 Well, thank you very much for the very kind introduction. The story I want to tell about today has to do with the evolution of a system before there was any tree on earth, any plant. When there was no oxygen, which is the evolution of the system, which is called photosystem 2, which is a complex protein that evolves oxygen by transforming water into the separation of charges that leads to oxygen evolution and reduction of quinone cofactors, which is one way of initiating photosynthesis. And in that process, we're going to learn what are the underlying principles of the functionality of natural photosynthesis so that we can replicate that in synthetic systems, in molecules that we can make in the lab running artificial photosynthesis for using solar light, water, and air to make fuel. So that's what photosynthesis does. And that's what we still don't know how to do today synthetically or artificially. So don't be afraid, there are no Hamiltonians and no quizzes at the end of the talk. But first of all, I want to present the people involved in the research in my group and particularly Crystal and Ka and Misha who are responsible for all the work on the study of the natural system. And the rest of the group works on artificial photosynthetic models as well as on the development of methods for analyzing both artificial and natural photosynthetic problems. So Crystal, Ka, and Misha are experts in yinke. And when they don't play yinke, they worry about photosynthesis. And so within any photosynthetic organism, if you zoom in into one of those green leaves, you might wonder, what is the chemistry that rules the functionality and the energetics of that system? So you might want to zoom into one of those cells. And what you see is that the leaves are green because the cells have these organelles, these blobs that are called chloroplasts that are green. And that's why the leaf is green. And those chloroplasts are like little bacteria that are embedded in the cells. Plants incorporated bacteria somehow transforming into these photosynthetic organisms or photosynthetic organelles within the cells. And so if you zoom into one of those chloroplasts, you will see that the chloroplast is green because it has internal membranes, like the internal membranes of bacteria that are green. So that's where the chlorophyll pigments are inside of these internal membranes of chloroplasts. And if you want to know where exactly, within that membrane, the pigment resides. So you have to zoom in a little bit better. And this is one of those lipid bilayers. Well, they happen to be loaded with these protein complexes, which are aggregates of proteins that operate in synergy. And within one of those complexes, called, the complex is called Photosystem II. But you have, you have zooming in a little bit more. This is that protein complex atomistic resolution. You have all kinds of chlorophyll pigments loading these proteins called the antenna proteins in the overall complex. So the absorption of visible light is due to these chlorophyll pigments that are loaded on the proteins on Photosystem II that are responsible for light harvesting. And the process of photosynthesis is initiated there. So going back one slide is initiated in that system within the antenna system of Photosystem II. So the energy is harvested by inducing an electronic transition, usually a pipi-star transition in the chlorophylls. And when one of these pigments gets electronically excited, in their way, it fills the interaction with nearby chlorophyll pigments of other groups nearby. So the energy sometimes is transferred by simple collisions into other chlorophyll pigments. And the energy is funneled into a special pair called the P680 pair. So that's the story of the initial energy harvesting in the antenna system of photosynthesis. So all the energy is finally congregated here in the P680 special unit, which is a special pair of chlorophyll species. And that is next to a chlorophyll unit that is missing the magnesium in the center, it's called pheophyting. So, and that pheophyting has a real exponential, it's downhill relative to the special pair. So what it happens over there is that when the special pair gets excited, it induces an electronic translocation. So an electron is transferred from the special pair onto the pheophyting, leaving the special pair oxidized and the pheophyting reduced. So that is the primary charge separation process that converts the solar energy into charge separation in the typical second time scale. Now, the electron that is now parked on the pheophyting sees the nearby, there is another redox cofactor which happens to be a quinone, which is quinone A, that can get reduced even more easily than pheophyting. So the electron gets transferred to the quinone and from there it goes to quinone B that can also be reduced even with more facility than quinone A. And in that way the electron migrates all the way to that side of the protein. And what I want you to think of this protein is this cluster that is embedded in the membrane, so here's where the membrane is positioned. So in a way, what you leave behind is a hole buried here in the special pair and an electron now parked on this quinone cofactor that is called the plastocuinone. So now, once the chlorophyll species gets oxidized, now it has oxidizing power. That means it can extract electrons from something that is nearby. So one possibility is to extract it from this quinone cofactor that was reduced. And sometimes that happens. But this is far away, so the rate at which that happens is very, very slow. So the recombination process is prevented by this charge separation process. So instead what it does is extracts an electron from a tyrosine residue that is near a little piece of manganese oxide that is buried inside of this protein that is highlighted in yellow, that is called the D1 protein subunit. So the tyrosine gets oxidized now. The redox state of the special pair is reestablished. And so now we have the hole in the tyrosine, the electron on the plastocuinone. And now the tyrosine can oxidize something. And where it oxidizes is that little piece of manganese oxide that is the catalytic site where oxygen evolves, where water reacts, and two water molecules come together to make O2. So water is taken from the other side of the membrane, as you see here, water is binds to that cluster. And when it gets sufficiently oxidized, it can react and make oxygen. And all the oxygen we are breathing at this very moment has been generated in this way. And so because as I mentioned, there was no photosystem II or oxygen 2.5 billion years ago on Earth. Now, as you realize, what you need is to extract four electrons from two water molecules in order to make one molecule of oxygen and four protons. So somehow you have to accumulate here in the catalytic center for missing electrons. And so far we have accumulated only one by this process. So when it happens, the whole process happens again. So another photon comes in, induces electronic translocation, and oxidizes the cluster one more time. After the second time, the plastoquinone that gets reduced twice, it affects the pK of nearby amino acid residue on this side of the membrane. There's a histidine residue, histidine 252, that is nearby this plastoquinone. And so it increases the pK of that histidine. Usually, histidine has a pK of 6.1, and it raises it to about eight. And now that histidine can recruit a proton from the other side of the membrane. So it increases the pH of this side of the membrane, it's called the stroma, by extracting proton. So now it gets reduced and protonated. And when plastoquinone gets reduced and protonated, it loses affinity for that niche where it is bound. So it comes off and it diffuses to the