Johann Deisenhofer (2016) - Photosynthetic Light Reactions, Revisited

Actually, I will talk quite a lot about the past. I work in this environment here in Dallas, Texas, and it’s a medical school. The people there are politely interested in photosynthesis but you immediately realise that they think, what kind of disease is related to photosynthesis? And there is none. Therefore the interest locally is quite limited let’s say. However, ever having worked on photosynthetic reaction centres it never leaves you. And so I am keeping up in the literature and I want to give you a review of one particular aspect of structure of molecules involved in photosynthesis. Now, Hartmut Michel already introduced the concept of cell respiration and he mentioned that the appearance of oxygen in the atmosphere was a major catastrophe for the then majority of living organisms. But nowadays, of course, photosynthesis is the most important chemical process on Earth. And it makes use of the fact that we on the Earth live in an environment that has an average temperature of 288 kelvin. And 8 light minutes out there is a hot body with for all practical purposes inexhaustible energy, that is 5800 kelvin and we receive the radiation. And the degradation of the radiation spectrum from this to that temperature is used for overcoming temporarily the entropy, and by energy input create a low entropy environment which is life. And the photosynthetic process can be chemically roughly divided into 2 parts. There are the dark reactions and the light reactions, meaning that light reactions are dependent on light and are the reactions that actually convert light energy to chemical energy. The dark reactions use that to fix carbon dioxide and make carbohydrates out of it. The light reactions use water as a source of electrons and transfer these electrons to the carbohydrate. And they release the oxygen in the atmosphere. And Hartmut talked about this. So let me continue talking about this. When we think of photosynthesis we think of green plants perhaps. And here is a microscope picture of a cross section of a so-called chloroplast. That’s where in green plants photosynthesis actually occurs. And you see here these fine lines, these are membranes in the chloroplast. There are lots of them. And these are in parts stacked and parts more loosely packed. And they carry the actual molecules that perform photosynthesis. These chloroplasts are thought to have originated from cyanobacteria that were incorporated into plant cells. In fact, nowadays still about half of the photosynthetic activity on the planet is done by cyanobacteria, following exactly the same principle. And here is a text book picture of the photosynthetic process with the input molecules in green, water and carbon dioxide, the output in blue, oxygen, carbohydrate and a proton gradient. That was also described in Hartmut’s talk, this general way of storing and providing energy to other processes in a cell. What you see here is light heading to big macromolecular complexes called photosystem II, photosystem I. In between there is a proton pump. This axis is, as far as electrons are concerned, a kind of an energy axis. Not exact but what the rising line here means, light is used to raise electrons to high energy then it loses some of the energy to drive the proton pump. It is delivered to the second, to the photosystem I, again lifted to high energy, and then delivered to an electron carrier molecule NADPH. And this is used in the carbon fixation cycle where carbon dioxide is taken up from the air. And this is also used for processes in this cycle. The oxygen is released into the atmosphere. This is a relatively complex system. And the pioneers of photosynthesis research 50, 60, 70 years ago, were looking for easier to treat systems as is the custom in many parts of biology and they found organisms that also do photosynthesis but on a much simpler scale. And this is outlined here. They have only one photosystem which is called photosynthetic reaction centre. They have a proton pump. They cannot extract electrons from water and therefore what they do is they just recycle the red electrons from the proton pump to the reaction centre. So they run a cyclic electron transfer system, driven by light and to produce the proton gradient. And these types of bacteria were sort of the guinea pigs of photosynthesis for a long time. And spectroscopy especially, spectroscopic methods, did a lot to clarify the steps after absorption of the photon, clarify the steps that happen to store the energy of the photon. But there was almost a crisis in the late 1970s because all the discussions always ended up asking what may be the structure of this thing. So here is a picture of a bacterium, a photosynthetic purple bacterium, that was one of the sources of reaction centre preparations. And as you can see it is also filled with membranes in its interior. It’s about 2 microns across. And it’s a rich source of reaction centres to do in-vitro experiments. And, as I said, structure was the real need that arose over time and the problem was psychological and also technical. Psychological because there was a widespread belief that one cannot crystallise proteins, they come out of biological membranes. Luckily the previous speaker did not believe that and published in 1982 this picture of wonderful crystals of photosynthetic reaction centres. And as he already said they were quite big, so they could be actually seen with the