Life (39) NuoH

This paper is almost completely dedicated to the function of NuoH and the CoQ binding pocket, AKA the Doohickey.

Redox-induced activation of the proton pump in the respiratory complex I

Here is Figure 1, it's the inset we're interested in:

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.

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:

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:

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)

taking an area of negative charge with it:

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:

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:

which repositions the polar amino acids like this:

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:

The final step involves these amino acids, mostly glutamates with an aspartate D72 at the end of the chain:

The conformation change in the protein structure moves these amino acids towards the source of protons 

and puts them in to a microenvironment which makes them highly avid to gain a proton, which they do:

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:

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.

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:

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.

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!).

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.

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.

Peter

Life (38) Water Wires

These people have ideas about the water channels for the fourth proton in modern complex I.

Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I

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:

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:

We can summarise this as a proton from bulk solvent:

"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:

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):

There is a convenient lysine to accept it. Tucked in behind the lysine is another glutamate which can next be protonated:

to open the water channel to the periplasm

and complete the transfer to the 4th proton:

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.

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.

Unlike NuoH. That's the next post. It's totally different.

Peter

Life (37) Just a gif of complex I

If we take the complex I from the doohickey paper

and also the mirror image of complex I from the water channels paper

and highlight the important bits, we get this

which can be rearranged to give this

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:

and then we can add in the orange-crossmarked pumped protons like this:

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.

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.

Peter

Life (36) Complex I. Protons are large

Now it's time to look at this paper:

Symmetry-related proton transfer pathways in respiratory complex I

We can start with this image of the membrane part of complex I from Thermus thermophilus 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:

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, H₃O⁺, 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.

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.

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:

I'll briefly go in to detail of the section marked within the red oval to look at the key mechanism:

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.

While Nqo8's amino acid is protonated it attracts the -ve charged glutamic acid and repels the +ve charged lysine.

When Nqo8 is de-protonated the lysine and glutamate return to their original electrostatic association. In detail it looks like this:

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.

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.

I've corrected these "blue +" symbols to open circles to make sense of it and have highlighted the repeats with ovals circles again:

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:

Nqo8's amino acid then protonates

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

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

This movement also opens the periplasmic water channels

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

As the protons exit, the associated conformational changes open the cytosolic water channels and the cycle is ready to repeat

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

"Despite a significant N-side entry channel, we observe no clear exit pathways to the P side of the membrane within Nqo8."

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.

Which gives us insightful constraints on where the system might have come from.

Peter

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