membrane and it goes to initiate further steps downstream in photosynthesis, leaving an empty cavity there that is refilled by a fresh plastoquinone because it's a reservoir of fresh plastoquinone here in a pocket inside of the membrane. That process of replenishing fresh plastoquinone is the rate-limiting step of photosynthesis. So if you want to design plants that grow faster, that have more efficiency of photosynthetic processes, that is the process you have to engineer. Now, once you park here a fresh quinone, then you are ready to repeat this process two more times. And after the second time you do that, then you have accumulated four holes in the oxygen-evolving complex. And those missing electrons are in the form of a high-valence state of manganese centers in this piece of manganese oxide. So all the manganese centers are four-plus now. They're all fully oxidized. And they can oxidize water by extracting four electrons from water to generate oxygen releasing four protons to the lumen, which is this other side of the membrane. So as a result, you reduce the pH of the lumen, you increase the pH of the stroma, creating a pH gradient that is the driving force for ATP biosynthesis further than the stream. So here's the ATPS where all that free energy gradient is transformed into ATP, into a chemical bond. So that's the way you make fuel for the plant from all the energy that is harvested from the sun. So what we've been studying a lot and what I'm going to be emphasizing is the nature of this catalytic site. Because that's where the magic happens. That's where water binds and reacts with very little effort, you see, because the light that is available from the sun is mostly visible light and infrared. You have very little ultraviolet. So these chlorophyll pigments absorb in the visible range. So you have very little energy. Nevertheless, you're able to oxidize water. This is a very demanding reaction that you know it doesn't happen when you put a glass of water exposed to sunlight. Just water evaporates, but never makes oxygen. So somehow if we want to transform water into oxygen, electrons and protons, and use perhaps the electrons and protons to make fuel as the plant does, or maybe just to make hydrogen, which is another form of fuel, we want to know how to make this kind of catalyst that we could sprinkle in a glass of water and make it produce oxygen driven with solar light. So that's what we decided to study. And there was, at that point, when we started with this work, breakthroughs in x-ray crystallography. So they were x-rays of PS2 for the first time with beautiful resolution of the structures. And as I showed here, you see, you have all of the proteins, all of the pigments, all of the amino acid residues were resolved. And even there was some level of resolution for the position where the oxygen evolving complex was. Here in blue, what you have is the electron density map that results from the scattering process of x-rays. But as you see, it has very low resolution. This was obtained at 3.5 angstrom resolution, which is not enough to detect where the individual atoms are. They're not individually resolved atoms or even individually resolved bonds. What you see is the whole blob. And inside of that whole blob, you can try to fit a model, like the one you have here in yellow, to see whether there is an atomistic model consistent with that broad electron density map that comes from the x-ray diffraction. And there were many possible models that would fit. This is just one. But at the moment, there were 44 models that would be just as consistent with this experimental data as this particular model. We favored this particular model because it was proposed not only on the basis of this electron density map, but also on the basis of information that came from high resolution spectroscopy, which is x-ray absorption and EPR spectroscopy. So with x-ray absorption is an alternative technique for exploring the nature of distances in the system because the x-rays are not scattering x-ray, but the x-rays being absorbed, in this case by the manganese interest. If you tune the energy of the x-ray, you can shine x-rays with energy that is sufficient to extract a core electron from manganese and make a free electron that comes off. So it's photoelectron detachment by x-rays. And when any of these manganese centers ejects an electron, it ejects an electron that looks very much like a wave, like a wave of water because the electron is a wave. And so the electron expands and when it finds a scattering center, something that is standing in its way, it bounces back. So the wave bounces off any of these other centers that are nearby, including other manganese centers that were nearby, very much like a wave of water scattering off a dock, right? So it interferes with itself. It creates an interference pattern that affects the probability of x-ray absorption by that center. So that probability, the intensity of x-ray absorption by that center has information on the distances of scattering centers nearby. So the coordination sphere of that center. So with that technique, I'm going to be talking a little bit more later about that technique, they had concluded that the distances between manganese centers were about 2.7 angstroms. They were, they have distances of 2.7 angstrom, sometimes two, sometimes three. And there were also distances that were long. There were about 3.3 angstroms that were determined with very high accuracy. And so they were determined with 0.05 angstrom resolution. So 0.05 angstrom error. So these are correct. So what Jim Barber did here was to try to fit a model where all the manganese centers had about 2.7 angstroms because of that reason. And it was also known from EBR and Endor data that the model structure that could fit in this was a little bit of a covoidal structure like a dice where the manganese centers were the edges of that dice. And also that it was a dangling manganese, the student involved in this project in the BR project with David Brett and co-workers at UC Davis called this one the dangling manganese. So that name is remain. And so this is the model that Jim Barber proposed in consistency with this high resolution X-ray data. But there were many aspects of this model that were unsatisfactory. For one reason was that first of all, the electron density map was very broad and there were many, many models that could be consistent with that data. So some of the experts in the field called a beautiful model called this work and this work, a beautiful model with very little data supporting. In addition, there was no water resolved in this structure. It's not clear where water binds, if it binds. It's not that it binds and reacts. And so there are parts that are missing. And the manganese centers, if you count each of these manganese centers according to EPR, you should be, manganese centers are high-valent. That means they have charge of plus three or plus four. And when manganese has that oxidation state, it has a covalency of five or six. So each of these might have five or six ligands. That is something you're learning in the organic chemistry class. And if you count here, you have fewer. So there is something wrong. So all these problems had to do with radiation damage. When you collect x-ray diffraction data, you expose the sample to so much x-ray radiation that you generate free electrons and the electrons go around and reduce whatever is reducible. And from the whole system, the part that is more easily reducible is the oxygen-evolving complex. It goes, that's the part that has high-valent metal centers, manganese four and manganese three. So these manganese centers are fully reduced in the x-ray diffraction data. They're all manganese two. And as you must have