Hartmut Michel (1998) - From Photosynthesis to Respiration: Structure and Function of Energy Transforming Membrane Protein Complexes

Thank you. As indeed was said I am going to talk about membrane proteins. You will see lots of membrane protein structures but I should make clear from the very beginning that we don’t do membrane protein structures in order to see how the structures look like. We would like to know how these machines work. And for this we have to know what the structure is. And this will then form the basis to understand the mechanism of action of these molecular machines. And I should say that membrane proteins in general perform many important roles. But the major problem is that we cannot study them in great detail because we don’t have much membrane protein, much of the membrane protein available from the amount. And also they are very difficult to handle and we have still a severe lack of knowledge about membrane proteins. Membrane proteins constitute about 40% of all proteins of your body. But we know at present about 20 membrane protein structures from 12 different proteins. And we know about maybe 5,000, 7,000 water soluble proteins. And this tells you where the challenges are. And the challenges in membrane proteins are primarily crystallisation. Crystallography is a method, we have many different methods. And one of the points of my lecture will be that you need many different methods nowadays in order to solve biological problems. And the first slide, I show you the biogenic system from purple bacteria, which is a form of photosynthesis. And we have here in the heart, the blue machine here is a photosynthetic reaction centre and that’s the one we were awarded the Nobel Prize in 1998. And the next, this actually, this machine gets the energy from the light harvesting complexes. You see here this one in pink, which surrounds the whole reaction centre and actually there are more light harvesting complexes which are in the membrane which transfer the energy from a light harvesting complex 2 to light harvesting complex 1 to the reaction centre where we get electron transfer. I will only shortly touch the reaction centre. The role of the reaction centre is to transfer electrons from a primary electron donor here across the membrane towards acceptor molecules which are quinones, here we’ve a QB and we have to transfer a second electron to get a double protonation. We have the hydronium ion diffuses towards, in the membrane towards the cytochrome BC1 complex, gets oxides there and the protons are transported across the membrane in this complex. And the electrons end up in cytochrome C2, diffuse spec, in the periplasmic space of the bacterium and reduce the primary electron donor again via bound heme groups. So we have a cyclic flow of electrons. And the purpose for that cyclic flow of electrons is to pump protons in the cytochrome BC1 complex across the membrane. And we will hear more about the cytochrome BC1 complex in the following talk given by Hans Deisenhofer. And the cytochrome BC1 complex also plays a very important role in respiration and you will see this complex in a similar scheme. The electrochemical proton gradient consisting of an electric field which is the more important component and the proton gradient drives proton spec through the membrane. And it’s now clear from the work of Paul Boyer who presented that part yesterday and John Walker and his colleagues in Cambridge, that you have here this water soluble complex for which they determined the structure. That the gamma subunit here rotates and the backflow of protons here drives the rotation of the gamma subunit. And per one rotation of the gamma subunit you get one ATP formed from ADP and phosphate. ATP is the general currency of life, you could call it the Euro of life. So we have already a unified energy currency in biology, we will have it in Europe, maybe in the world at the end also. Now I start with the light harvesting complex. You see here the structure of a light harvesting complex determined by us recently in Frankfurt. And you see here in green chlorophyll molecules. We have two rows of chlorophyll molecules in overall. There are 8 chlorophylls in this complex, there are 16 of this type up here and they are actually, they are linked, they are bridged by carotenoid molecules. Carotenoids are very important molecules in life. First, they have a photoprotective function. And they prevent the damaging effect of activated oxygen species. And also they quench triplet states of chlorophylls which could generate such excited oxygen molecules. And second, they absorb light and they transfer the energy to the chlorophylls and the chlorophylls then transfer the energy to the next light harvesting complex and then to the reaction centre where some work is done. This here is the same complex viewed from the top on to the membrane. You see here 2 helical membrane proteins. This is the so called alpha subunit, this is the beta subunit. And in between we have in green the chlorophylls, one circle and another chlorophyll we have here. And you see again here the carotenoids with their double role. And this kind of circular