Photosynthetic Light Reactions, Revisited
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Deisenhofer, Johann
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.
 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, and the people there are politely interested in photosynthesis, but you immediately realize that they think what kind of disease is related to photosynthesis, and there's none. Therefore, the interest locally is quite limited, let's say. However, ever having worked in photosynthetic reaction centers, it never leaves you, and so I'm 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 Michl 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 eight light minutes out, there's a hot body for all practical purposes in a sourceable energy that is 5,800 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 two 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 Hartmann talked about this, so let me continue talking about this. When we think of photosynthesis, we think of green plants perhaps, and here's 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. This textbook picture of the photosynthetic process with the input molecules in green, water and carbon dioxide, the output in blue oxygen, carbohydrates, and a proton gradient that was also described in Hartmann's talk, this general principle of way of storing and energy and providing energy to other processes in itself. What you see here is light hitting two big macromolecular complexes called photosystem 2, photosystem 1. In the middle, in between, there's a proton pump. This axis is for 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 rise 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 1, again lifted to high energy and then delivered to a electron carrier molecule NADPH. 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 were in 50, 60, 70 years ago, they're looking for easier to treat systems as is the custom in many parts of biology. They found organisms that also do photosynthesis, but on a much simpler scale. Photosynthesis is outlined here. They have only one photosystem, which is called photosynthetic reaction center. They have a proton pump. They cannot extract electrons from water and therefore what they do is they just recycle the electrons from the proton pump to the reaction center. So they run a cyclic electron transfer system driven by light and produce the proton gradient. And this was this type of bacteria where 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, photosynthetic purple bacterium that was one of the sources of reaction center preparations. And as you can see, it was also filled with membranes in its interior. It's about two microns across. And it's a rich source of reaction centers 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 crystallize 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 centers. 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 determine the structure of the reaction center. And here's a model in two representations. One 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 center consists of four protein subunits called H, M, L and cytochrome shown in different colors. And I need to say that many of the relatives of this bacterium plus the chlorisverides 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 hems of the cytochrome, four bacterial chlorophylls in green, bacterial pheophytans in blue, carotenoid, non-hem iron and two 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 two-fold symmetry, an approximate two-fold symmetry. So there is an axis running here through the iron and you can rotate around. 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? Okay. 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 three picoseconds to the right side only, not here, just to this side, almost exclusively. And then within 0.7 picoseconds it moves on to the field 15 in 200 picoseconds to the quinone, by then it has crossed the whole thickness of the membrane. And then in this case, in the blasterchloris viridus 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, a much, much longer time, 150 microseconds. And this whole thing has to happen twice, then this quinone is fully reduced, as we say. It picks up protons and moves out. And is a transfer, or transfer is 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 threw this picture again to show how the protein subunits, this L in brown and 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 one, two, three, five, four. One, two, three, five, four. And also I would like to point out that the place where the photons are first absorbed and the electrons coming from is a pair of parallel, almost parallel chlorophils. And these we will see in the following again and again. Okay, 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 crystallizing all these major components of the chloroplasts or the cyanobacterial photosynthetic system. And here I have given a short list of some of the most important papers starting with 2001, 3.8-angstrom structure by the Berlin group around Wolfram Sanger and Horst de Beerswit. And the so far best structure came from Osaka City University, Yumeina Edal in 2011 at 1.9 angstrom. And I will quote short briefly from this structure. Here is the picture of the whole complex and that is just one of two parts of a dimer, two identical parts of a dimer. And you can see here that this is very much more complicated structure than the bacteria for the synthetic reaction center. And I want to point out that there are many proteins up unit 20, but in the middle there are two that have the same color as the ones I showed for the reaction center and one brown and one blue. And if we show these, okay, 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 center, a pair of chlorophylls, so-called accessory chlorophylls, pheophytines, 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 bacteria reaction center 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 Umeina Edal showing four manganese and a calcium and oxygens and waters. And this does a very, very demanding process like taking two water molecules in succession, extracting one electron at a time. And after four 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 2. And you again, you already may notice a similarity also of the protein arrangement to the bacteria reaction center. And if you look perpendicular to the membrane, we see almost exactly the same