learned from inorganic chemistry, when you reduce the oxidation state of the manganese center, it loses affinity for the ligands. So the ligands come off and the cluster disassembles. And so that's why it creates the structural disorder. And that's why you have a blob with very low resolution. Here, each of these metal centers have an arrow of more than one angstrom. So it could be anywhere within a sphere of one angstrom from where they were placed. Nevertheless, we thought this was the best empirical model available at the time. It was about 13 years ago. And we decided to redefine the oxidation state of the manganese centers according to EPR spectroscopy. So put them back in manganese four and manganese three and re-optimize the structure of ligands to see whether we can find out how the ligands bind and whether there is water. There is any room left for water to bind because somewhere water has to bind and react. And also, it was known that if you depleted the PS2 system from chloride because chloride binds somewhere, then it stopped producing oxygen. So chloride had to be central to the mechanism of oxygen evolution, to that reaction by which two water molecules react to make oxygen. Nevertheless, there was no chloride or any hint of chloride nearby the OEC. So we decided to start with that and implement the method we discussed yesterday and the quantum chemistry class with a combination of quantum mechanics and molecular mechanics techniques. So the OEC and its surrounding ligands is described with DFT and everything else is described with a force field at the molecular mechanics level. And with Gaussian, that method was already implemented in the form of electronic embedding. So the distribution of charges surrounding the OEC could influence the electronic density in the core, in the oxygen evolving complex. So that was a nice feature because you had polarization effects. But this method doesn't include the reaction, the polarization of the distribution of charges in the surrounding cavity due to the distribution of charge in the oxygen evolving complex. So we developed a method for doing that, which is this DFT-QMM self-consistent protein polarization approach that we also discussed yesterday, where each of the fragments in the cavity is polarized one at a time. And the charges are recomputed with a QMM approach. And when we come back, and we try to recompute the charges of that fragment, the surrounding environment is updated with the distribution of charges and geometry in the previous round of calculations. So next time we reparameterize the charges of that fragment, all this is going to be taken into account. And after repeating this a few times, five times usually, the process converges. And you have a self-consistent description of the distribution of charges in the cavity consistent with the distribution of charge in the core. And mathematically, that's how we do it in terms of the electrostatic potential due to the DFT description with the DFT density. And here the electrostatic potential with the distribution of single-point atomic charges that we fit to reproduce that electrostatic potential from DFT calculations. So the trick was to do this right. And the way we did it was each time we polarized a fragment, we kept it with fragments and that are not polarized. So that the effect of clipping bonds when we define a fragment is not an artifact of the repolarization effect because it's far away from the core that it's impolarized. And then we move the center to a nearby amino acid ratio, we repeat the process over and over until we get a complete description of polarization effects. And when we do that, we end up having, as we mentioned yesterday, corrections over the charges of atomic centers, sometimes more positive, sometimes more negative, and of about 15 to 20% corrections on the overall charge of the center. So they're not big charges, big changes, but there are many. And when you accumulate all those corrections over the interface of the quantum mechanics region and the surrounding environment, then that leads to large correction of about 15 to 20 kcal per mole. And those corrections are necessary when one is trying to elucidate the structure of the OEC and its surrounding ligand environments. So by implementing that method, we came up with this model, which is, yes, a covoidal structure was quite consistent with these calculations, although it's slightly different from what the crystallographic data predicted, because this one has water. So their water molecules bound to calcium and to that dangling manganese. We have an additional myoxobreach, and we have a complete coordination of each manganese center with five or six ligands. We also have chloride bound nearby. And we suggested that the mechanism by which oxygen forms has to do with the reaction of these two water molecules that bind like terminal water molecules. So an intermediate step in that process is the deprotonation and oxidation of one of the two water molecules to make this oxyl radical. And then this nucleophilic attack of one water molecule onto that oxyl radical to make this hydroperoxone intermediate. This is subsequently oxidized one more time and deprotonated to make O2. And that's why we breathe. So other people disfavor this mechanism because there was no evidence for water bound to the OEC. And because our calculation were based on an empirical model, they had huge amounts of error. And they favor instead the reactivity of myoxobreaches that perhaps form a similar intermediate by direct condensation of those myoxobreaches. Today, we don't really know what the mechanism is. But what I want to mention, and that's the subject of this presentation, is that today we have a way of assessing which one is more consistent with experiments. And so that's a little bit of the end of the story for the oxygen evolving process. One of the reasons why people favor this mechanism versus the terminal water molecules in addition to the lack of evidence for terminal water molecules in the OEC is that when you place any of these complexes with terminal water molecules in water, then the terminal water molecules exchange with water in the bulk very quickly in the picosecond time scale. But the reaction time for oxygen evolution as it would happen for these two water molecules as measured by X-ray fluorescence experiments in this system is the millisecond time scale. So it takes about one to two milliseconds for each pair of water molecules to react and make oxygen. So they have to stay bound to the catalytic side for about a millisecond. So it just orders of magnitude slower than the rate of exchange of terminal water molecules with these kind of transition metal complexes. So that was another one. And so for that reason they favor this one because myoxobreaches stay for a lot longer than the exchange at a much slower rate. What we found through calculations with QMM calculations, and by the way we're awarded that methodology was awarded the Nobel Prize of Chemistry in 2013 to Warshel Levitt and Carplus who developed QMM methods, was that the exchange rate for this particular set of water molecules is much slower than when we place the water with this complex in solution because the surrounding cavity doesn't have a complete salvation shell of water. So removing these water molecules has a free energy penalty and the water molecules that are detached from the cluster has very much frustrated salvation and stabilization that leads to a barrier, free energy barrier of about 16 kcal per mile consistent with a millisecond time scale. So even the rate of exchange of water molecules for this model of ours is in the millisecond time scale. So that sort of points at one of the tricks that nature must have evolved in