Oxygen Reduction and Energy Conservation by Membrane Integrated Terminal Oxidases
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Michel, Hartmut
Molecular oxygen appeared in the atmosphere about three billion years ago, due to the oxygenic photosynthesis invented by the ancestors of the cyanobacteria. Oxygen forms “reactive oxygen species”, which are highly toxic. To deal with the threat nature developed two membrane integrated enzymatic systems which reduce oxygen to water and, at the same time, use the enormous energy of this reaction to produce biologically important energy carriers. These enzymes are the proton pumping haem-copper terminal oxidases, e.g. cytochrome c oxidase, and the bd oxidases. The atomic structures of representative members of both enzyme families will be presented, and their mechanisms of action will be discussed. These evolutionary unrelated enzymes apparently use the same mechanisms to conserve energy and to prevent the formation of toxic reactive oxygen species, although the bd oxidases do not pump protons across membranes.
 So, thank you very much for the introduction and it's of course always a pleasure for me to be here and to talk to you about my research and some general aspects. I do work at the Max Langley Institute of Biophysics in Frankfurt and there are about 200 people and we focus on membrane proteins. So there must be something particular with membrane proteins and we consider them to be the most interesting important proteins on earth. So to give you some idea, because membranes are barriers, you have to transport substances across membranes and as already outlined in the previous talks, they are involved in biologetics. They perform the biological electron transfer in photosynthesis as well as in cellular respiration. Cells also have to communicate, so have many signal receptors which are located in the membrane and they transduce the signal across the membrane and amplify the signal. And some of the membrane proteins are enzymes involved in the biosynthesis of hydrophobic vitamins and they use hydrophobic substrates. And what makes membrane proteins interesting for the application is that most of the marketed drugs which are used when you are ill or sick act on membrane proteins. We got the Nobel Prize as mentioned by Astrid 1988 for the determination of the three-dimensional structure of a photosynthetic reaction center and there were four points which I wanted to line out here. It laid the foundation to understand the first steps in photosynthesis and as already mentioned in the previous talk, it was considered to be the most important chemical reaction on Earth. It also was the first atomic structure of a membrane protein and it was determined by X-ray crystallography and this work was not considered to be possible because there was the misbelief that it is impossible to crystallize membrane proteins. And it, I could show that this actualism is belief was able to get the first well-refracting crystals. I left to work on photosynthesis rather soon after that work and turned towards cellular respiration was already mentioned in the talk given by John Walker and the oxygen which you have in the atmosphere is the product of photosynthesis. And this leads to some problems on Earth. It caused the oxygen catastrophe or the oxygen crisis and this was probably the most significant extinction event in Earth's history. And this was caused by the invention of the oxygenic photosynthesis by the ancestors of the present-day cyanobacteria. So the evolution of Earth's atmospheric oxygen concentration through time is shown here. So Earth's life probably became into existence about 4 billion, close to 4 billion years ago and then after photosynthesis, anaerobic photosynthesis was started pretty soon after life was created and oxygenic photosynthesis occurred about 3 billion years ago. But the released oxygen didn't live long because there was lots of hydrogen sulfide and other reducing agents iron-2 on Earth so it didn't give rise to an oxygen concentration in the atmosphere. This changed about 2.5 billion years ago and suddenly we had the first increase in the oxygen concentration to a rather low level and at this rather low level it stayed for another close to 2 billion years and only about 600 million years ago where the second rise in oxygen concentration to the present-day level. Why is that a catastrophe? It was mainly because oxygen forms reactive oxygen species and it was already outlined in Thomas Lindel's talk that this is dangerous and I can show you here that the reactive oxygen species are dangerous. Here they have the oxygen which is present in the triplet state. It's pretty harmless but upon energy transfer from excited days it dies. It can form singulate oxygen which is oxidizing very dangerous. If you put one electron onto the oxygen you get superoxide radical. Superoxide radical likes oxidized close to everything. Second electron and two protons you get hydrogen peroxide. Hydrogen peroxide itself is not so dangerous but it becomes very dangerous if you have iron-2 present because then you put another electron onto the peroxide and you get the hydroxyl radical. The hydroxyl radical is the most damaging oxygen species which we know and this after the fourth step of electrons you have to form water. So there was a real threat when you had oxygen appeared and the living organisms had to cope with the threat and some of these organisms could survive in oxygen-free niches which still existed and you could try to remove dangerous intermediates e.g. by developing enzymes like superoxide, disneytases or by developing catalases. And another solution was to remove the molecular oxygen via reduction to water by the oxygen reductases. There are a number of oxygen reductases known in biology. There are flavine containing oxidases which occur in oxygen-tolerant methanogenic archaeas or some archaea producing methane. They are able to survive because they can reduce oxygen to water by using that enzyme. Many plants and many microorganisms have cyanide insensitive alternative