arrangement is quite remarkable and maybe nature invented some kind of synchrotron before man did it. So nature maybe is always first. And how the whole structure is arranged in the membrane, photosynthetic membrane is in here. We have here the light harvesting complex 2. That’s the light harvesting complex 1 which surrounds the reaction centre. You see here in yellow the reaction centre. And the energy, only the energy is transferred from here to the next and from here to the primary donor of the reaction centre. You see the respective chlorophylls here vaguely in red and there you get electron transfer. And electron transfer then is chemistry. We start off with first light absorption, energy transfer and then electron transfer which is chemistry. And at the end we generate ATP, the ATP is used to fix carbon dioxide, to synthesise carbohydrates in the dark reaction. And this is the food where we all live from. And of course we eat the food, we degrade food and at the end we have the citric acid cycle, we have glycolysis. And we end up in the splitting of our food stuff into hydrogen, in some biological form of hydrogen which is then converted further. And this is done in the respiratory chain. And the respiratory chain is shown on this slide. You see here the respiratory chain of a bacterium called paracoccus denitrificans, which is quite well studied. It has the advantage that you can use genetic methods in order to study the role of these components. And also it appears to be closely related to the ancestor of your own mitochondria. And everything what I tell you here is also valid for your own mitochondria in your own body. First you generate NADH, in the cyclic acid cycle. And this is the bound form of hydrogen in biology. And what the respiratory chain does, it converts it with oxygen to form water. And it has developed a quite complicated machinery. Not only to prevent that you have a detonating gas reaction to prevent explosions in your body all over and the loss of energy. And also we like to make use of the energy and the principle is the same as in photosynthesis. We have here four complexes, NADH is first oxidised here in this complex called complex 1. And protons are translocated across the membrane in addition to some electrons, to reduction of quinone molecules. So we reduce quinone molecules in the reaction centre. Complex 1 does the same. The hydroquinone diffuses in the membrane towards the cytochrome BC1 complex. And we will hear all the details of that complex in the subsequent talk. Electrons there are transferred towards cytochrome C, cytochrome C diffuses towards cytochrome C oxidase, this is this complex. And this complex is of particular interest because it is the one where oxygen is reduced, water is formed. And here we have some kind of vectorial reaction. These kind of vectorial reactions were first described by Peter Mitchell in his so called chemiosmotic hypothesis for which he was awarded the Nobel Prize too. And this is a very important concept and it was very tough to get the concept brought through and he fought about 20 years. And I think that Boyer yesterday had a slide where he showed you the rate of acceptance of his hypothesis among his colleagues. But I think now nearly everybody accepts it. And cytochrome C oxidase there is the terminal enzyme. So electrons from cytochrome C are transferred to a binuclear copper A centre. So we have here some inorganic chemistry going on, reduction of two copper atoms. Electrons are transferred further to a first heme A molecule. Heme A which is in this case called simply heme A. And the electron further transferred to a second heme A which is now called heme A3, for historical reasons. And pretty near the heme A3 iron we have a copper B bond and the active site, the business unit here is between the iron and the copper B. That’s the place where oxygen is found, where electron reduced the oxygen, where protons are taken up. Protons are taken up exclusively from the inside and water is formed. So we get creation of an electric field by having the electrons from the outside and the protons from the inside. And in addition nature has invented a trick to transport the same amount of protons as there are consumed by water formation across the membrane. And this doubles the energy yield in our cytochrome C oxidase. The consequences of course that you need only half of the food in order to get the same amount of ATP at the end. Nowadays of course in Europe we have the opposite problem. And there certainly are people which would like to switch off the proton pump in order to have less efficient energy conversion and to fight obesity in the body. But certainly this is not the approach, not the goal of our research to abolish the proton pump in cytochrome C oxidase. We tried again the method of crystallography to get the structural information which we need to understand the mechanism of this enzyme at the end. So we tried again to crystallise it, isolating this complex first from the membrane in the form of detergent micelles and trying to crystallise. This didn’t work from the beginning and we used another trick. We used monoclonal antibodies. So