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.

Peter

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 CoQ^2-. 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 CoQ^2- 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.

Peter

Life (33) Transient hunger

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

pH 6:

CO₂ + 2e- + E(in) -> CO + O^2-

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

The next step is:

CO + O^2- + 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) -> Ferredoxin^2-

If we take out all intermediate steps we can summarise the generation of Fd^2- near the origin of life like this

H₂ + Fd -> Fd^2- + 2H⁺

Fd^2- (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 Fd^2- 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 Fd^2- 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 Fd^2- 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 Fd^2-.

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 Fd^2- 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 Fd^2- 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^2- 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:

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

also figure 6 and the associated legend.

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.

Peter