oxidase which is attached to the membrane and used for that purpose. But the most prominent enzymes are the hemecopa containing terminal oxidases and also the cytochrome bD oxidases oxidizing keynotes. I will focus on these two membrane-integrated enzymes. So it's cytochrome bD oxidizing keynotes and the members of the hemecopa containing terminal oxidases. And this enzyme used the enormous energy inherent in the oxygen reduction reaction for the generation of biological energy via building up electric voltages also called membrane potential and pH gradients across membranes. This is the mitochondrial respiratory chain. I'm due to the presentation by Jules Borgher. I don't have to spend much time on it. So we have here something like fixed hydrogen NADH and then electrons are transferred along these blue lines onto cytochrome oxidase, the hemecopa oxidase, cytochrome c-oxidase. And three of these complexes actually translocate pump protons across the membrane. And so I focus on these enzymes here. And of course, John has beautifully outlined the role, the mechanism of the ATP synthase. The hemecopa terminal oxidases perform the four electron reduction. And this actually implies a separation of charges because the electrons are delivered from genomes or cytochromes from the outer surface of the mitochondrion membrane, whereas the protons which are consumed for water formation come from the inner surface of the membrane. And this process gives, of course, separation of four elementary charges. And in addition, the hemecopa terminal oxidase developed a mechanism to pump four addition protons across the membrane. And I have to say, we don't really understand this process. I will not discuss it further during my talk. These enzymes are rather old. And one wonders actually why they are so old and where they are. Probably before the existence of molecular oxygen in the atmosphere. Probably they performed some other reaction and then they were somehow modified in order to be able to reduce oxygen. They form three families. One is called the A family. The mitochondrial enzymes are all here and also many bacterial enzymes. And we work on an enzyme from a bacterium called panococcus denitrificans, which is closely related to the boron enzyme, as you see here, which is a little bit surprising. And we also work on the C family enzyme, which are the outliers in the tree, but have some fundamental differences, their fundamental differences between the A family enzymes and the C family enzymes. So this is a short sketch, how they look like. We have the A type, AGPases. They contain two copper atoms here and they contain two hems here. And near the second heme, we have a copper. It's called copper B and actually here is the binding site for the oxygen, where you consume also protons in water formation. And this occurs in the central catalytic subunits and this is conserved, the central subunit between all three families. But A type family here has a copper binding protein, which receives electrons from cytochrome C and electrons are moved towards this heme and the copper here. And the B type family are pretty similar, whereas in the C type family, we have a completely different subunit too. So this subunit is replaced by cytochrome subunit containing heme C and there are some other differences. One not well understood difference is that the A type, AGPases have two pathways for proton transfer across the membrane, one straight one directly to the active site and another one, a second one. Actually it turns out that this second pathway is used for the transport of six protons and this only for two protons. And we don't understand really the sequence of these events. The other two enzyme families here have just one, the short pathway towards the center, nevertheless they are able to pump protons. Also this process is not understood. Because we do crystallography and our major challenge, as I already said, is getting crystals. We succeeded here with the help of antibody fragments and could determine the structure. You see here the central subunit here in yellow. Then we have here the subunit two with the copper atoms getting electrons from cytochrome C up here and here we have another subunit and the four subunits whose function is not also not precisely known. If we go into the atomic details of the structure up here, we have the two copper atoms and the copper atoms, the electron is transferred towards the first team, the heme A and then what the electron goes on to heme A3 and here's the copper B where the oxygen is bound. If you look for the electron density, one surprising event was that actually near the active site, the binucleoside where you have the copper B and where you have here the heme A3, there is a histin ligand and the histin ligand contains a tyrosine covenantly linked and actually this was completely unexpected but this provides a hint that there is a radical reaction involved in that during the turnover of the enzyme. The complete simplified picture of subunit looks like this. Here we have the cytochrome C bound, electron transfer here towards the heme A3, uptake of protons, two protons, two via the k-pathway and pumping of the protons here, out here and also some transport from protons here towards the active site to be involved in information of water. The proton transfer is supposed to occur according to the Grotus mechanism, so we have here a chain of hydrogen bonds. What you then do is you shift the protons and you change the hydrogen bonding pattern and then you can at the end you can here release a proton and then you have to release a proton and then you have to rotate back via these bonds and then you are back at the original state but you have transferred a proton. We find such chains actually in the enzyme, this in this D-part which is used for pumping. You see here a nice chain of water molecules appear to a glutamate residue where we have transfer of protons for pumped up here and towards the active site down here. But actually there are blocks in this proton transfer pathway and this