we used methods from immunology in order to get crystals. We used methods from gene technology to produce crystals. We produced monoclonal antibodies first, which means we take a mouse, immunise it with this complex, the mouse develops antibodies against the cytochrome C oxidase. We sacrifice the mouse, take the spleen cells, fuse them with cancer cell lines. Get a hybridomas cell lines which produces antibodies against the cytochrome C oxidase. Then we continue, isolate the genes for these antibodies and express these genes in bacterium E coli. Then we go on in our stuff, make the complex of cytochrome C oxidase in the part of the antibody fragment and crystallise this complex. These are now the crystals. They have a length of about 1 millimetre, diameter is about 0.3. Then we use crystallography and the most important point when you get crystals is that they should diffract electrons well. The diffraction here was I would say, was motivated best, you see here diffraction. Taking a synchrotron and the synchrotron actually was in Japan, in Tsukuba. And this synchrotron was particularly helpful because it had a mode of data collection which could be used for less well diffracting crystals and also for adjacent sensitive crystals, much better than the European ones. And the diffraction limit here is about 2.8 angstrom but perpendicular to it, it’s only about 4 angstrom which is just the limit of getting a protein structure. Nevertheless you see here now this structure of the entire complex. So this is the cytochrome C oxidase. This is the antibody fragment which we used for getting the crystals. It helps to form the crystal lattice and without this addition to watch the cytochrome C oxidase, we would not get crystals because most of the protein here is actually buried in a detergent micelle. And in a detergent micelle this part of the protein is not available for crystallisation. So this is why we had to use this antibody fragment. And we tried it also with a PC1 complex, it worked well and at present the method, we have a 100% success rate. And I hope that we will be able to determine more membrane protein structure in the near future. And I hope you can hear about more membrane proteins 3 years from now in the same chemistry symposium here. Here you see here in purple, subunit 2, you barely can see here carbon atoms bound to the protein. You can see here innovate now a heme A which is bound towards the yellow subunit 1. You see here heme A3 and here is the copper B. So electron transfer is restricted to this rather narrow part here. Cytochrome C binds towards this corner of the enzyme and transfers the electron towards here and then the electron is transferred here and then here. Protons are taken up from the inside. And protons are pumped across the membrane and it is of great interest to understand how the transfer of electrons and protons are coupled. What's also of particular interest is the distribution of amino acid residues. And I show you here the distribution of both residues which are frequently charged in nature. Of course argenine and lysine can be positively charged, aspartic acid and glutamic acid can be negatively charged. And we have only very few in the hydrophobic environment of the membrane. And they are there for either structural or functional reasons, these are there for structural reasons. This is, we don’t know it yet for structural reason. This is here a glutamic acid residue and when you change this by genetic methods, so you have to use genetic methods, site directed mutagenesis to change it, even to a glutamine which is a rather minor change. The enzyme is dead, it doesn’t work. And this is therefore of great importance. There is another residue of great importance which is this lysine here in blue. And if you change this to a neutral residue, you get the same phenotype, the enzyme is dead, it doesn’t function. And we presume that these residues are involved in proton transfer towards the active site. And this may be also involved in transferring those protons which are transported across the membrane. And I will also discuss some more residues later on. I simply, and I will describe individual protein subunits. We have here subunit 3, subunit 3 has an open V-shape arrangement. We have two transmembrane alpha helices here, we have here five transmembrane alpha helices. And we have some bound lipid molecules here in the cleft. And the role of subunit 3 is not well understood. There are two suggestions, first you can delete the gene in our bacterium and what you get is you get a reduced amount of cytochrome C oxidase consisting of two subunits. This 2 subunit cytochrome C oxidase is still active in proton pumping and in the turnover of the enzymes. So it can have nothing to do, the subunit with proton pumping or with the oxygen reduction. We think actually that this subunit has some role in oxygen diffusion and I will talk about this point later on a little bit. Here I show you subunit 2, subunit 2 has an N terminus up here. Here it goes to the C terminus and we have an irregular protein fold. Then we have two membranes spanning helices. Then we have two here, the kind of protein