picture. And especially I want to point out 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 sequence, the amino acid sequences are very different. Now briefly, photosystem 1 structures, again the Berlin group was leading the efforts and they published a 2.5-uncim resolution in 2001. And I will quote from this structure. So this is the photosystem 1. Here again we have a brown and a blue subunit, but they are much bigger than in the bacterial reaction center. If you look perpendicular to the membrane, we see that these proteins are actually covering the space that in the photosystem 2 are covered by light harvesting proteins. And they have many, many, many chlorophylls bound to them. And it turns out that these two 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 two-fold symmetry arrangement, chlorophylls, two accessory chlorophylls, two more chlorophylls. These are now not pheophytins, but chlorophylls, then two quinones and three iron-silver clusters. And 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 percent preferred and the other one only 30 percent. And the electrons end up on these iron-silver clusters are then picked up by transport proteins and delivered to their destination. And if we add here helices, which are directly in contact with the cofactors, we again find five, five brown, five blue. If we look perpendicular to the membrane, we see again a two-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, this and this 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 the purple bacteria reaction center for the system 2 and for the system 1 look from this direction at least, very, very similar. The amino acid sequences are unrecognizable, have unrecognizable similarities or similarity is not recognizable. And 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 the 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 Hartman and I picked the same figure from a review about the evolution of atmospheric oxygen content. And as he already pointed out, there are two major events in this, at least in this respect in the concentration of oxygen. So this year, both axes are logarithmic, but this is the pressure of oxygen in units of a present atmospheric levels, PAL means present atmospheric levels. And this is the partial pressure of oxygen. And for quite a long time in Earth history, oxygen was very, very low, like six orders of magnitude less concentrated than nowadays. And here is this great oxygenation event, which Hartman described as, rightly described as a catastrophe. And then it rose again, it rose, but stayed in the order of 1% 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 variation. But then, around 540 million years ago, there was another increase, and to the current levels, approximately. And this coincides with a, the appearance of organisms as we, more or less as we know them today. They have, they are multicellular, they have external shells and so on. They predate, they eat each other, and once more similarities. And human history is essentially inside this line here. It's not, 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, 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'll find an answer. Thank you. Thank you.
 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, and the  people there are politely interested in photosynthesis, but you immediately realize that they think  what kind of disease is related to photosynthesis, and there's none.  Therefore, the interest locally is quite limited, let's say.  However, ever having worked in photosynthetic reaction centers, it never leaves you, and  so I'm 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 Michl 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 eight light minutes out, there's a hot body for all practical  purposes in a sourceable energy that is 5,800 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 two 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 Hartmann talked about this, so let me continue talking about this.  When we think of photosynthesis, we think of green plants perhaps, and here's 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.  This textbook picture of the photosynthetic process with the input molecules in green,  water and carbon dioxide, the output in blue oxygen, carbohydrates, and a proton gradient  that was also described in Hartmann's talk, this general principle of way of storing and  energy and providing energy to other processes in itself.  What you see here is light hitting two big macromolecular complexes called photosystem  2, photosystem 1.  In the middle, in between, there's a proton pump.  This axis is for 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 rise 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 1, again lifted to high energy and then delivered  to a electron carrier molecule NADPH.  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 were  in 50, 60, 70 years ago, they're looking for easier to treat systems as is the custom  in many parts of biology.  They found organisms that also do photosynthesis, but on a much simpler scale.  Photosynthesis is outlined here.  They have only one photosystem, which is called photosynthetic reaction center.  They have a proton pump.  They cannot extract electrons from water and therefore what they do is they just recycle  the electrons from the proton pump to the reaction center.  So they run a cyclic electron transfer system driven by light and produce the proton gradient.  And this was this type of bacteria where 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, photosynthetic purple bacterium that was one of the sources  of reaction center preparations.  And as you can see, it was also filled with membranes in its interior.  It's about two microns across.  And it's a rich source of reaction centers 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 crystallize 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 centers.  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 determine the structure of the reaction center.  And here's a model in two representations.  One 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 center consists of four protein subunits called H, M, L and cytochrome shown  in different colors.  And I need to say that many of the relatives of this bacterium plus the chlorisverides 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 hems of the cytochrome, four bacterial chlorophylls in green, bacterial  pheophytans in blue, carotenoid, non-hem iron and two 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 two-fold symmetry, an approximate  two-fold symmetry.  