block says to have to be relieved during turnover. Another dispute was for quite some time what is in between the heme A3 iron and the Kappa B and the original idea was that there was a water or a hydroxide iron but actually it turns out that the peroxy bridge fits the electron density much better but this actually was not expected. This is a whole truth for this enzyme and also for other enzymes but now the existence of the peroxy bridge in the isolated enzyme is now firmly established using a free electron laser in Japan and our Japanese colleagues could really show that such a peroxy bridge is not caused by a radiation damage which was argued before that experiments. Just to show you here catalytic cycle of cytochrome C oxidase. We have the oxidized form as isolated enzyme. You put in the first electron and the first electron in a proton then you get the one electron reduced form then you get the two electron reduced form. And this binds oxygen, this two electron reduced form is supposed to bind oxygen and the oxygen actually immediately then is split here and originally there was the belief that there is a peroxy form but that's not the case. You already get the split oxygen and you get an oxoferyl form which was completely unexpected. Then you get a third electron here and a proton. You get a so called oxoferyl form. Then you got a fourth electron and a proton and you get to come back to the oxidized form. This is what we have some problems with that catalytic cycle because as I said we have already a peroxide, dienion, bridging the H3, iron and copper B. I already mentioned that the P so called P state actually is already an oxoferyl iron that is due to Raman spectroscopy and it's also obvious that three of the electrons which you need for the splitting of the dioxygen bond can be provided by the H3 iron and the copper B and the missing fourth electron is believed to originate from the crossing tyrosine procedure which I outlined. And so we have a simultaneous transfer of four electrons and this prevents the formation of the partially reduced oxo-scent species. Now I turn towards the C-type family and actually they have been discovered by geneticists because deletion mutants were inactive in nitrogen fixation in rhizobia and they were termed for that species fix n, fix o, fix p before it was discovered that they are actually cytochrome C oxidases. And their primary role appears to be to remove the oxygen in order to maintain anaerobic conditions which is required for the nitrogen fixation. The, from applied aspects the CBB3 oxidases are essential enzymes for the patogenicity of many bacteria like helicobacter and campylobacter and they are the only oxidases there. So if you block them, you have a, if you are able to block them, you can cure stomach ulcers and also gut diseases. And as I already, the only coming sub-unit here is the central sub-unit here. And we work with CBB3 cytochrome C oxidase for bacteria called pseudomonas stutzerei. So that is how the structure look like. Central sub-unit looks pretty similar. Then we have here a first cytochrome C sub-unit bound to it. And there's a second cytochrome C sub-unit containing two hemcrophs up here. And if we compare these sub-unit once, we have some differences mainly up here but not in the central part. We have smaller loops connecting the transmembrane helices and sometimes separate different conformations. The electron transfer here occurs from a cytochrome C to a first hemcorp, goes on to the next hemcorp, two hemcorp here, then to the hemB, then to the hemB3 with a nearby carbobie. The distances here are shorter than in the A and B type oxidases. And this may lead to a faster electron transfer and a better oxygen trapping leading to a lower Km value for oxygen. There are some differences in the details here. So this looks pretty similar. But we have here a glutamate residue unexpectedly which is bound here to a histin ligand to this Ccmb. And there are more tryptophan residues up here. Also from a perspective, there's calcium ions bound towards the negatively charged side chains of the hemcorp involved in the charge neutralization of the negative charges here on the propionate side chains of the hem. Was another unexpected feature. And but also, but this was already known from mass spectroscopic experiments is that this cross-linked tyrosine in the A family enzymes, in the A3 oxidase and in the C family enzymes originate from different transmembrane helices. In the A3 oxidases, it comes from here. This is the tyrosine, cross-linked to the histidine here. Whereas in the C family oxidase, it comes from transmembrane helix number seven stretches over here. And I think that this cross-link was a later invention in evolution from an original function which we don't know, which allowed them to reduce oxygen. If we change this tyrosine or try to exchange here the position of the tyrosines, we get an inactive enzyme. So conclusions on that is that the Cb3 oxidases possess a completely different electron supply systems. Distances are shorter. They possess only one proton transfer pathway. I didn't show you this in detail, but we also know that they pump protons and this is difficult to understand. And it's also quite clear that the nitric oxide reductases are actually very similar to the C family enzymes, although these enzymes do not pump protons and take protons from the outer phase of the membrane. Now I turn towards the BD oxidases. They are widespread among brokaryotes and they are also essential for the pathogenicity of many home human pedigins. And John Walker has already outlined that micro bacteria are a big threat and actually BD oxidases are the only term of oxidases of micro bacteria. So if we block the BD oxidase, we might be able to cure tuberculosis. These BD oxidases do not pump protons. They generate electric voltages and pH gradients by releasing protons from the green wall to one side of the membrane and take up the protons from the water formation from the opposite side of the membrane. That was a original collaboration with Professor