Thank you very much for the introduction and I also would like to say that it’s always a great pleasure to be here.
We now change gears and we go more into chemistry and biology and primarily structural and mechanistic chemistry.
I start off by showing you the place where I work.
That’s the Max Planck Institute of Biophysics in Frankfurt, and we focus on membrane proteins,
membrane-bound proteins, which have a number of many different functions.
Today I will focus, however, on the enzymes which reduce oxygen in your body,
or in the body of bacteria or other microorganisms, and generate energy.
Let me start by asking you what was the biggest catastrophe ever on Earth.
Actually, it was the so-called Great Oxygenation Event, also called the oxygen catastrophe or oxygen crisis.
And it was the most significant extinction event in Earth’s history.
It was caused by the living organisms, primarily by the ancestors of the present-day cyanobacteria.
These are little small algae that invented photosynthesis,
the oxygenic photosynthesis, which causes the release of oxygen.
This was the big catastrophe.
You see here that the original atmosphere on the Earth was reducing.
Earth was created about 4.5 billion years ago and life probably started to be around 4 billion years ago.
Oxygenic photosynthesis started about 3 billion years ago but the oxygen produced didn’t do much harm
because there was lots reducing substances around.
So there was no free oxygen available.
Only after about 2.5 billion years ago we got a significant increase of the oxygen level in the atmosphere.
And this was the catastrophe because what we have now in the atmosphere is molecular oxygen.
That’s triplet oxygen, that’s what we breathe in and what we use.
But of the other oxygen species you see here 4 are highly damaging.
If you get energy transfer on to the triplet oxygen by some dyes, excited dyes you get singlet oxygen
which oxidises most organic compounds.
If you put one electron onto the dioxygen you get a superoxide radical which is highly oxidising.
The second electron plus 2 protons gives you the hydrogen peroxide which is also very oxidising.
You use it for sterilising.
If you put a third electron and a proton on it, you get 2 hydroxyl radicals.
Hydroxyl radicals are the most damaging oxidising substances around.
And at the end, after the 4th electron and adding a proton, you get 2 water molecules.
These reactive oxygen species caused this death, the killing of most organisms at that time.
And of course the organisms had to cope with the threat.
And of course some of them could try to survive in oxygen-free niches like mines and so on.
There are also ways to remove dangerous intermediates,
for instance, by superoxide dismutases and peroxide catalases.
And the best way, of course, is to remove molecular oxygen via reduction to water by oxygen reductases.
And this lists the known oxygen reductases in biology.
In so-called methanogenic archaea we have di-iron flavoprotein which does this job.
Not much is known about it, I will show you some structural data on it.
Plants and many microorganisms contain a cyanide insensitive alternative oxidase
which is attached to energy-converting membranes but doesn't span it.
And then we have the 2 major classes: the heme-copper containing terminal oxidases
and the cytochrome bd oxidases which oxidise quinols.
I show you here the structure of the di-iron flavoprotein from methanogenic archaea.
You see there a di-iron centre, and you see here 2 flavin molecules
which are used to supply electrons for the reduction of oxygen to water.
We can go back to a crystallography in detail: You see here the 2 iron atoms, you see the ligands.
You see here the bridging carboxylate group into protein
and you have some rough ideas where the oxygen could bind and be reduced.
This has to happen very fast in order to prevent the formation of these reactive oxygen species.
But, as I said, not much is known about that enzyme.
Let’s go to the membrane-integrated terminal oxidases.
This is, as I mentioned already, cytochromes bd.
We have the members of the heme-copper containing terminal oxidases
and what they do is they not only detoxify the oxygen by reducing it.
They use the enormous energy at least with respect to biology and chemistry
which is inherent in the oxygen reduction reaction for the generation of biological energy forms
via building up electric voltages across membranes and building up pH gradients across these membranes.
The prime example is here the mitochondrial respiratory chain - that’s what you have in your body.
And the mitochondrial respiratory chain here starts off with NADH - NADH is, I should say, fixed hydrogen.
If you would have hydrogen available it would diffuse away and would at the end disappear into space.
And we don’t like that. So hydrogen in biology is fixed to coenzyme.
This is called NADH.
And here it actually is oxidised.
The electron is transferred on to quinols.
And then the electron goes further on.
It goes on, on a so-called complex 3, which is this complex.
And in this complex you get proton pumping across the membrane.
And you also have proton pumping across the membrane in complex 1.
Finally here in the respiratory chain you have the complex 4, cytochrome c oxidase which oxidises cytochrome c.