the evolution of this system, which is designing a little dielectric environment that stabilizes water bound to the OEC so that they would stay for milliseconds. And that might also give structural stability to the OEC because many times when we put this kind of complex in water they just disassemble by salvation because the ions can fully solvated and they are unstable. But instead by placing this piece of manganese oxide buried inside of the D1 protein subunit, then the whole stability of the cluster is enhanced and the stability of substrate water molecules is also further enhanced. So in 2011, Chandra and Shannon Coworker finally resolved the structure of PS2 at 1.9 angstrom resolution where you can already see bonds with the electron density maps. And this is the model they proposed. This was a poster that was submitted to the International Conference in Photosynthesis that was in Beijing in 2011. I was not there, I was in Boston at an ACS meeting at the time this happened, but Gary was here and he tells me that the poster was promoted to a small talk because the work was finished by the time that the conference was organized. And when he showed this data, this work was received with the standing of Haitian in five minutes of loss. And we were, he emailed me immediately, look at that. Finally, for the first time there is evidence for the termine of water molecules we had suggested even the presence of chloride next to the OEC and a complete coordination of manganese centers. There were some small changes that needed to be addressed. For instance here, this aspected residue we had suggested it was a monodentate to the dangling manganese and came out as breaching between the dangling manganese and calcium. Here is the breaching. So with those corrections we could go ahead and re-optimize and test whether the model confirmed the experimental data base on X-ray absorption. And that's what we did. We calculated that pattern, the interference of the intensity of X-ray absorption as a function of the energy of X-rays. Here is reported as the kinetic energy of the photo-detached electron. But you see this is the interference phenomenon that if you fully transform, you get in reduced space, the positions of the first coordination sphere, second coordination sphere, and that position of those bands is what it is determined at very high accuracy, at very high precision of 0.05 angstrom. And here this is not a good figure, but when you see there are two lines, the red line is the experimental data from our collaborators, Holger Dao and Michael Hauman, who are from the Frey University in Berlin and did this work at the Grenoble synchrotron. And in black, our calculations of the multi-scarrying process of the electron that gets bounced back from the scanning center, comes back and interferes with itself. And as you see there, right on top of each other. And this is isomorphous and data that means it's an average of our opus orientations of incidence of the X-ray beam. And this is the same calculation now, comparing to data coming from the Berkeley group, Vitalia Chandra and Junco Yano from the Kensauer group that grew single crystals of PS2 and run the same experiments now along the principal axis of the single crystal. This is the most detailed pieces of experimental data available. And again, the QMM model passes a very stringent test in structural characterization. So with that in hand, we decided to go ahead and see whether we could explore the activation process by which that wide discatalyst is able to oxidize water. And so it has been known and observed for a long time now that if you analyze that X-ray absorption spectrum before and after flashing the system with a pulse of light, then that the features change quite dramatically. For instance, this bimodal distribution collapses into a single band. So that has been observed for a long time, but there was no understanding of why that happens and why this is necessary for activating the catalyst, for getting the catalyst in a state that is able to oxidize water. So what we decided to do is to prepare the system in that most reduced state in S zero and induce the oxidation and the protonation because it's known that in this advancement of the oxidation state, the system also deprotonates to see whether we could reproduce these changes in spectroscopy. And as you see, the calculations do reproduce the bimodal distribution changing into a single band. And since we have agreement with experiments, now we can go and see what happened to the structure of the OEC in this transformation. And where we see the following, that from all of the manganese centers that can get oxidized, the one that gives off its electron is this manganese three that changes from manganese three to manganese four. It comes from blue to green. And when that happens, it's more possibly charged. So now it attracts more, this ligand, that is the ligand of calcium, that is an OEC ligand, that now gets closer to the manganese center and forms this additional myoxobridge that was not existent here because this dotted line is too far away from making a bond. So now there is a demyoxobridge here and in that process of getting close to this Lewis acid, then it gets deprotonated. So that's why this process involves oxidation of the manganese center and deprotonation of an OH ligand. One drives the other one. And if you compute the free energy change of the transformation, it is lower than the free energy change that is required to either oxidize that center or deprotonate that ligand. So removing an electron or removing a proton costs more energy than doing both at the same time. And the reason for that is that the surrounding environment is a low dielectric environment. So advanced accumulating charge in the system is more costly than keeping the whole system with the same amount of charge. So that's one of the tricks that nature discovered, which is called proton-coupled electron transfer. And any design of artificial photosynthetic systems must explode, exploit these, you should not explode anything, you should exploit that trick which enables the transformation and accumulation of oxidizing equivalents without accumulating charge. It's a little bit counterintuitive that that is possible but in fact it is possible because you also deprotonate the system. So that's a bottom line of the mechanism of advancing the oxidation state. And the origin of this transformation in the spectroscopic signature has to do with the fact of forming an additional myoxo bridge because now with a demyoxo bridge, the manganese-manganese design is no longer 2.9 becomes 2.7. So that peak that was 2.9 shifts towards 2.7 and now the coalescence into a single band. So this spectroscopic change is reporting on a PCET that leads to formation of the demyoxo bridge, critical for stabilizing the high oxidation state of the manganese center, in this case, manganese stream. In addition, with these models, we were able to explore how water binds to the OEC, substrate water molecule, this terminal water molecule that we fabled. And what we find is that water gets into and binds to the catalytic side through these carousel mechanisms, the rearrangement of water molecules around this manganese center, that in the process of doing that, it forms this additional myoxo bridge and separation of this part of the cluster from the rest. So the cluster sort changes from close to open in this transformation. And as a result, the electronic density in the center of the cluster is decreased. And that was, again, a question that had puzzled the community for many years because when you accumulate oxidizing equivalents, when you make manganese centers that are all four-plus now, you would expect the cluster would be more