Junji Sakamoto from Japan for more than 10 years. And at the end it was one graded student, Shara Safarian, who was able to determine, to improve the crystals and to determine the structure. And the knowledge about BD oxidases is before we got the crystal structure summarized here, so we have here a green hole binding site and the green hole is oxidized, it releases the protons to the periplasm. The electron was supposed to be transported to a first team, B558 to a B595 and then to the HMD and their oxygen is bound and the protons are accessed here, accessed here from the side of the plasm and you get water formation. Everything was supposed to be concentrated here and this B558, B595D was the expected order. The structure brought up many surprises. First we discovered there's another protein subunit which wasn't known. We realized that the two major protein subunits actually share the same protein fold, which reminds me of the photosynthetic reaction centers where we had discovered the same feature unexpectedly. But the hems are only bound to one subunit and the green hole binding site is here in this quite irregular loop. And the order of the hems, as you see it's triangular, it's not a linear arrangement and the order is B558, HMD and B595. So just the position of these two hems is exchanged from the results which were provided by the spectroscopists. And also we found here quite interesting the triptophanosidium between this HMB558 and the HMD. We have an access channel for oxygen towards the oxygen binding site and we also find two potential proton transfer pathways unexpectedly and one has to find out now the role of the two proton transfer pathways and for the moment this is still speculative and there is still a lot to do. If we now compare the center post-added groups we come up with some similarities. The BDOxidases as I said we have here the HMA and then we have here the HMA3 and the COB and here is the oxygen binding site and here we have a triptophanosidium and once the oxygen is bound and you have the reduced, you have these two metals are reduced, you transfer four electrons simultaneously onto the bound oxygen, one from COB, two from the HMA3 ion, the missing one is supposed to come from this tyrosine residue. If we now come to the cytochrome B oxidases we have here the equivalent of this HMA is the HMB558 here and then surprisingly we have a triptophanosidium in a rather similar position. We have here the oxygen binding HMD and here we have the HMB559 and it looks like that the HMB595 plays a similar role as the COBB in the HMCOB oxidases. So and you easily can transfer four electrons onto the bound oxygen here from the iron and also one is supposed to come from the macrocyle and you have a fast transfer of electrons then from the accompanying heme via the triptophanosidium and it looks like that via this mechanism the formation of the reactive oxygen species can be prevented and the same mechanism was thus invented twice by mother nature. Present situation of protein crystallography. I have to say we experienced in the last two or three years a dramatic increase in the methods involved in determining protein structure. But X-ray crystallography in most cases probably became obsolete because of the resolution, revolution and single particle electron cryo microscopy which allows to determine atomic structure and this revolution is actually caused by the development of novel detectors for the diffracted electrons and in order to get atomic structures you don't have to crystallize your proteins and you need much less material. And with electron microscopy you can be pretty sure that you will get a result as protein crystallization is still a gamble. You have heard about John Walker's attempts to crystallize the entire ATP synthase for how was it for close to 30 years and we tried to crystallize our proteins for 10 years and when you are able to crystallize to use cryo electron microscopy to get your structures you just isolate your enzyme go to the electron microscope and try to determine structure. And so what I think the crystallization of proteins, particularly of membrane proteins which is the most challenging object can go to the histories of Tanghib. Finally I want to see how to get a Nobel Prize. And this is a question which we were some time which you are quite often asked and you have to do something which is considered to be highly important but thought to be impossible or very difficult. And in particular there are many important problems in biomedicine and John Walker has already outlined the neurological diseases Alzheimer's is a big problem and the hypothesis that it's the amyloid accumulation that causes Alzheimer's didn't find support and many companies invested billions, already close to billions of dollars in order to cure Alzheimer's why it is very didn't work out. So we have to work Alzheimer's. We want to know why do we sleep and what happens during sleep and also aging is a thing which we have seriously considered. What is the cause of aging and how can we stop it? Actually this was the cause why I started biochemistry in order to find it out but I gave it up. The next way to get a Nobel Prize is to make a serendipitous discovery and primarily you have to think very carefully on unexpected results not forget them to think about start thinking then. And the most direct way to get a Nobel Prize is to develop a novel method to solve an important problem for the development of all new major methods. Nobel prizes have been awarded. I just want to remind you of X-ray crystallography, nuclear magnetic resonance, electron microscopy, DNA and protein sequencing, mass spectroscopy, super resolution microscopy. And most importantly you have to be at the right place at the right time and to rely on your own personal strength. I don't want to finish without providing the acknowledgments because you need people to do the work. This is the team which work mainly on the IEA3 oxidase. It's how on the CBP3 oxidases you can get the X-ray crystallographer and actually Zabid Bushman spent 10 years of her life trying to get the crystals or the well-effected crystals of that enzyme. The people involved in the BD oxidase is mainly Shara Safarian at the end with the help of Julia Breu and our mass spectroscopist Julian Langer. And of course many opportunities to do exciting work. So thank you very much for your attention.