Cytochrome c is reduced by complex 3, and this is the terminal oxidase here.
So these complexes here work by transferring protons across the membrane.
This is here the membrane which is electrically tied.
So you can build up these electric voltages.
The electric voltage and the pH created drives protons back through the ATP synthase.
This is a very small rotatory engine and this engine creates ATP,
which is the general energy currency of life, from ADP and inorganic phosphate.
This happens continuously in your body.
Your body produces about 70 kilograms of ATP every day.
So the heme-copper terminal oxidases of the superfamily use electrons from cytochrome c,
some from hydroquinols or other from HIPIPs which are iron sulphur proteins.
They all come from the external side of the membrane.
They use protons from the internal membrane side.
They pump additional protons across the membrane in order to increase the yield of energy.
The heme-copper terminal oxidases in general have this reaction.
You have, of course, the dioxygen molecule.
You have 4 protons from the inner side of the membrane
and you have 4 cytochrome molecules from the outer side of the membrane.
And you have the oxygen diffusing in the membrane.
Oxygen is enriched in the membrane, diffuses in the membrane. Here we have the active side.
You get the electrons from the outer side and you get the protons from the inner side.
In addition, this enzyme family has developed a mechanism to pump 2 to 4 additional protons across the membrane
to increase the electric voltage – also called membrane potential – and to increase the pH gradient.
We want to understand actually how this machinery works.
I would like to mention here that this enzyme actually is well-known since many, many years.
It was a Scottish spectroscopist who discovered it, originally in 1887.
But we still do not know how it works.
Otto Warburg in Germany, he took up again this project.
He got the Nobel Prize in Physiology or Medicine in 1931
for his discovery of the nature and mode of action of the respiratory enzyme.
So you could believe that everything is already known
but I have to say we do not know actually how the machinery works.
It is still a challenge and I hope that some of you intelligent students
can contribute to our further understanding of this machinery.
Otto Warburg also, I have to say, did other important things.
He proposed that cancer cells generate energy by glycolysis.
This is only the breakdown fermentation of sugars but not by cellular respiration.
This cellular respiration is by effect of close to 10 more efficient in energy conservation than glycolysis.
And glycolysis also produces protons.
It is now generally accepted that cancer cells actually live primarily by glycolysis and not by cellular respiration.
He presented this hypothesis in detail here in 1966, so 50 years ago.
Tomorrow would be the 50th anniversary of his announcement.
The heme-copper oxidases are quite widespread in all of the living organisms.
We have 3 families within the super family and this is the type-A family.
Here we have the animals, the mammals and close to it we have bacteria which have very closely related enzymes.
Here we have the type-B family and the type-C family.
Actually in the lab we started type-A and type-C families.
The 3 families are diverse with respect to electron donors, subunit compositions and the heme types.
The A-family has here the central subunit which contains the bi-nucleoside
which reduces where the oxygen is bound and reduced.
And it has 2 channels, pathways for the transfer of protons.
One leads directly to the active site – this way, which is here –
and the other one actually leads nearby but also to the active site.
It must somehow divert protons towards the active site and to the outer site for protons to be pumped.
The B-type oxidase have only one of these proton transfer channels, otherwise they are pretty similar.
The C-type oxidases are very different.
They do not contain this yellow subunit,
instead, it’s replaced by some cytochrome molecules which contain heme groups
and not a carbon centre here for the delivery of electrons back to the active site.
I also should mention there is a paradox, because they appear to have been present in the living world
already before the photosynthetic oxygen evolution was invented.
The question then is, what was the original function of these oxidases?
One speculation was that they were derived from nitric oxide reductases.
But you can discard this because the nitric oxide reductases are closely related to the type-C heme-copper oxidases
and probably appeared really late in evolution.
We use X-ray crystallography to try to understand, to get the structure and to get functional hints.
And still the bottleneck is getting crystals.
It has become much easier nowadays.
Actually, when I worked in the photosynthetic reaction centre we needed crystals of at least 0.5 mm in diameter.
Nowadays people do this with 10 micrometre crystals, X-ray crystallography at the synchrotron.
And what’s also quite astonishing, it took me 4 months to collect one data set.
Nowadays it is done in 8 seconds at the synchrotron.
So this tells you really how technology development has speeded up.
With this cytochrome c oxidase we got the crystal to publish the structure, first structure of this type in 1995.
Here you see the central unit in yellow which is the subunit one which contains here the heme groups
and the copper-B which is where the oxygen is bound and reduced.