bare eye. And they were very well suited for X-ray crystallography. So together with a number of colleagues, who are listed here as authors of our first or second paper, we determined the structure of the reaction centre. And here is a model in 2 representations, 1 where the proteins are in cartoon representation, and here where they are in spheres representation without showing the hydrogen atoms. So almost half of the atoms in this structure actually are missing but that’s because at the resolution of the X-ray crystallography we did, these were invisible. And the reaction centre consists of 4 protein subunits called H, M, L and cytochrome, shown in different colours. And I need to say that many of the relatives of this bacterium, plus the chloris viridis, do not have the cytochrome. It is also possible to operate this system without, and the cytochrome essentially is a sort of a storage for electrons that fill holes when they are created by absorption of light. Here is a picture of the cofactors alone, there are 14, the hemes of the cytochrome, 4 bacterio-chlorophylls in green, bacterio-pheophytins in blue, carotenoid, non-heme iron and 2 quinones. And one of the biggest surprises for many people was the fact that this structure, especially when you leave out the cytochrome and the carotenoid, has a 2-fold symmetry, an approximate 2-fold symmetry. So there is an axis running here through the iron and you can rotate around and the structure is unchanged almost. Of course, there is chemical difference between this and this. But this was a big, big, big surprise and I was asked many times, are you sure that this is really true? And the reason why I was, or we probably were asked this, is the following. Spectroscopy found out, established a sequence of events, that after photons are absorbed by this pair of chlorophylls an electron is transferred within 3 picoseconds to the right side only. Not here, just to this side, it’s almost exclusively. And then within 0.7 picoseconds it moves on to the pheophytin, in 200 picoseconds to the quinone, and by then it has crossed the whole thickness of the membrane. And then in this case, in the Blastochloris viridis’ case, an electron is coming from the nearest heme group in the cytochrome within 130 nanoseconds. So that means the hole here is already filled up again. There is an extra charge here. And this charge then moves parallel to the membrane surface to the second quinone in a much, much longer time, 150 microseconds. This whole thing has to happen twice. Then this quinone is fully reduced as we say. It picks up protons and moves out. And it transfers its electrons and its protons through the membrane back by this proton pump. And eventually the electrons come back here. So this is the cyclic electron flow that I mentioned before. And the isometry is definitely caused by the protein. And I drew this picture again to show how the protein subunits, this L in brown, M in blue, how they form a scaffold to hold the cofactors together. And not only do they do that, they also exert subtle influences on the electronic properties of the cofactors, so that this pathway is always followed. And if we look from this side on the structure we see the symmetry including the cofactors and the helices of the protein. All the parts outside of the membrane have been left out and I specifically want to point out the sequence of the helices as they appear in the amino acid sequence. And there is 1, 2, 3, 5, 4; 1, 2, 3, 5, 4. And also I would like to point out that the place where the photons are first absorbed and where the electron is coming from is a pair of parallel, almost parallel chlorophylls. And these we will see in the following again and again. So after that structure had been published and all the events that followed people began to look at chloroplasts again. And they were successful in actually crystallising all these major components of the chloroplast or the cyanobacterial photosynthetic system. And here I have given a short list of some of the most important papers, starting with 2001, And so far the best structure came from Osaka City University, Umena et al. in 2011, at 1.9 angstrom. And I will quote briefly from this structure. Here is a picture of the whole complex and that is just one of 2 parts of a dimer, 2 identical parts of a dimer. And you can see here that this is a very much more complicated structure than the bacterial photosynthetic reaction centre. And I want to point out that there are many protein subunits, 20, but in the middle there are 2 that have the same colour as the ones I showed for the reaction centre, 1 brown and 1 blue. In the very middle there are these cofactors and if you look just at this group then you see essentially the same arrangement as in the reaction centre, a pair of chlorophylls, so-called accessory chlorophylls, pheophytins, quinones. And these cofactors are actually making electronic contact to the light-harvesting groups that are out here. And what is not found in the bacterial reaction centre is this, the so-called oxygen evolving complex. And this was actually the highlight of this paper. And here is a structure directly taken from the paper by Umena et al. showing 4 manganese and a calcium and oxygens and waters And that’s a very, very demanding process, like taking 2 water molecules in succession, extracting one electron at a time and after 4 electrons have been extracted, dioxygen is released. And many spectroscopists, among