 So, thank you very much for the introduction and it's of course always a pleasure for me  to be here and to talk to you about my research and some general aspects.  I do work at the Max Langley Institute of Biophysics in Frankfurt and there are about  200 people and we focus on membrane proteins.  So there must be something particular with membrane proteins and we consider them to  be the most interesting important proteins on earth.  So to give you some idea, because membranes are barriers, you have to transport substances  across membranes and as already outlined in the previous talks, they are involved in  biologetics.  They perform the biological electron transfer in photosynthesis as well as in cellular  respiration.  Cells also have to communicate, so have many signal receptors which are located in the  membrane and they transduce the signal across the membrane and amplify the signal.  And some of the membrane proteins are enzymes involved in the biosynthesis of hydrophobic  vitamins and they use hydrophobic substrates.  And what makes membrane proteins interesting for the application is that most of the marketed  drugs which are used when you are ill or sick act on membrane proteins.  We got the Nobel Prize as mentioned by Astrid 1988 for the determination of the three-dimensional  structure of a photosynthetic reaction center and there were four points which I wanted  to line out here.  It laid the foundation to understand the first steps in photosynthesis and as already mentioned  in the previous talk, it was considered to be the most important chemical reaction on  Earth.  It also was the first atomic structure of a membrane protein and it was determined by  X-ray crystallography and this work was not considered to be possible because there was  the misbelief that it is impossible to crystallize membrane proteins.  And it, I could show that this actualism is belief was able to get the first well-refracting  crystals.  I left to work on photosynthesis rather soon after that work and turned towards cellular  respiration was already mentioned in the talk given by John Walker and the oxygen which  you have in the atmosphere is the product of photosynthesis.  And this leads to some problems on Earth.  It caused the oxygen catastrophe or the oxygen crisis and this was probably the most significant  extinction event in Earth's history.  And this was caused by the invention of the oxygenic photosynthesis by the ancestors of  the present-day cyanobacteria.  So the evolution of Earth's atmospheric oxygen concentration through time is shown here.  So Earth's life probably became into existence about 4 billion, close to 4 billion years  ago and then after photosynthesis, anaerobic photosynthesis was started pretty soon after  life was created and oxygenic photosynthesis occurred about 3 billion years ago.  But the released oxygen didn't live long because there was lots of hydrogen sulfide and other  reducing agents iron-2 on Earth so it didn't give rise to an oxygen concentration in the  atmosphere.  This changed about 2.5 billion years ago and suddenly we had the first increase in the  oxygen concentration to a rather low level and at this rather low level it stayed for  another close to 2 billion years and only about 600 million years ago where the second  rise in oxygen concentration to the present-day level.  Why is that a catastrophe?  It was mainly because oxygen forms reactive oxygen species and it was already outlined  in Thomas Lindel's talk that this is dangerous and I can show you here that the reactive  oxygen species are dangerous.  Here they have the oxygen which is present in the triplet state.  It's pretty harmless but upon energy transfer from excited days it dies.  It can form singulate oxygen which is oxidizing very dangerous.  If you put one electron onto the oxygen you get superoxide radical.  Superoxide radical likes oxidized close to everything.  Second electron and two protons you get hydrogen peroxide.  Hydrogen peroxide itself is not so dangerous but it becomes very dangerous if you have  iron-2 present because then you put another electron onto the peroxide and you get the  hydroxyl radical.  The hydroxyl radical is the most damaging oxygen species which we know and this after  the fourth step of electrons you have to form water.  So there was a real threat when you had oxygen appeared and the living organisms had to cope  with the threat and some of these organisms could survive in oxygen-free niches which  still existed and you could try to remove dangerous intermediates e.g. by developing  enzymes like superoxide, disneytases or by developing catalases.  And another solution was to remove the molecular oxygen via reduction to water by the oxygen  reductases.  There are a number of oxygen reductases known in biology.  There are flavine containing oxidases which occur in oxygen-tolerant methanogenic archaeas  or some archaea producing methane.  They are able to survive because they can reduce oxygen to water by using that enzyme.  Many plants and many microorganisms have cyanide insensitive alternative oxidase which  is attached to the membrane and used for that purpose.  But the most prominent enzymes are the hemecopa containing terminal oxidases and also the  cytochrome bD oxidases oxidizing keynotes.  I will focus on these two membrane-integrated enzymes.  So it's cytochrome bD oxidizing keynotes and the members of the hemecopa containing terminal  oxidases.  And this enzyme used the enormous energy inherent in the oxygen reduction reaction for the generation  of biological energy via building up electric voltages also called membrane potential and  pH gradients across membranes.  This is the mitochondrial respiratory chain.  