If you look more into the atomic details then you see here 2 copper-A atoms.
The copper-A atoms receive the electrons from cytochrome c, from here.
And then the electrons hop down onto this heme A that’s known from spectroscopy.
Then the electron is transferred further to the heme A3 and nearby we have the copper-B
where the electronhere can equilibrate between the 2 metals.
And oxygen is bound here when the 2 metals are in their reduced form.
That is how the electron density looks like.
What came as a surprise was that nearby we have a crosslink of a tyrosine, a tyrosine molecule,
covalent cross-linked to a nearby histidine.
The histidine is ligand here of this copper-B molecule
which provides maybe some hint that radicals are involved in the reaction
because radicals, tyrosine radicals, can form such kind of cross links more or less spontaneously.
Functionwise the structure, a different representation we have here,
now showing you the transmembrane, the helixes transmembrane, the strength of the protein.
We have here the active site.
The K-pathway leading straight towards the active site
and here the D-pathway being in a more curved way and which transfer both protons.
On the other side we have cytochrome c binding, cytochrome c provides here the electron for the reaction.
We have 8 protons here involved in the whole business, we have 1 oxygen, we have 4 cytochrome c molecules.
So it’s 13 substrates but we have no idea about the precise order of these 13 substrates
at which they arrive at the bi-nuclear site and how they are used.
Proton transfer is supposed to occur according to the Grotthuss mechanism.
So we have a hydrogen-bonded chain like this.
You can add one proton at one end of the chain and then you shift the proton.
You then get electron release here.
You can then rotate along this bond back and you have release of a proton on that side.
You have to transfer it by this mechanism, the proton from here to there.
This may actually happen in the system because you see here a very nice water chain
with some amino acid residues contributing.
This is the D pathway where a water chain is here, leading up here to glutamate residue.
But actually it’s interrupted here by some asparagine residues, it’s blocked.
Probably this prevents some bad side reaction and we actually have to remove that block for the proton transfer.
Now what about proton pumping?
The most convincing principle so far is the charge compensating principle.
Basically it says that each electron transfer into the hydrophobic part of the enzyme is charge compensated
by the uptake of one proton from the opposite side of the membrane.
So you form some kind of ion pair within the protein which is supposed to be energetically more favourable
than having a single negative charge of the electron in the membrane face.
The protons taken up via the D-pathway in order to compensate the negative charge of the reducing electrons
are those which are being pumped.
Then you get another electron coming in directly to the active site and this electrostatically repels the protons
which have taken up first and which are consumed at the active site.
This is probably the mechanism accepted by most people but there are still some doubts on it.
One question which was quite long under debate was, actually,
what is in the active site between the heme iron and the copper-B?
In 2006 there was another structure published.
It was pretty clear that the peroxide already here fits well into the oxidised form
but it’s not expected in the oxidised enzyme.
We also had analysed our structure more carefully
and we also found that the best fit at our 2.2 Angstrom resolution structure into that bridge is a dioxygen molecule.
So we believe that peroxide-dianion is there but this actually was not accepted by the community.
For me peroxide in the active site is attractive
because it’s the best fit for the electron density in the structure-determined enzyme.
Bridging peroxide dianion would provide the best electrostatic compensation
for positive charges at the iron of the heme a3 and the copper-B .
It could be singly protonated upon the first reduction, and receive a second proton upon the second reduction.
It could leave the binuclear site and make it available for dioxygen binding.
This sequence would explain why a singly reduced active site does not bind dioxygen.
A few words about the catalytic cycle of cytochrome c oxidase.
When you isolate the enzyme normally you have it in the oxidised form.
Then you start to reduce the enzyme, normally by cytochrome c.
You take up a proton at the same time and you get one electron in reduced form.
Then you put in the second electron, a second proton, you get the reduced form.
And if 2 electrons are at the binuclear site you can bind oxygen.
It forms an adduct, similar as myoglobin or haemoglobin does, and then the reaction goes on.
That was the belief that actually this forms a peroxide by a transfer of one proton on to the oxygen.
But actually this was ruled out, and it’s clear that already in this state we have here an oxoferryl state.
We have a split oxygen bound here.
Put in the third electron and you get something called the oxoferryl state, fourth electron and you are back.
The problem with this catalytic cycle is that the purified oxidised form already contains a peroxide dianion
bridging the heme a3 iron and Copper-B.
And this is now known for sure because our Japanese colleagues used the radiation of the free-electron laser in Japan
where you have a 20 femtosecond X-ray pulse.