fold which was already known, it corresponds to that of the type 1 copper proteins. And here there are some differences. Primarily we have here, we have two copper atoms. You see them here at the tip together with the mode of binding. I don’t go into further details, due to the time limits. And why we have here two instead only of one copper atoms, is not well understood. It might be due to the reorganization energy and what this actually is, probably we will hear tomorrow by Rudi Marcus. And he would develop a theory of electron transfer and the reorganization energy is certainly an important parameter and might be the reason why we here have two and not only one copper atom. This is here subunit 1. Subunit 1 has a rather regular appearance, looks like a cylinder consisting of 12 transmembrane helices. And in here we have the binding site of the porphyrin rings, the hemes, we have here the heme A3. And quite interesting, the hydrophobic tail here is bent away and this allows the access of protons towards the active site here. And I will discuss this point also further. And here you see subunit 1 now from the top and this is actually the most interesting view. You see the 12 transmembrane helices, they are all tilted. They are rather long, much longer than was expected. Here we have helix number 1, the brown one, number 2 is the green one, number 3 the blue, 4 is purple, 5 is the red, this red one is 6, they continue like this with counting. It’s a sequential simple topology, sequential arrangement of helices. However 4 helices each in projection form a half circle. So here we have one half circle, here we have another half circle and here we have the third half circle. And in this kind of arrangement you generate three pores. So we have here some kind of pore. And we think that this pore is used for proton transfer to the centre of the membrane. And then the proton is diverted towards the active site. Here we have the second pore and this pore is used for proton access towards the active site partly, controlled proton access to the active site. And then the pore is blocked by the heme A3 which is seen edge on. Here you see the copper B nearby. And here is the place where the oxygen is bound. And the tail here is bent away again, as I mentioned already in order to allow access of protons towards this place. Here we have the third pore and this appears to be tightly blocked by the heme A which is the first electron acceptor which transfers the electron towards this other heme. The entire cytochrone C oxidase can be seen here from the periplasmic space. Again in a simplified truncated version. Subunit 1 in yellow, subunit 2 in blue, together with a beta-strand (in red) rich area. And you can see here this part covering here additionally the heme A 3 and the copper B which is in this position. And you see also very nicely here subunit 3. And what we actually think is, we have here identified a hydrophobic channel from the binding site of the lipids towards the active site here. And we think that this channel actually is used for the diffusion of oxygen towards the active site. And you are probably not aware of the fact that oxygen is a hydrophobic entity. It likes to be enriched in membranes and you know from spectroscopy that it is enriched in the membrane by a factor of 7. And therefore it’s very likely and it makes good sense if we have oxygen diffusion from the lipidic milieu from the membrane towards selective site. And if we here have loosely bound lipid molecules, we are able probably to enrich oxygen here further. So this may be an oxygen trap and the oxygen then can diffuse from this oxygen pool towards the active site. Here you see more details of this oxygen diffusion channel, of this presumed oxygen diffusion channel I should say. You see primarily hydrophobic residues, phenyl rings, lining up, tryptophan residues. And at the end you here have valine. Here we have the binding site for the copper B together with a ligand. Here we have the heme A3. And it is known from spectroscopy that when oxygen diffuses in, it first becomes temporarily bound towards copper B and then it is transferred to the heme A3. It also makes sense from the channel structure. And site directed mutagenesis, genetic method has shown that when you change this valine to the slightly larger isoleucine, you get a reduction of the diffusion speed which means that the KM value for the enzyme for oxygen is increased by a factor of 10. But maximum to an over number of the enzyme is still the same if you simply have a 10 fold higher oxygen concentration. So this makes sense with the assignment of this channel to an oxygen diffusion channel. Now I come towards the most complicated thing in my talk. This is the environment of the binding site of the heme A3. You see the heme A3 light blue together with the protein. The heme groups have two propionate side chains. So here we have one propionate side chain and this is charged, neutralised by forming an iron pair with an arginene here. We have the second propionate here and this is not charged, neutralised. The charge there gets stabilised by accepting hydrogen bonds from neighbouring residues