compact, right? But instead, from the analysis of XF data, X-ray absorption, they saw that some distances got longer. So that was difficult to interpret. What we see here is that the manganese center gets so positive that it now enables binding an additional water molecule that gets deprotonated and forms this additional motif that separates from the cluster. So it is because it is more positive that it opens up the structure. And here we are comparing our calculations of the difference in electron density between one oxidation state and the element of the OEC with recent results from the Petra from and coworkers and from Arizona State University who were able to run these experiments called femtosecond X-ray crystallography experiments, which are the latest developments in the field. So now one is incredible that you can collect the diffraction pattern of X-rays, but using pulses that are only 50 femtoseconds long. So pulse of X-rays extremely intense, but goes through the sample with its width of 50 femtoseconds. And so the whole information is collected within 50 femtoseconds. And of course, it has so much intensity that ends up evaporating the sample. But by the time that happens, you already have a spectrum collected, the image, the X-ray and diffraction pattern. So before any motion of the nuclear centers can even start, you already have collected the data of X-ray diffraction because they are so short. So in original formulations, it was presented as diffraction without destruction. So these problems that I mentioned at the beginning of radiation damage are nonexistent here. We've recently published a couple of papers questioning that, but that's a detail that we can discuss offline and or further if you have questions. Bottom line is that with that methodology, you can run the experiments before and after flashing and therefore you can collect the X-ray diffraction difference at the electron density difference without any problem of radiation damage. And we can do the same thing with our models and confirm this carousel mechanism for sub-survive water binding. In the same way we could validate that mechanism by exploring how ammonia binds to the cluster because there's data, X-ray absorption data, that we have been able to match by binding ammonia also to that terminal water molecule where we suspect this after-water molecule binds and that also agrees with experiments. Therefore we have supported that carousel mechanism and the addition of one water molecule to the OEC in the S2 to S3 transition. So now the big question is how the O bond forms and whether these mechanisms of the terminal water molecule is consistent with experimental data or not. As I mentioned, X-ray fluorescence experiments have been performed on the OEC to measure the fluorescence of manganese centers. So if you have, it's very simple, this experiment is very simple. Well, not to run it, I mean to interpret it, is that if you have more electrons, you have more fluorescence. And if you oxidize the system, the fluorescence decays. That's already this experiment reports. And as you see here, for instance, if you start with the S1 state and you give a flash of light, then the fluorescence decays. What is happening? They're losing electrons from a manganese center. They're oxidizing the cluster. And from this profile, you can fit that to an exponential and get the rate for the oxidation process. And the time scale for that oxidation is 70 microseconds. So that's the way they determine that oxidation of the cluster from what is called the storage state one to the storage state two takes about 70 microseconds. You give another flash of light, another flash of light. The S2 gets oxidized again in a similar fashion, now in 190 microseconds. So that's how you accumulate the second oxidizing equivalent in the OEC. And if now you flash for the third time, the intensity of extra fluorescence increases. So what's going on? Any ideas? Somehow the cluster has been reloaded with electrons. Those electrons come from water that was found either from the myoxobruses or from terminal water molecules are now replenishing the electrons into the manganese centers. And that's why the fluorescence increases because now the manganese centers have more electrons. And that's the process of water oxidation. That's the process by which oxygen evolves leaving behind the electrons and reestablishing the redox state of the cluster in the most reduced form. And then the cycle goes on and on. So each time you go through that process you form one unit of oxygen. Now that rate, you see it has two features that are very interesting. One is the lag face. If you take a look at that lag face is between 100 and 200 microseconds. So for 100 to 200 microseconds nothing happens in terms of the oxidation state. It doesn't get reduced or oxidized. But after that the oxidation state of manganese is changed so that now we have more electrons. And that takes about a millisecond. Someone between one and two milliseconds is this process. So whatever mechanism we propose it has to be consistent with these kinetic data. And one aspect that was analyzed is the dependency with pH. So if you change the acidity of the medium in contact with and in the lumen whether you can change that rate or not the rate of one to two milliseconds. What you see is very very sensitive to the value of pH in this range. That means that if there is a deprotonation present and there must be a deprotonation at some point it has to happen after the oxidation or at the same time as the oxidation. It cannot happen before because if it happened before then the rate would be determined by that rate limiting step. One can slow it down by reducing the pH enough. And contrary to that the rate is independent of the pH. So that's one of the constraints that we have. So our work has been in the last year so it has been focused on that process and see whether we can nail this experimental data. So trying to understand the molecular rearrangements that lead to bone formation at the slowest step of the cycle and to explore whether there's three 2S4 transition involves like the S2S1 deproton capital deproton transformation or if it is an electron transfer followed by deprotonation. And whether the mechanism that is most consistent with experimental data is this terminal water mechanism that we proposed or the oxyloxal mechanism. Our proposal for the oxyloxal for the terminal water mechanism was previously suggested with model systems by Vincent Pecoraro, Gary Bradwick and Jim Barber in different times in different parts of the study of photosystem 2. And we found that mechanism to be most consistent with our QMM models that happen to be validated with this wealth of experimental data including X-ray absorption. So these are some of the transformations that we found but in a nutshell the main message that I want to emphasize is that the lack phase that lack phase that has to do with that is about 100 to 200 microseconds before formation of the oxygen molecule is ascribed here in our mechanism to deprotonation of an OH-Ligan of the dangling manganese that forms that oxyl radical that is susceptible to nucleophilic attack by water molecule. And that's why it is pH dependent that lack phase where it's independent of pH is the O-bond formation. So this is the nucleophilic attack of the water molecule and the oxyl radical forming a hydroperoxy intermediate and deprotonating this water molecule to the sundial ion. So it forms the sundial ion so there is a proton transfer concerted with the O-bond formation. And the rate at which this O-bond formation takes place is determined by the energetics. Here is the free energy profile of that O-bond formation process. And as you see you have a free energy of activation