You hit the crystal position once and you get a diffraction pattern before radiation damage can occur.
They did it by shifting the crystal several thousand times
and got a complete data set and could later identify that there is a peroxide bridge between heme a3 iron and Copper-B.
And this will cause us to rethink the mechanism and role of cytochrome c oxidase.
I want to mention that the so-called P-state contains already an oxoferryl-iron.
The dioxygen bond is split.
This was done by resonance Raman spectroscopy mainly in Japan by Kitagawa
and later by the Babcock group in the United States.
If you think about the splitting of the dioxygen bond then you need 4 electrons for that.
3 electrons can be provided from the heme a3 iron and Copper-B, actually 2 from the heme a3 iron and 1 from Copper-B.
The missing fourth electron is postulated to originate from that cross-link tyrosine residue
which I think is a convincing proposal but it’s clear that there is no peroxide-state during the cycle.
Now I change gears a little bit and tell you about cbb3 cytochrome c oxidases.
The C-family, they have been discovered rather recently, in the 1990s.
They are essential enzymes for nitrogen fixation in Rhizobia.
They remove the oxygen in order to maintain anaerobic conditions which are required for nitrogen fixation.
In the presence of oxygen the nitrogen fixation apparatus is destroyed.
They are also essential for the pathogenicity of some bacteria:
Helicobacter – causing stomach ulcers – and Campylobacter.
They only share the subunit 1 which is called fixN from fixation, nitrogen fixation with the A- and B-type terminal oxidases.
They use the firmly bound cytochrome c subunits for electron supply.
We have determined the structure of the Pseudomonas stutzeri enzyme.
That is now the structure: This is the subunit one.
You have the second subunit, a cytochrome c, a third subunit which contains here 2 heme groups.
When you compare the architecture of the central subunit it’s pretty similar with respect to the transmembrane part.
But it’s dissimilar when you look at the loops on both sides of the membrane.
With respect to electron transfer it’s most likely this heme gets an electron from a soluble cytochrome here.
Electron transfer occurs and then this down here.
Then final step here towards the active site with containing the heme b3 now and Copper.
In general we have shorter distances for the electron transfer than in the A-type oxidases
which means faster electron transfer and better oxygen trapping low Km value for the oxygen binding here.
So more efficient enzymes.
Looking into details you can see here we have the Copper-B, we have the heme a3 iron,
we have also a cross-linked tyrosine here but this is very different.
Then we have here some differences towards other residues.
We have glutamate residue here which was also quite unexpected.
We also have here a calcium ion bound which is ligated here to 3 of the heme propionates.
So it neutralises an arginine.
So we have 3 positive charges, 1 arginine, 2 calcium charge compensating the free negative charges
on the heme propionates here which appears to be important.
Another important difference to the A-family enzymes
is that the cross-linked tyrosine residue originates from a different transmembrane helix.
In the A-type it’s this green one, from this helix here,
and in the type-C family it originates from this one and they crosslink which you see in the same residue here.
So this was of course not expected, but the crosslink is very well established also by mass spectrometry.
A different view of the same you have here of the type-A families.
You have it from this helix, the tyrosine residue in the C-type family, you have the tyrosine residue over from this family.
We have a K-pathway equivalent which allows proton transfer towards an active site.
We also can clearly show that the D-pathway is absent.
We have blocks here.
The D-pathway, hydrophobic a big aromatic residue, tryptothane phenylalanines, tyrosine residue,
which clearly block proton transfer towards this site that we had in the A-family enzyme.
So conclusion here is that the cbb3 oxidases possess a completely different electron system.
The distances are shorter leading to the faster electron transfer.
We have only one proton transfer pathway.
Nevertheless the enzymes pump protons,
and that’s difficult to understand in the framework of that electrostatic repulsion hypothesis.
The 2 heme propionates are not available for proton pump loading because of calcium binding.
And I also say the NO reductases are structurally very similar to the cbb3 oxidases although they do not pump protons
and take the protons from outer face of the membranes.
So the NO reductases use 2 cytochrome c molecules, 2 NO molecules, 2 protons from the outside and 1 oxygen molecule.
They get oxidised cytochrome c, N2O and water by the NO reductases but structure is pretty similar.
Now I turn gears and come to the bd oxidases.
They are widespread among the prokaryotes.
They are essential for the pathogenicity of many human pathogens also.
In particular the mycobacteria which causes tuberculosis need a functional cytochrome bd oxidase.
