew-wei Tan said...

Yeah, you get both CO and H2 generated from CO2 just using some metals and light.

Another related paper 'Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane' --

This was specifically using a liposome with:

(1) an F-type ATPase embedded in the membrane
(2) presence of a carotene-porphyrin-naphthoquinone (C-P-Q) triad
(3) presence of lipophilic Quinones (Qs, which was fairly simple -- 3 benzenes, with 2 Oxygens to the doubly bonded to the central benzene at position 1 and 4, and the other 2 benzenes bound to the central benzene via single bonds at positions 3 and 5)

Then the following cascade happens:

(a) 633nm light caused excitation of the C-P-Q to give [C+]-P-[Q-]
(b) a Qs gains an electron from the [Q-] in the [C+]-P-[Q-] triad (now we have [C+]-P-Q)
(c) Qs- gets protonated by aqueous H+ (or H3O+) to form semiquinone (HQs)
(d) HQs delivers proton and electron to [C+]-P-Q , forming C-P-Q again (and ready for another photoexcitation)
(e) because the Carotene end is aligned toward the inside of the liposome, the proton carried by HQs is ejected inside the liposome. This generates a concentration of protons inside the liposome

Some commentary:

- We see nitrogen-containing rings coming up again as a key in converting photons to electrons. Porphyrin is basically an arrangement of 4 nitrogen rings. This arrangement is highly conserved in photosynthetic cores (around Mg2+), as well as in hemoglobin (around Fe2+), so I assume it does its job well.

- We need a lipophilic Quinone to work as an electron shuttle in a lipid membrane. This is a fairly simple molecule

- Orientation of the C-P-Q is key. Carotene must be pointing inwards towards the center of the liposome.

How likely is this set of conditions? I dunno, but it's within the realm of possibility.

If it were possible, then we have a light-mediated way of generating the needed osmotic gradients for ATP synthesis. if an ATPase of any form finds itself in such a membrane arrangement, it may actually have the right environment to kickstart ATP synthesis.

Then if you believe Ling's work, some of that ATP will find itself on other proteins as an Electron Withdrawing Cardinal Adsorbent, and allow for potentially more osmotic gradients to form (by generating EZ water near the unfolded protein surface).

What these gradients then go on to do is a separate matter of course, and I'm still thinking about that ....


- Sidenote: Proton-Coupled Electron Tunnelling (PCET) --

I made some speculative thoughts on the Kruse forum --

This is again another example of quantum mechanic tunnelling of both protons and electrons after light excitation. Again you'll see the same porphyrin + metal arrangement turn up.

What is surprising about those setups is that with both collinear and orthogonal PCET, you still get long range quantum tunnelling (up to 35 Angstroms, which is far higher than the 14 Angstroms normally thought to be an upper limit).

I have no conclusions yet from this, except that these factors must be considered in the discussion of ATP formation, because they drastically lower the energetic barriers needed to for ATP-generating reactions to occur.

I'm basically saying that there are "magic geometric arrangements" of molecules that suddenly allow for lower energies than conventionally expected to occur, and that these arrangements are likely to have happened in the many many years before the emdosymbiosis event.


Peter said...

My interest is in the origins of metabolism. CO2 + H2 giving CO which will combine in a test tube with CH3~HS to give acetate It's the generation of CO which is difficult under abiotic conditions. After that we are talking evolution. I'm interested in where it started. I don't see exclusion zones generating CO from H2. How important they might become is another question but nothing is convincing me that they are capable of reducing CO2 using H2. This is the sticking point.


Jack Kruse said...

Peter then you need to read Kaufman. He is the king abiotic chemistry and autocatalysis.

Peter said...

Jack, no, I don’t need to read Kaufman. I want to know how you put an electron on to an FeS moiety at a redox potential which will convert CO2 to CO in the presence of H2. Self organisation of compounds derived from this generation of activated acetate reacting with nitrogen compounds is undoubtedly fascinating but does not get the process started. Had you suggested extra terriestrial H-CN, hydrated to foramide, as a side step to this conundrum or Zn based photosynthesis as a route in, OK. But Kaufman will not put an electron on to FeS to reduce CO2 to CO.
Formamide and the origin of life.
Open questions on the origin of life at anoxic geothermal fields. of first cells at terrestrial, anoxic geothermal fields.

On the origin of life in the zinc world: 1. Photosynthesizing, porous edifices built of hydrothermally precipitated zinc sulfide as cradles of life on Earth.

Jack Kruse said...

I'll find his work on this and post it for you.....its not on my laptop and I think its in a file or my work computer.