I'm due to the presentation by Jules Borgher.  I don't have to spend much time on it.  So we have here something like fixed hydrogen NADH and then electrons are transferred along  these blue lines onto cytochrome oxidase, the hemecopa oxidase, cytochrome c-oxidase.  And three of these complexes actually translocate pump protons across the membrane.  And so I focus on these enzymes here.  And of course, John has beautifully outlined the role, the mechanism of the ATP synthase.  The hemecopa terminal oxidases perform the four electron reduction.  And this actually implies a separation of charges because the electrons are delivered  from genomes or cytochromes from the outer surface of the mitochondrion membrane, whereas  the protons which are consumed for water formation come from the inner surface of the membrane.  And this process gives, of course, separation of four elementary charges.  And in addition, the hemecopa terminal oxidase developed a mechanism to pump four addition  protons across the membrane.  And I have to say, we don't really understand this process.  I will not discuss it further during my talk.  These enzymes are rather old.  And one wonders actually why they are so old and where they are.  Probably before the existence of molecular oxygen in the atmosphere.  Probably they performed some other reaction and then they were somehow modified in order  to be able to reduce oxygen.  They form three families.  One is called the A family.  The mitochondrial enzymes are all here and also many bacterial enzymes.  And we work on an enzyme from a bacterium called panococcus denitrificans, which is  closely related to the boron enzyme, as you see here, which is a little bit surprising.  And we also work on the C family enzyme, which are the outliers in the tree, but have some  fundamental differences, their fundamental differences between the A family enzymes and  the C family enzymes.  So this is a short sketch, how they look like.  We have the A type, AGPases.  They contain two copper atoms here and they contain two hems here.  And near the second heme, we have a copper.  It's called copper B and actually here is the binding site for the oxygen, where you  consume also protons in water formation.  And this occurs in the central catalytic subunits and this is conserved, the central subunit  between all three families.  But A type family here has a copper binding protein, which receives electrons from cytochrome  C and electrons are moved towards this heme and the copper here.  And the B type family are pretty similar, whereas in the C type family, we have a completely  different subunit too.  So this subunit is replaced by cytochrome subunit containing heme C and there are some other  differences.  One not well understood difference is that the A type, AGPases have two pathways for  proton transfer across the membrane, one straight one directly to the active site and another  one, a second one.  Actually it turns out that this second pathway is used for the transport of six protons and  this only for two protons.  And we don't understand really the sequence of these events.  The other two enzyme families here have just one, the short pathway towards the center,  nevertheless they are able to pump protons.  Also this process is not understood.  Because we do crystallography and our major challenge, as I already said, is getting crystals.  We succeeded here with the help of antibody fragments and could determine the structure.  You see here the central subunit here in yellow.  Then we have here the subunit two with the copper atoms getting electrons from cytochrome  C up here and here we have another subunit and the four subunits whose function is not  also not precisely known.  If we go into the atomic details of the structure up here, we have the two copper atoms and  the copper atoms, the electron is transferred towards the first team, the heme A and then  what the electron goes on to heme A3 and here's the copper B where the oxygen is bound.  If you look for the electron density, one surprising event was that actually near the  active site, the binucleoside where you have the copper B and where you have here the heme  A3, there is a histin ligand and the histin ligand contains a tyrosine covenantly linked  and actually this was completely unexpected but this provides a hint that there is a radical  reaction involved in that during the turnover of the enzyme.  The complete simplified picture of subunit looks like this.  Here we have the cytochrome C bound, electron transfer here towards the heme A3, uptake  of protons, two protons, two via the k-pathway and pumping of the protons here, out here  and also some transport from protons here towards the active site to be involved in  information of water.  The proton transfer is supposed to occur according to the Grotus mechanism, so we have here a  chain of hydrogen bonds.  What you then do is you shift the protons and you change the hydrogen bonding pattern  and then you can at the end you can here release a proton and then you have to release a proton  and then you have to rotate back via these bonds and then you are back at the original  state but you have transferred a proton.  We find such chains actually in the enzyme, this in this D-part which is used for pumping.  You see here a nice chain of water molecules appear to a glutamate residue where we have  transfer of protons for pumped up here and towards the active site down here.  But actually there are blocks in this proton transfer pathway and this block says to have  to be relieved during turnover.  Another dispute was for quite some time what is in between the heme A3 iron and the Kappa  B and the original idea was that there was a water or a hydroxide iron but actually it  turns out that the peroxy bridge fits the electron density much better but this actually  was not expected.  This is a whole truth for this enzyme and also for other enzymes but now the existence  of the peroxy bridge in the isolated enzyme is now firmly established using a free electron  laser in Japan and our Japanese colleagues could really show that such a peroxy bridge  is not caused by a radiation damage which was argued before that experiments.  