They do not pump protons.
They generate electric voltages and pH gradients by releasing protons from quinols to one side of the membrane
and take up protons for water formation from the opposite side.
I would like to say bd oxidases differ also from the copper oxidases by their inhibitor sensitivity.
I haven’t heard so far anything about Schrödinger’s cat.
Schrödinger’s cat actually is killed by the cyanide which blocks the A-type cytochrome c oxidase.
But it wouldn’t work if the cat would have a bd oxidase because the bd oxidase is not blocked by cyanide.
And bd oxidase containing organisms probably survives in your gut
because you produce some stinking compound, mercaptoethanol, it’s sulphide, disulphide.
The sulphide in the gut actually blocks the heme-copper oxidase of many bacteria
and the bd oxidase containing organism in your gut have a survival advantage.
Ok, I have to say this was a 10 year venture together with Junshi Sakamoto from Japan.
But we could determine the structure finally and publish it just 2 months ago.
And the proposed model of the cytochrome b oxidase is shown here,
known from spectroscopy with a genesis of indirect experiments.
So you have here the quinol, the quinol here is oxidised.
Electrons are transferred to cytochrome B558, then to B595, and then to heme D
which is called bd oxidase which binds oxygen.
You get water formation.
Protons are taken up from the cytoplasm whereas the protons from the oxidation of the quinol are released here
which again leads to electric voltage across the membrane.
The reality is shown here.
To our big surprise we have the 2 big protein subunits
and the 2 big protein subunits have the identical structure, identical folding.
This reminds me of the photosynthetic reaction centre
where we had at the first membrane protein structure exactly the same finding.
The role of the 2 subunits was changed.
Here we have all the pigments are bound only to one subunit
and the other subunit is there with the identical protein fold but does not contain any pigments.
The binding site for the quinol is located here in this area where we have an extra loop which is not present on the other side.
In addition, we discovered another short protein subunit in the enzyme which was unknown before our work.
Now, the big surprise was that actually hemes hook up pretty well in the membrane, not at the outer surface.
And also the order of electron transfer must be just the opposite as believed.
So most likely the B558 gets first an electron but then it’s pretty clear that the heme D must get the electron.
Then the electron can equilibrate the heme B559.
These 2 are just inverted in the sequence as was expected from spectroscopy.
We also found an oxygen entry site towards the heme D molecule.
We also found proton transfer pathways.
To our surprise there appear to be 2 proton transfer pathways, one in the A-subunit and the other one in the D-subunit.
But, of course, their meaning, their importance-role still has to be worked out.
When I compare now the central prosthetic groups of the bd oxidases and the heme-copper oxidases we find some surprises.
For instance, for comparison we have here the first heme, heme A, and here the heme B558
which gets the electron from the quinol here, from the cytochrome, from the copper-A centre.
Then we have tryptophan residue in here.
We have a similar tryptophan residue in here.
And tryptophan residues actually can help you very much in the electron transfer,
actually, they also can be electron donors.
And then we have here the oxygen binding heme.
We have here the oxygen binding heme in the middle.
And here we have the other electron supplier which is here, copper-B.
And here it’s the heme B5595.
So the oxygen-binding heme posesses in both cases a central location.
This feature allows the rapid electron transfer from both sides on to the central heme irons and the oxygen molecule.
It appears to be that for the breaking of the oxygen bond
4 electrons are transferred on to the oxygen molecule at the same time, simultaneously.
By that you do not get formation of super oxide, peroxide or hydroxyl radicals.
You have no damaging side reaction in this kind of enzymes.
So obviously the same mechanism was invented twice by Mother Nature to prevent the formation of this reactive oxygen species.
With that I am at the end, but I really do recognise there is still much to do.
I will tell you about the people who did most of the work.
That is the team which recently worked on the heme a3 type oxidases.
We got crystallography done by Juergen Koepke, spectroscopy by Iris von der Hocht.
The people doing the cbb3 oxidase are listed here.
Sabine Buschmann actually worked for 10 years trying to get the crystals - after 10 years she succeeded.
Structure was done by Ulrich Ermler. Julian Langer and Hao contributed biochemistry and mass spectrometry results.
And most recent the bd structure it was Schara Safarian, an outstanding student.
As you see he’s a weight lifter, he lifts heavy weights as was the structure of the bd oxidase.
And he got help from Julian Langer and with the mass spectrometry also from Julia Preu
and Hanne Mueller from the biochemical side.
With that I want to end and thank you for your attention.
(Applause)