here. Quite interestingly, we also had from the very beginning, we could identify the copper B down here. And from site directed mutagenesis work it has been known that there are mostly like three histidine ligands. But we could only identify two histidine ligands and the other one, for the third one we had no electron density at all. And there had been the proposal in the literature that a histidine might change its protonation stage during the term of the enzyme being negatively charged meaning imidazolate form in one state of the enzyme becoming imidazole and imidazolium. The positively charged form in the enzyme and the imidazolium form cannot be ligand to copper B. So the histidine could shuttle between two positions depending on the portonation state. And it could carry over protons by switching between the two positions and this might be the mechanism of how a proton pump works. But this I have to say is speculation and there is at present no evidence for that. And when we repeated the whole experiment in the absence of azide with the oxidised and reduced forms, we could not discover the absence of the histidine ligand. We could clearly see the ligand of the histidine as being still a ligand to copper B. And it stays there upon oxidation and reduction of the enzyme. And then we think that the absence of the histidine ligand was an artefact due to the presence of azide. So you have to be pretty careful. As I mentioned we had some problems with our original crystal form and a graduate student in the lab Christian Ostermeier who also had made the first crystal form, worked very hard to get better crystals. He succeeded now with the 2 subunit form of the enzyme, only two proteins of unit presence, he got these crystals. They don’t look as nice as the other ones, the protein is pretty unhappy, you see some denature protein under the crystallisation conditions, but the crystals are much bigger than our first version. So we could determine an improved structure and the improvement was largely, we had a much better definition of the position of the atoms and we could start to identify bound to water molecules. And each of these green balls means the position of a water molecule. And much to my surprise you see many water molecules up in the upper half of the enzyme but only very few below them. Which probably means that we have also water molecules here but they are much less ordered whereas the water molecules here are much better ordered, much more highly ordered. And this could have some functional significance. This here, coming back to mechanism, shows you the electron density of the copper B, this is the copper B electron density. And this is the missing electron density for histidine ligand. And this could be interpreted in the form of a mechanism of proton pumping when you consider that actually you have an iron atom here, a copper B atom, the iron has a formal charge of +3, the copper of +2. And most likely you have an OH-, a hydroxyl group between both atoms cannot be resourced by x-ray crystallography at our resolution. But there is spectroscopic evidence for that. And I think also otherwise if you would have only the positive charges here, you would get a too strong electrostatic repulsion, the distance is only about 5 angstrom. And the whole thing nearly would explode if you wouldn’t have a negative charge between both atoms. So the OH here makes good sense. The basic idea however in this histidine cycle mechanism is when you start from the oxidised form of the enzyme, you get reduction of the enzyme from the periplasmic place. The electron is transferred first to the iron, then it hops over, jumps over on to the copper. And this then disturbs the charge balance in the whole environment. And in order to regain the charge balance, you take up a proton, very simply. And this then converts upon the first reduction, this copper B, this ligand from the imidazolate to the imidazole. Then you take up the second electron which stays on the iron. You neutralise this additional charge by taking up a proton. You convert this ligand to the imidazolium form and the imidazolium can no longer be a copper B ligand and it flips over in this position. Then you bind oxygen, this is known that only after full reduction you bind oxygen here. And then the chemistry, the oxygen starts. You get uptake of protons to form the first water molecule. When you take up protons you disturb again the charge environment and the charge of the protons then expels these two protons up here. And so the uptake of the two protons to form water would expel the protons already here in order to have the same charge environment. And this hypothesis I think is the most simple one for explaining a pump mechanism but it certainly cannot be phrased in the term of a histidine cycle and we have to think of alternatives. This is now the electron density for the oxidised and reduced form. And in the histidine cycle, in the reduced form we should not have all the histidine ligands present. They are there and this I think is one of the fundamental experiments which tells you that the histidine cycle mechanism is unlikely. In addition I have great