of about 13.2 kcal per mole that is consistent with a millisecond time scale. So this process is consistent with the kinetic data from X-ray fluorescence. We could also compute the changes in this free energy profile by changing oxygen 16 to oxygen 18 that is called the kinetic isotope effect. How much the barrier changes when you make a heavy water attack the oxyl radical. And what we find is that there is a kinetic isotopic effect of 25 per thousand, which is very consistent with the experimental data not yet reported but recently measured by Bradwick and coworkers. So that is another test that is consistent with his mechanism. And third, he has a deuterium isotope effect because as I mentioned, there is deprotonation formation of that zandelion. So also the deuterium isotope effect is 2.5 in experimental data, our calculation is two very much consistent with one another. Now in contrast, so here is something to note is that these proposed intermediate structure form upon deprotonation, the first intermediate, should be detectable by vibrational spectroscopy. And a similar structure was proposed by Mark Johnson and coworkers for clusters of waters. So it's not an unreasonable structure to suggest as a way of stabilizing that proton and an early state of the translocation of the proton to the lumen. Now, the issue of the kinetic isotope effect has to do with the doll effect. I'm sure that you're all familiar with the doll effect, which is that the isotopic composition of oxygen that we are breathing is different from the isotopic composition of water in the ocean. There's more oxygen 18 in the oxygen we breathe than the isotopic composition of water in the ocean. So, and the reason for that is that the process of respiration consumes oxygen 16 faster than oxygen and that's why we enrich the atmosphere with oxygen 18. Photosynthesis shows no kinetic isotope effect. So the ratio of oxygen 16-16 versus 16-18 is one. So it doesn't distil oxygen 18 or oxygen 16 relative to the isotopic composition of the water that is provided as a substrate. But that is under normal conditions because the rate limiting step, as I mentioned, is the acceptor side, that quinone that is being exchanged by a fresh plastic quinone. So that's the rate limiting step. So the process of all bone formation might or might not have a kinetic isotope effect. But it's not detected because that's not rate limiting. So what Gary has done recently was to see whether we could measure the kinetic isotope effect index in the donor side where the all bone formation is formed. And so what he did was to increase the concentration of that plastic quinone. Because usually we have eight plastic quinones per PS2. So what he did is to increase it to 400. So he puts a lot of electron acceptors. So now the acceptor side would not be rate limiting. And also he slowed down the deprotonation by reducing the pH. And when you do those two factors here, you increase the concentration of the electron acceptor and you reduce the pH, you start seeing a kinetic isotope effect of about 25 per thousand. And that's fully consistent with the measured kinetic isotope effect that we reported. Now, how does the oxyloxyl captive mechanism work? In that case, we have, that is just the all bone formation of the oxyloxyl radical with the oxo bridge that has a kinetic isotope effect of 40 per thousand. So it's much higher. And there is an inverted deuterium isotope effect that is inconsistent with experimental data. So for the first time, we are able to favor one of the two proposals for the all bone formation by direct comparison with kinetic isotope effects and kinetic data. And this is the summary of the data I just described. And with that understanding, now we can start analyzing biomimetic synthetic models that operate under the same principle. So one of those models is a center, is the manganese terpidimer that was reported by Encrubtri and Bradwick in 1999 in science, which is able to split war and form oxygen once the manganese centers are activated with a primary oxygen with something that extracts the electron from it. In that case with oxon, the primary oxygen responsible for activation of the manganese terpidimer. As you see here, you have a manganese, two manganese centers with demioxa bridge very much like in the OEC. And what we found computationally is that the mechanism responsible for all bone formation driven by this complex also involves an oxyl radical just like the dangling manganese and a nucleophilic attack of the water molecule into oxyl radical just like in the natural system. So that's why this complex we thought is truly biomimetic mimic of what this is done to. This is, these are the intermediates that can be computed with Gaussian for that manganese terpidimer showing that in fact the intermediate is an oxyl radical and that that oxyl radical is attacked by water to form the hydroperoxy intermediate as we propose in the oxygen evolving complex that leads to formation of the superoxy that then forms triplet oxygen that is what we breathe. So one aspect of these calculations was also to reveal that one of the steps in the cycle is manganese two. As I mentioned that manganese two is very unstable because it doesn't have affinity for the ligands. So these catalysts are very fragile and both radicals is a fabricier. So you touch it and breaks, right? Perhaps that's another lesson that we should learn from nature and from biomimetic models of natural systems which is for these catalysts to be functional to be able to catalyze water oxidation they have to be very unstable. In fact, they always see in plants gets a reform every 20 minutes inside of the inside of the telecoid memories of green plant chloroplasts while the pigments survive for the whole season. Now they're coming off the trees but the whole there. So instability leads to kinetics, favors kinetics versus thermodynamics and somehow manganese centers with ligands that are very labile are good for this process. So the turnover numbers for this catalyst is only three or four. So it goes three or four times if it breaks into pieces and has to be reformed. One solution to that is to put a high concentration in excess of ligand in the solution so that it can reform very quickly. But another aspect that is very important is the functional role of ligands. So here I showed that there's an acetate ligand that is not in the original formulation of this catalyst. But it happens to be that the catalyst is shown to work much better when you put in an acetate buffer. And what we find is that the acetate moiety replaces one of the water molecules into a manganese center. And that's two things very better. It reduces the oxidation potential for that center so it can be oxidized more easily. And also it works as a buffer so it can pick up the protons that are extracted from the water that is undergoing the nucleophilic attack. So those carboxylate residues are holding the OEC in place, might be serving the same role, might be stabilizing the structure of the OEC and might be facilitating the oxidation of the manganese centers. And the carboxylate residues in the second coordination sphere might be responsible for picking up the proton that has to come off the substrate water molecules in the process of deprotonation towards the lumen. Both of those design principles should be, again, exploited by artificial and synthetic models. What we have shown is that these manganese terpidimer can be bound to TiO2 surfaces. This is another QMM model. And we should still operate well. We found that the elbow formation does go well with this arrangement. And that motivated my co-workers and colleagues to work on this design, to actually try this in the lab. And in fact, they were able to stabilize