Just to show you here catalytic cycle of cytochrome C oxidase.  We have the oxidized form as isolated enzyme.  You put in the first electron and the first electron in a proton then you get the one  electron reduced form then you get the two electron reduced form.  And this binds oxygen, this two electron reduced form is supposed to bind oxygen and the oxygen  actually immediately then is split here and originally there was the belief that there  is a peroxy form but that's not the case.  You already get the split oxygen and you get an oxoferyl form which was completely unexpected.  Then you get a third electron here and a proton.  You get a so called oxoferyl form.  Then you got a fourth electron and a proton and you get to come back to the oxidized form.  This is what we have some problems with that catalytic cycle because as I said we have already  a peroxide, dienion, bridging the H3, iron and copper B.  I already mentioned that the P so called P state actually is already an oxoferyl iron  that is due to Raman spectroscopy and it's also obvious that three of the electrons which  you need for the splitting of the dioxygen bond can be provided by the H3 iron and the  copper B and the missing fourth electron is believed to originate from the crossing tyrosine  procedure which I outlined.  And so we have a simultaneous transfer of four electrons and this prevents the formation  of the partially reduced oxo-scent species.  Now I turn towards the C-type family and actually they have been discovered by geneticists  because deletion mutants were inactive in nitrogen fixation in rhizobia and they were  termed for that species fix n, fix o, fix p before it was discovered that they are actually  cytochrome C oxidases.  And their primary role appears to be to remove the oxygen in order to maintain anaerobic  conditions which is required for the nitrogen fixation.  The, from applied aspects the CBB3 oxidases are essential enzymes for the patogenicity  of many bacteria like helicobacter and campylobacter and they are the only oxidases there.  So if you block them, you have a, if you are able to block them, you can cure stomach ulcers  and also gut diseases.  And as I already, the only coming sub-unit here is the central sub-unit here.  And we work with CBB3 cytochrome C oxidase for bacteria called pseudomonas stutzerei.  So that is how the structure look like.  Central sub-unit looks pretty similar.  Then we have here a first cytochrome C sub-unit bound to it.  And there's a second cytochrome C sub-unit containing two hemcrophs up here.  And if we compare these sub-unit once, we have some differences mainly up here but not  in the central part.  We have smaller loops connecting the transmembrane helices and sometimes separate different conformations.  The electron transfer here occurs from a cytochrome C to a first hemcorp, goes on to  the next hemcorp, two hemcorp here, then to the hemB, then to the hemB3 with a nearby  carbobie.  The distances here are shorter than in the A and B type oxidases.  And this may lead to a faster electron transfer and a better oxygen trapping leading to a  lower Km value for oxygen.  There are some differences in the details here.  So this looks pretty similar.  But we have here a glutamate residue unexpectedly which is bound here to a histin ligand to  this Ccmb.  And there are more tryptophan residues up here.  Also from a perspective, there's calcium ions bound towards the negatively charged  side chains of the hemcorp involved in the charge neutralization of the negative charges  here on the propionate side chains of the hem.  Was another unexpected feature.  And but also, but this was already known from mass spectroscopic experiments is that this  cross-linked tyrosine in the A family enzymes, in the A3 oxidase and in the C family enzymes  originate from different transmembrane helices.  In the A3 oxidases, it comes from here.  This is the tyrosine, cross-linked to the histidine here.  Whereas in the C family oxidase, it comes from transmembrane helix number seven stretches  over here.  And I think that this cross-link was a later invention in evolution from an original function  which we don't know, which allowed them to reduce oxygen.  If we change this tyrosine or try to exchange here the position of the tyrosines, we get  an inactive enzyme.  So conclusions on that is that the Cb3 oxidases possess a completely different electron supply  systems.  Distances are shorter.  They possess only one proton transfer pathway.  I didn't show you this in detail, but we also know that they pump protons and this is difficult  to understand.  And it's also quite clear that the nitric oxide reductases are actually very similar  to the C family enzymes, although these enzymes do not pump protons and take protons from  the outer phase of the membrane.  Now I turn towards the BD oxidases.  They are widespread among brokaryotes and they are also essential for the pathogenicity  of many home human pedigins.  And John Walker has already outlined that micro bacteria are a big threat and actually  BD oxidases are the only term of oxidases of micro bacteria.  So if we block the BD oxidase, we might be able to cure tuberculosis.  These BD oxidases do not pump protons.  They generate electric voltages and pH gradients by releasing protons from the green wall to  one side of the membrane and take up the protons from the water formation from the opposite  side of the membrane.  That was a original collaboration with Professor Junji Sakamoto from Japan for more than 10  years.  And at the end it was one graded student, Shara Safarian, who was able to determine, to improve  the crystals and to determine the structure.  And the knowledge about BD oxidases is before we got the crystal structure summarized here,  so we have here a green hole binding site and the green hole is oxidized, it releases  the protons to the periplasm.  