problems to see how a positively charged histidine would not deliver back its proton towards a reduced oxygen species at the binuclear site. Now I show you some more interesting new details. Again it’s the heart of the enzyme. You see here an unbiased electron density map, calculated by a technique called simulated annealing omit map, you omit from the model all these residues and start to, and do simulate annealing to get a way of bias, use the new phases, calculate an unbiased electron density map and this is this electron density here and you rebuild the model. You see here the model of the heme A3, this is the iron, it’s a histidine ligand to the iron. You see here the copper B and you see here the histidine ligands towards copper B. This is the third one and much to the general surprise what you find is that a nearby tyrosine is covalently crosslinked with the histidine. So we have here an unexpected covalent crosslink. And of course this kind of thing, these modifications cannot be discovered from DNA sequences. You still have to do some further experiment apart from DNA sequencing. And there is some meaning about this and I would think that the principle meaning is that you get such a high oxidative power during the reaction that it extracts an electron from the environment. You generate a radical and it’s known that tyrosine can form this kind of radicals. And then in a radical mechanism you simply, you get this kind of crosslink. And such kind of cross links also have been observed, not with histidines but with for instance cysteines and other residues in peroxidases. So peroxidase in general have enough oxidative power to generate radicals which then leads to crosslinks in proteins. And this means we can have the same type of reaction here. Now I think it becomes more systematic. This is now the catalytic cycle of cytochromes C oxidase. And this is simple, we start off with oxygen, the iron is in +3 form, copper +2. When we put on the first electron which goes on to the copper, converting one to copper 1 and this is known that this is accompanied by the uptake of a proton, this is the one electron reduced form, take up the second electron accompanied by the uptake of a proton, you get the reduced form iron +2, copper +1. Then you take up the oxygen only and you get formation of the so called compound A, which was discovered by Britton Chance in Philadelphia more than about 20 years ago. And then you form a compound called P, P was for a long time thought to be a peroxy compound. At least a former peroxy compound, iron +3, copper +2, but the electrons from the metals are transferred onto the oxygen. I always considered this as a very unlikely structure and I wondered how this could be stable. And actually my scepticism was, I should say was satisfied by recent experiment in Japan by Kitagawa, who by resonance in spectroscopy got clear cut evidence that you actually have already in the P state split oxygen compound. And you have actually oxo-ferryl. This means an oxygen bound to the iron in a double bonded way, iron +4, oxygen -2 in this form and you have the copper +2. But this would then mean that you miss an electron here. And this compound has to steal an electron. And the possibilities are that it’s stolen, the electron is stolen from the porphyrin, it’s stolen from the residue and the tyrosine is a good explanation. People discuss also that the copper might become copper +3. And also people discuss that the iron might be +5. But these are all less likely than this explanation where you steal the electron from a tyrosine creating a tyrosine radical. But this is under debate but I think that this root became much less likely within the last year. So this is a rather new experiment. The major problem that we then have is however that it is known primarily from Wikström’s work in Helsinki that the proton pumping is of course only in the transition from the P state to the F state. This state here is well characterised and everybody agrees that this is oxo-ferryl state here. And it’s also known that the next two protons are pumped in the conversion, in that way of conversion. And if we have this kind of mechanism we might, we should have observed proton pumping already and we have to think about a way out if we don’t have formation of water molecules and the incoming protons expel the protons which had been taken up during reduction at that time. So now I just want to come back towards the structure. You see here in red together with the histidine ligands, the heme A, this is the heme A3. And the point I want to discuss is where do the protons go which are taken up upon reduction? I think actually that the protons which are taken up are stored on the propionates in this area up here. And there appears to be a rapid equilibration of protons in that area. And later you get uptake of the protons and I have formulated a mechanism which I think would be in agreement with all kind of mechanisms. Actually they are in the protein which I didn’t say so far, they are the two identifiable proton transfer pathways, this one with lycene involved and this one here which has an aspartic acid at the beginning and a