the manganese terpidimer on TiO2 surfaces. And when activated with a primary oxygen, in this case, was cerium. That doesn't work for the homogeneous case, but it works when it is bound on the surface. It still works well and generates a lot of oxygen. So the catalyst remains active when bound to TiO2 surfaces as predicted by calculations. What we haven't been able to do is to induce this oxidation of water with the catalyst bound to TiO2 surfaces with light. We're able to do it with a primary oxygen like cerium, but we still have not been able to do it with light. And that is still a challenge that I want to talk a little bit more about. But what I'm showing here is the moving of the evolution of the wave function, the distribution of charge. This is the electronic density. Right after photo excitation of that complex, the manganese terpidimer bound to the surface. Let's see whether this one can play again here. It is right after photo excitation. The electronic density in gray is now being injected into the TiO2 surface. And you see here the d-orbitals of titanium picking up that electron leaving an oxidized terpiligand. That is the same thing as the oxidized chlorophyll and the special pair of chlorophyll erasives. The electron, instead of going to fear-fighting QA and then QB, it has been injected into TiO2. But it's leaving behind an oxidized group just like a chlorophyll species that can oxidize an oxomanganese core. That is like the OEC in natural photosynthetic systems. So this is an artificial model of what actually happens inside of a green leaf. And what this calculation show is that the timescale for this process of electron injection into the TiO2 surface is ultra-fast. So it's in 100 femtoseconds, 10 to the minus 15 seconds is 100 femtoseconds. In 100 femtoseconds, the process is over. So the electron is all injected. And that's exactly what you want to have in ultra-fast process of charge separation like in the primary process of photosynthesis where you translocate an electron from the special pair into fear-fighting in about two picoseconds. I think that photons rain from the sun at a rate of 2000 photons per second per nanometer square. That's under normal average solar radiation. That means that you have to wait for about a millisecond. If you have a nanometer square where you have one of these molecules sitting on the surface for the next photon to strike. So you don't want the process of electron injection to take milliseconds because you'd be wasting photons. But what you show here is that that would not happen with this arrangement. This is very good for that kind of processes. And in fact, these kind of models have already been exploited in solar cells and they're called the Gretzel cells where you have similar kind of transition metal complexes bound to TiO2 surfaces. And TiO2, you might be familiar with the TiO2. If you like M&Ms, the M&M is written with TiO2. That's why it's white. And TiO2 doesn't absorb visible light, it absorbs only in the ultraviolet. That's why it's white. It just scatters light. And it's non-toxic, it's cheap and it's natural. So this is a great material and it also has great porosity. So it has a lot of surface where you can put lots of these molecules attached to it in an electrode. So that's another design principle. So I'm running out of time but I want to show you just a couple of slides of these experiments. This is the photocatalytic cell that is operating in Gary Bradwick's group with these electrodes designed with those principles. This is in the pink TiO2 is when you decorate them with these complexes of M&M centers, it becomes pink. And when you shine light, here's the emulator of a solar spectrum coming through the fiber optics, hitting the photo-ionode and here the complex is oxidized. And what we're running here is the oxidation of isopropanol into acetone. That works. What it still hasn't worked is to replace isopropanol by water so that to generate oxygen instead of acetone. And the reason for that, we think, and here's the photo-current generated as a function of time when we turn on the light, we establish a photo-current and we switch off the light. Photo-current disappears when you switch it on back. It is re-established. So it's clearly light driving this reaction. And the electrons that are being collected on this other side, they're generating hydrogen, which is a fuel. So this is converting solar light into fuel from, in this case, extracting the electron from isopropanol. This is not a good design because you're consuming a chemical. So you're consuming a fuel. But if that chemical happens to be water, that would be ideal because you can consume water and when you make hydrogen react with oxygen, you regenerate that water. In this case, if you make hydrogen react with oxygen, you would not regenerate isopropanol. So you would be good if these are fuel cells that where hydrogen would be used to reduce acetone into isopropanol. And that would be a design of fuel cells that would make good use of solar energy in this way. But there are still challenges in terms of the membrane and catalyst for hydrogenation of acetone that could exploit this principle. But the reason why this doesn't work with water, and this is where I would like to stop, is the following, that for two electron reactions like this one, everything works well because the first photon comes in, photoexcites the ligand, injects an electron, and that oxidized ligand oxidizes the manganese center, evolving it from two to three. Then that happens again, it goes from three to four. The third time around is when things get difficult because the photon comes in, excites the ligand, and then the photoexcited electron has the choice of getting injected like a previous two, or going back and reducing a manganese four center. And as you know, manganese four center is thirsty for electrons, they might attract that electron. So the electrons go in the wrong way and undo what the previous two electrons have done. Electrons have done, so reduce the center instead of advancing the oxidation state of the center further. So what we need here is to induce directionality of charge transfer to prevent the electron from going in the wrong way. And so we have to design molecules that enable that directionality of charge transfer. As I mentioned, in photosystem two, that is enabled through the redox gradient. So the redox cofactors that are positioned, strategically positioned so that they pick up the electron going downhill in free energy. But here we don't have that, and we don't want to do that either. We don't want to copy all of the aspects of photosystems. We don't want to waste voltage. What we wanted to design molecular rectifiers that enable directionality of charge transfer without losing voltage. And that is possible, and I'd be glad to discuss what the work we have done in the design of those rectifiers. But with that, I would like to stop here by first showing a picture of my colleagues. This is Bob Crabtree. We've been talking a lot about Bob today. We're the students. He's a great colleague and extremely inspiring and true scientist in all possible ways. It's a very big, fantastic colleague as well, as Charlie Schmuttmeier, with whom we've been working for more than 13 years now on this project. And these are some of the students and postdocs who work on the artificial photosynthetic materials. They are responsible for most of the ideas and the work that resolve the difficulties that we face. And for that, we are extremely grateful for their commitment and her thinking and innovation into this project. And here's Misha, Crystal, and Kaa, who are responsible for all the work, all the most recent work on photosystem 2, the natural system. And with that, I would