The electron was supposed to be transported to a first team, B558 to a B595 and then to  the HMD and their oxygen is bound and the protons are accessed here, accessed here from  the side of the plasm and you get water formation.  Everything was supposed to be concentrated here and this B558, B595D was the expected  order.  The structure brought up many surprises.  First we discovered there's another protein subunit which wasn't known.  We realized that the two major protein subunits actually share the same protein fold, which  reminds me of the photosynthetic reaction centers where we had discovered the same feature  unexpectedly.  But the hems are only bound to one subunit and the green hole binding site is here in  this quite irregular loop.  And the order of the hems, as you see it's triangular, it's not a linear arrangement  and the order is B558, HMD and B595.  So just the position of these two hems is exchanged from the results which were provided  by the spectroscopists.  And also we found here quite interesting the triptophanosidium between this HMB558 and  the HMD.  We have an access channel for oxygen towards the oxygen binding site and we also find two  potential proton transfer pathways unexpectedly and one has to find out now the role of the  two proton transfer pathways and for the moment this is still speculative and there is still  a lot to do.  If we now compare the center post-added groups we come up with some similarities.  The BDOxidases as I said we have here the HMA and then we have here the HMA3 and the  COB and here is the oxygen binding site and here we have a triptophanosidium and once  the oxygen is bound and you have the reduced, you have these two metals are reduced, you  transfer four electrons simultaneously onto the bound oxygen, one from COB, two from the  HMA3 ion, the missing one is supposed to come from this tyrosine residue.  If we now come to the cytochrome B oxidases we have here the equivalent of this HMA is  the HMB558 here and then surprisingly we have a triptophanosidium in a rather similar position.  We have here the oxygen binding HMD and here we have the HMB559 and it looks like that  the HMB595 plays a similar role as the COBB in the HMCOB oxidases.  So and you easily can transfer four electrons onto the bound oxygen here from the iron and  also one is supposed to come from the macrocyle and you have a fast transfer of electrons then  from the accompanying heme via the triptophanosidium and it looks like that via this mechanism  the formation of the reactive oxygen species can be prevented and the same mechanism was  thus invented twice by mother nature.  Present situation of protein crystallography.  I have to say we experienced in the last two or three years a dramatic increase in the  methods involved in determining protein structure.  But X-ray crystallography in most cases probably became obsolete because of the resolution,  revolution and single particle electron cryo microscopy which allows to determine atomic  structure and this revolution is actually caused by the development of novel detectors  for the diffracted electrons and in order to get atomic structures you don't have to  crystallize your proteins and you need much less material.  And with electron microscopy you can be pretty sure that you will get a result as protein  crystallization is still a gamble.  You have heard about John Walker's attempts to crystallize the entire ATP synthase for  how was it for close to 30 years and we tried to crystallize our proteins for 10 years and  when you are able to crystallize to use cryo electron microscopy to get your structures  you just isolate your enzyme go to the electron microscope and try to determine structure.  And so what I think the crystallization of proteins, particularly of membrane proteins  which is the most challenging object can go to the histories of Tanghib.  Finally I want to see how to get a Nobel Prize.  And this is a question which we were some time which you are quite often asked and you  have to do something which is considered to be highly important but thought to be impossible  or very difficult.  And in particular there are many important problems in biomedicine and John Walker has  already outlined the neurological diseases Alzheimer's is a big problem and the hypothesis  that it's the amyloid accumulation that causes Alzheimer's didn't find support and many companies  invested billions, already close to billions of dollars in order to cure Alzheimer's  why it is very didn't work out.  So we have to work Alzheimer's.  We want to know why do we sleep and what happens during sleep and also aging is a thing which  we have seriously considered.  What is the cause of aging and how can we stop it?  Actually this was the cause why I started biochemistry in order to find it out but I  gave it up.  The next way to get a Nobel Prize is to make a serendipitous discovery and primarily you  have to think very carefully on unexpected results not forget them to think about start  thinking then.  And the most direct way to get a Nobel Prize is to develop a novel method to solve an important  problem for the development of all new major methods.  Nobel prizes have been awarded.  I just want to remind you of X-ray crystallography, nuclear magnetic resonance, electron microscopy,  DNA and protein sequencing, mass spectroscopy, super resolution microscopy.  And most importantly you have to be at the right place at the right time and to rely on  your own personal strength.  I don't want to finish without providing the acknowledgments because you need people to  do the work.  This is the team which work mainly on the IEA3 oxidase.  It's how on the CBP3 oxidases you can get the X-ray crystallographer and actually Zabid  Bushman spent 10 years of her life trying to get the crystals or the well-effected crystals  of that enzyme.  The people involved in the BD oxidase is mainly Shara Safarian at the end with the help of  Julia Breu and our mass spectroscopist Julian Langer.  And of course many opportunities to do exciting work.  So thank you very much for your attention.
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