glutamic acid at the end. And I think that the protons are delivered from this glutamic acid on to the propionates towards this area during the pumping. And when electrons come in towards forming the oxygen, they are then expelled from that area to the outside, this would explain pumping. But I cannot go further into details in order to explain that cycle. And I would take too long now to explain these details and you have to contact me privately in order to discuss it further. But just to say, we need different methods in order to prove this and what we did now, we made a biosynthetic deficient mutant for heme A and fed isotopically labelled precursors in a way that only these carbon atoms are labelled with 13C of the heme A and did Fourier transform infrared spectroscopy. So we now use even another method and we look what changes in the vibrational bands upon reduction. And you see this for labelled and unlabeled, you see the differences for labelled and unlabeled cytochrome C oxidase. And there are clear differences which tell you that there are changes of protonation states and conformation of the propionates upon reduction. And this at the end can be summarised in some kind of mechanism which purely operates on electrostatic grounds. And this is now our working hypothesis and we work very hard either to prove or disprove such kind of mechanism in detail. But at least you get an idea. The whole thing is complicated. And still we have to get an idea, we have to go into details and we need all the methods. And I think the message for you is, for the students is, that you have to know all the methods in order to use them efficiently to solve biological problems. And the people at the end I would like to acknowledge are here from my own group, Gerald Kleymann establishing the method of the antibody fragment production in the lab. Christian Ostermeier did most of the crystallisation work and also determined the second crystal structure. Hanni Müller isolated most of the material, So Iwata isolated in a very, sorry solved the protein structure in a very short time. Axel Harrenga then improved his structure and got now a much better structure. Aimo Kannt did electro study calculations which, theoretical calculations which I didn’t mention. And Julia Behr is involved in this Fourier transform infrared study together with Werner Mäntele and Petra Hellwig from the University of Munich. Some mutant work was done at Frankfurt University by our biochemical collaborator Bernd Ludwig and Heike Witt. Now I thank you for your attention.

Hartmut Michel (1998)

From Photosynthesis to Respiration: Structure and Function of Energy Transforming Membrane Protein Complexes

Hartmut Michel (1998)

From Photosynthesis to Respiration: Structure and Function of Energy Transforming Membrane Protein Complexes

Comment

For many years the Royal Swedish Academy of Sciences gave a Nobel reception on the 7th of December. At this reception, the members of the Academy met the Nobel Laureates in Physics and Chemistry and the Economic Sciences Laureates. The members of the Academy are the ones who earlier during the autumn have made the formal decision about the new Laureates, most members usually without ever having met the Laureates in person. So there is usually a lot of personal curiosity to be satisfied this particular evening. The first time that I participated in this reception was in 1988 and I still remember shaking hands with the three Physics (Lederman, Schwarz and Steinberger) and the three Chemistry Laureates (Deisenhofer, Huber and Michel). At that time, I was not aware of the yearly Lindau Nobel Laureate Meetings, so I could not know that the three German Chemistry Laureates would be invited to give lectures every year, starting in 1989! If they had all accepted, every year, we would now have a set of about 50 lectures by them. In reality, of course, they also have other meetings and commitments, so what we have so far is “only” around 25 lectures. In the early Lindau lectures, German is the dominating language. But with increasing internationalization, also the German Nobel Laureates turned to English. The present one is the first Lindau lecture that Hartmut Michel gave in English and the topic is very close to the one for which his Nobel Prize was awarded. In particular, in the lecture Michel describes some of the very tricky problems involved in getting crystals of large biomolecules, a prerequisite for X-ray diffraction studies of the structure of the biomolecules. I must admit that I was very impressed by the method used to get crystals, a “high-tech” method in which even gene technology plays an important role! Anders Bárány

Abstract

Nearly all energy available to mankind is the result of photosynthesis. Coal, mineral oil, and natural gas are derived from the products of photosyntheses. During photosynthesis, first the light of the sun is absorbed by the antennae pigments of the light-harvesting complexes. Next, energy is transferred by excitonic interaction to the so-called photosynthetic reaction centre, where the primary change separation takes place and an electron is transferred across the photosynthetic membrane.