like to thank you again for your invitation and be glad to entertain questions. Yes, so here's one specific application that would be revolutionary. If we could generate hydrogen in this way by using water here as the source of electrons and protons, then we would be transforming water into fuel. And in fuel, that when the fuel could be put directly into a fuel cell in a car and run an electric car or could be used for hydrogenation of liquid so we could transform oil into butter or we could put it into a liquid that is been loaded in the tank of the car and send to a fuel cell where it gets dehydrogenated and again generates electricity for running the car. So in a way, we are transforming that energy, solar energy into chemical energy to making bonds. And it's wonderful because it doesn't pollute the atmosphere, doesn't increase the carbon footprint, simply because when hydrogen reacts with water, sorry, with hydrogen reacts with oxygen, it generates a lot of energy and water. So water is the outcome of that combustion of hydrogen, if you want to call it a combustion. And so you see a lot of smoke, but it's just steam, right? And so that would be one way of making use of this technology that would be really transformative. So if we imagine that formulation of what we call the virtual hydrogen technology, where you have fuel that you load into your car and gas stations, but instead of being the normal fuel, it's this fuel that is some liquid that is hydrogenated. For instance, benzene that happens to be hydrogenated and making it cyclohexane. So what you load is cyclohexane. That is what goes to the engine, gets dehydrogenated and makes benzene. And as a result, the protons and electrons that I extracted from cyclohexane have been used for running that fuel cell, reacting with oxygen, and therefore generating electricity. And then all the benzene that is produced in that way can be dumped in the gas station. You can put more cyclohexane. And you never pollute the environment. You see, you only have to rehydrogenate it. Or perhaps you can plug the car and you can hydrogenate benzene in reverse by running that in reverse. So it would be a technology very similar to the one we have now, but with fuels that don't burn. They just get hydrogenated and dehydrogenated. And there's a wide range of applications along the lines of that technology. But bottom line is that we want a sustainable approach in the sense that we don't want to be consuming chemicals, like we are consuming oil now. With this technology, we extract the electrons from water, but we generate water again. So we never consume a chemical. So that's why we call it a sustainable approach. And also, the amount of CO2 in the atmosphere will remain the same because it would not increase in the carbon footprint. There are alternative ways of doing that, but this is one of the viable solutions. We have a good, yeah, that's a great question. So the efficiency of plants is very low. So most of the energy is wasted by plants because plants evolve by responding to an evolutionary pressure. So long as they outcompete, the competition, they survive. And so most of the photons, and that's where they have a lot of leaves. And so they capture the photons. But not all of the photons are being used for growth, but nothing grows under them. So that's a little bit that not every single step in the process of photosynthesis has been optimized to its limit because what is being optimized is something beyond that process, which is the evolutionary aspect. So less than 1% is the efficiency of, if you measure the biomass that is generated based on the amount of energy of the photons that have been absorbed by those plants. So we think we can do much better than that. And in fact, Jacob, which was one of the centers in the California, and focused on this problem, had set a challenge of producing in the next five years 10% efficiency, 10 milliamps per centimeter square of electricity. So that is achievable. Whether there is an ultimate efficiency limit, depends a lot on the application. So same thing with solar cells. But the aspect that I think is most important is the design principle based on earth-abundant materials. So like manganese that is abundant and is not a precious metal, and that can be scaled. And so you can produce a lot of that. And perhaps you don't have to go to the ultimate limit of efficiency that is necessary for making it transformative. Now both are excellent questions. So something that I, when I presented the process of initiation of photosynthesis, I stopped here in PS2, right? So apparently someone took a biochemistry class here. And knew that there was a PS1 down the hill because now PS1, unfortunately it's called PS1 where it comes after, but this one was discovered first. That's why it's called PS1, and this one is called PS2, but photosystem starts at PS2. So the conjunction of, so both of them are necessary to have enough free energy gradient here for driving ATP biosynthesis. So what it's called, there is indeed a synergy between the two protein complexes. Through a mechanism that is called a Z-scheme. So because it's like a Z, a letter Z, here it goes up, down, and then up again. There's a Z that is 90 degrees rotated. In the first part of the Z is one process of photoactivation. So it's one photo and it's absorbed here. Then it loses a lot of energy, goes down hill, and here another photo is absorbed. So it's a two-photon process that leads to enough pumping of protons from the stroma to the lumen as necessary for having enough driving force for ATP biosynthesis. So Z-schemes have been a challenge that many people have been trying to develop as biomimetic versions of photosynthesis for the last five to 10 years. And it might be one of the keys to this process of both oxidizing water and reducing CO2, for instance, on the other side. Because doing both processes on one center, let's say on one TiO2 functionalized with complex, might be too difficult. So it could be that on one piece of TiO2 with complexes, one oxidizes water and transfers the electrons to the other one that also absorbs the photon and creates enough reducing power to reduce suffering. Perhaps TiO2 on the other side is reduced to methanol. So if we want to both fix TiO2 and oxidize water, maybe we have to design models that mimic that Z-scheme of photosynthesis. Now, your question, I think, might also have pointed at another possibility, which is to engineer plants, engineer perhaps PS1 to produce fuel. And that's another line of research that people like John Goldbeck and Penn State are exploring. So what they're doing here, one of the functionalities of PS1 is again absorb light, separate charge, just like I was having PS2 and transfer those electrons all the way to freodoxin that is the way that electron is used for a reduction process. So one approach is to shortcut that electron transfer chain with another electron acceptor here, which might be a little piece of a metal cluster or even a polymer that is attached to PS1 that has a catalyst that enables formation of hydrogen from reduction of protons. So that by shortcutting PS1, we might be able to place there a wide range of catalysts that produce fuels. So these will be plants or algae that would generate hydrogen in addition to generate oxygen. So they would generate a mixture. And other schemes like Danosera recently has been trying to overexpress hydrogenases. So which are enzymes that would be able to generate hydrogen. So that's yet another approach that could be done without making any kind of materials. It is just engineering, modifying natural systems to produce fuel in a slightly different way as we are thinking along the lines of semiconductor materials functionalized with molecules.