So there is an axis running here through the iron and you can rotate around.  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?  Okay.  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 three picoseconds to  the right side only, not here, just to this side, almost exclusively.  And then within 0.7 picoseconds it moves on to the field 15 in 200 picoseconds to the  quinone, by then it has crossed the whole thickness of the membrane.  And then in this case, in the blasterchloris viridus 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, a  much, much longer time, 150 microseconds.  And this whole thing has to happen twice, then this quinone is fully reduced, as we say.  It picks up protons and moves out.  And is a transfer, or transfer is 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 threw this picture again to show how the protein subunits, this L in brown and  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 one, two, three, five, four.  One, two, three, five, four.  And also I would like to point out that the place where the photons are first absorbed  and the electrons coming from is a pair of parallel, almost parallel chlorophils.  And these we will see in the following again and again.  Okay, 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 crystallizing all these  major components of the chloroplasts or the cyanobacterial photosynthetic system.  And here I have given a short list of some of the most important papers starting with  2001, 3.8-angstrom structure by the Berlin group around Wolfram Sanger and Horst de Beerswit.  And the so far best structure came from Osaka City University, Yumeina Edal in 2011 at 1.9  angstrom.  And I will quote short briefly from this structure.  Here is the picture of the whole complex and that is just one of two parts of a dimer,  two identical parts of a dimer.  And you can see here that this is very much more complicated structure than the bacteria  for the synthetic reaction center.  And I want to point out that there are many proteins up unit 20, but in the middle there  are two that have the same color as the ones I showed for the reaction center and one brown  and one blue.  And if we show these, okay, 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 center,  a pair of chlorophylls, so-called accessory chlorophylls, pheophytines, 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 bacteria reaction center 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 Umeina Edal showing four manganese  and a calcium and oxygens and waters.  And this does a very, very demanding process like taking two water molecules in succession,  extracting one electron at a time.  And after four 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 2.  And you again, you already may notice a similarity also of the protein arrangement to the bacteria  reaction center.  And if you look perpendicular to the membrane, we see almost exactly the same picture.  And especially I want to point out 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 sequence, the amino acid sequences are very different.  Now briefly, photosystem 1 structures, again the Berlin group was leading the efforts and  they published a 2.5-uncim resolution in 2001.  And I will quote from this structure.  So this is the photosystem 1.  Here again we have a brown and a blue subunit, but they are much bigger than in the bacterial  reaction center.  If you look perpendicular to the membrane, we see that these proteins are actually covering  the space that in the photosystem 2 are covered by light harvesting proteins.  And they have many, many, many chlorophylls bound to them.  And it turns out that these two 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 two-fold symmetry arrangement, chlorophylls,  two accessory chlorophylls, two more chlorophylls.  These are now not pheophytins, but chlorophylls, then two quinones and three iron-silver clusters.  And 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 percent preferred and  the other one only 30 percent.  And the electrons end up on these iron-silver clusters are then picked up by transport proteins  and delivered to their destination.  And if we add here helices, which are directly in contact with the cofactors, we again find  five, five brown, five blue.  If we look perpendicular to the membrane, we see again a two-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, this and this 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 the purple bacteria reaction center  for the system 2 and for the system 1 look from this direction at least, very, very similar.  The amino acid sequences are unrecognizable, have unrecognizable similarities or similarity  is not recognizable.  And 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 the 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 Hartman and I picked the same figure from a review about the evolution  of atmospheric oxygen content.  And as he already pointed out, there are two major events in this, at least in this respect  in the concentration of oxygen.  So this year, both axes are logarithmic, but this is the pressure of oxygen in units of  a present atmospheric levels, PAL means present atmospheric levels.  And this is the partial pressure of oxygen.  And for quite a long time in Earth history, oxygen was very, very low, like six orders  of magnitude less concentrated than nowadays.  And here is this great oxygenation event, which Hartman described as, rightly described  as a catastrophe.  And then it rose again, it rose, but stayed in the order of 1% 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 variation.  But then, around 540 million years ago, there was another increase, and to the current levels,  approximately.  And this coincides with a, the appearance of organisms as we, more or less as we know  them today.  They have, they are multicellular, they have external shells and so on.  They predate, they eat each other, and once more similarities.  And human history is essentially inside this line here.  It's not, 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, 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'll find an answer.  Thank you.  Thank you.
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