Photosynthesis of the purple bacteria is well understood. This knowledge is based on the fact that the light-harvesting complexes and photosynthetic reaction centres of these bacteria could be isolated and crystallized, so that their structures could be determined by X-ray crystallography. The light-harvesting complexes of purple bacteria are highly symmetric ringlike oligomers of a basic unit that consists of two short protein chains, two or three bacteriochlorophylls and one carotenoid molecule. In the case of the purple bacterium Rhodospiriflum mo/ischionum eight of these basic units form the light-harvesting complex 2. The carotenoids possess a dual function: First, they serve as light-harvesting pigments covering spectral ranges different from that of the chlorophylls, and second they have a protective function against the damaging effects of light by quenching the triplet states of bacteriochlorophylls and preventing the formation of the very dangerous singlet oxygen. The ring-like arrangement of the pigments appears to optimal for energy storage and subsequent energy transfer of the reaction centre. There electrons are released from the "primary electron donor" which consists of a non-covalently linked dimer of two bacteriochlorophylls. The electron is transferred via a bacteriopheophytin and a first quinone molecule to a second one. Stable reduction of the second quinone requires two electrons, during or after the second electron transfer two protons are taken up from the cytoplasm. In subsequent reactions the hydroquinone is oxidized again, and adenosine -5'-triphosphate (ATP), the universal energy currency of life, is generated. ATP, and biologically fixed hydrogen are needed for the synthesis of sugar molecules from carbon dioxide.

The sugar molecules are taken up and metabolized by other organisms. Carbon dioxide is formed again together with biologically fixed hydrogen. The fixed hydrogen is converted to water in the "respiratory chain" of the mitochondria or a aerobic bacteria. During this process protons are "pumped" across membranes. Therefore, an electric field across these membranes is formed, which drives electrons back through the ATP-synthase leading to ATP-synthesis.

Cytochrome c oxidase is the terminal enzyme of the respiratory chains. It oxidizes cytochrome c and transfers the electrons to molecular oxygen. Water is formed. The protons needed for water formation originate from the cytoplasm of the bacteria or the interior of the mitochondria. Simultaneously the same number of protons are pumped across the mitochondrial (or bacterial) membrane. This fundamental enzyme could be crystallized from the soil bacterium Paracoccus denitrificans, using cocrystallization with an antibody Fv-fragment as a novel approach for membrane protein crystallization (Ostermeier et al., Nature struct. biol. 2, 842-846 (1995)). The structure could be determined at 2.8 A resolution (lwata et al., Nature 376, 660-669 (1995)), and then at 2.7 A resolution with a new crystal form (Ostermeier et al., Proc. Natl. Acad. Sci. USA 94, 10547-10553 (1997)). The arrangement of the prosthetic groups (three Cu-atoms, two haem A groups, one being called haem a and the other haem a3), their interaction with the protein surrounding and the structure of the four protein subunits are therefore known. Subunit I contains 12 membrane spanning helices in a rather regular arrangement and binds both haem groups and copper B. The oxygen is bound to the Fe-atom of haem a3, which is 5.2 A away from copper B. Subunit 11 possesses two membrane spanning helices and binds a binuclear copper A centre in a globular plastocyaninlike domain. Two possible proton transfer pathways could be identified in subunit I. Possible mechanisms of coupling water formation to proton pumping are discussed: Evidence for an involvement of a histidine residue undergoing protonation changes ("histidine cycle") is weak. The propionate side chains of the haem groups might be critical for proton pumping. They undergo structural and/or protonation changes upon reduction of the enzyme as indicated by Fourier Transform infrared spectroscopic experiments using cytochrome c oxidase, specifically 13C-labelled at the haem propionates (Behr et al., Biochemistry 37, 7 7400-7406 (1998)). A model for proton pumping, based on long range electrostatic interactions, will be presented.

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