them our session chair, have done important work to clarify the steps. But I want to go on and show you now the core of the photosystem II. You already may notice a similarity also of the protein arrangement to the bacteria reaction centre. And if you look perpendicular to the membrane we see almost exactly the same picture. And especially I want to point out that the order of the helices as they appear in the sequence, 1, 2, 3, 5, 4, is exactly the same. So there is a relation, even though the amino acid sequences are very different. Now, briefly the photosystem I structures. Again the Berlin group was leading the efforts and they published a 2.5 angstrom resolution in 2001 and I will quote from this structure. So this is the photosystem I. Here again we have a brown and a blue subunit but they are much bigger than in the bacterial reaction centre. If you look perpendicular to the membrane we see that these proteins are actually covering the space that in the photosystem II are covered by light harvesting proteins. And they have many, many, many chlorophylls bound to them. And it turns out that these 2 proteins, the brown and the blue, are fusions of light-harvesting proteins and core proteins. And when we look at the cofactors, again, we see a 2-fold symmetry arrangement, chlorophylls, 2 accessory chlorophylls, 2 more chlorophylls. These are now not pheophytins but chlorophylls, then 2 quinones and 3 iron sulphide clusters. And in this structure is now accepted that after the photon has been absorbed electrons can go, in principle, both ways, even though one of the pathways is 70% preferred and the other one only 30%. And the electrons end up on these iron sulphide clusters, are then picked by transport proteins and are delivered to their destination. And if we add here helices which are directly in contact with the cofactors, we again find 5 brown, 5 blue, if we look perpendicular to the membrane we see again a 2-fold symmetry picture with a pair of chlorophylls and the helices now appearing in the order 7, 8, 9, 11, 10 in a sequence. And again these and these are interchanged with respect to their appearance in the sequence. So it’s exactly the same principle. And if we show these pictures together you can see that purple bacteria reaction centre, photosystem II and photosystem I, look – from this direction at least – very, very similar. The amino acid sequences are unrecognisable or similarities not recognisable, but the order of helices is exactly the same. And this in my view very much supports the idea that photosynthesis, or the ability to do photosynthesis, was developed only once on planet Earth. And, of course, for some organisms this was a catastrophe but we would not exist without it. And I find it amusing that Hartmut and I picked the same graph from a review about the evolution of atmospheric oxygen content. And as he already pointed out there are 2 major events in this, at least in this respect, in the concentration of oxygen. So both axes are logarithmic but this is the pressure of oxygen in units of present atmospheric levels, PAL, it means present atmospheric levels. And this is the opposite, the partial pressure of oxygen. And for quite a long time in Earth’s history oxygen was very, very low, like 6 orders of magnitude less concentrated than nowadays. And here is this great oxygenation event which Hartmut rightly described as a catastrophe. Then it rose again, it rose but stayed in the order of between 1 and 2% of the atmosphere for a very long time. Like on this picture it’s in the order of 1.8 billion years. And the authors of this review, they want to argue that of course there was a variation. But then, around 540 million years ago, there was another increase and to the current levels approximately. And this coincides with the appearance of organisms as we, more or less, know them today. They are multi-cellular, they have external shells and so on. They predate, they eat each other and many more similarities. And human history is essentially inside this line here. It’s really sobering to look at this because I think we can say that we are actually parasites of photosynthetic organisms. We make use of their production. And I think we should sometimes keep this in mind. And, of course, there are speculations now and you can see, I mean from the question alone, that there is a lot of uncertainty in what caused it. And I recommend this article, it appeared in 'Nature' in February of this year and perhaps you find an answer. Thank you.

Johann Deisenhofer (2016)

Photosynthetic Light Reactions, Revisited

Johann Deisenhofer (2016)

Photosynthetic Light Reactions, Revisited

Abstract

Photosynthesis, the conversion of light energy from the sun into chemical energy, has been the basis of life on Earth for more than 3 billion years. The first steps of this conversion are light-driven electron transfer reactions catalyzed by the so-called photosynthetic reaction centers (RCs), which are complexes of protein subunits and pigments located in biological membranes. This lecture will present an overview of known three-dimensional structures of RCs from purple bacteria and from cyanobacteria and green plants (Photosystems I and II). Despite different sizes and compositions, the RCs have at their cores arrangements of transmembrane helices and pigments that are amazingly similar. This supports the assumption that they all derive from a common ancestor.

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