 Hi, are you ready for today's class on photosystem 2? I hope so. So, let us look at the photosystem 2. In the last class we have discussed that reduction of molecular oxygen takes place at heme copper oxidase and that is the cytochrome C oxidase. And also we were mainly discussing the water oxidation by which two molecule of water will come close together and form the oxygen molecule that is in photosystem 2. Oxygen evolving center is involved and 4 manganese 1 cluster is involved right. These two reactions are almost microscopic reverse of each other right. Oxygen going to water and water coming to oxygen this is what perhaps one would dream of in their wildest life right. So, this enzyme which is involved into water to oxygen is called the oxygen evolving complex OEC or OEC for the center right. Four manganese center one calcium is involved for such transformation and we have seen earlier that cytochrome C oxidase can be converted to converted to water from oxygen right. So, overall these two are very important enzyme over and they are involved in multi electron processes. Now the crystal structure of OEC is very complex as you have discussed in the last class, but major interesting point for us is this OEC oxygen evolving complex of oxygen evolving center. This structure has many problems, but it can all give rise to a situation where all these steps are happening very happily perhaps. And the crystal structure does not give the clear evidence or clear indication what exactly the manganese structure is, but these are many different proposition that exist in the literature all of them are valid, but it is going to be only one center that is involved what that is I think nobody knows for sure 100 percent. Now, these are the one or these are the structure which got support from the exact study. If you look at the crystal structure in that science paper this is the center where you see the four manganese center 1, 2, 3, 4 are situated as is here calcium is bound in between, but more importantly this structure people believes that is not the right structure because X-ray is damaged damaging these manganese calcium and manganese oxygen bonds right. So, nonetheless although this is not reliable data or reliable structure for four manganese 1 calcium, but still people believe that it would be some combination of these species that is involved into the oxygen evolving center. Although this structure is quite interesting, but we have to keep this in suspense right now. But one thing for sure throughout the literature what we learned is S0 the fully reduced form will be oxidized to S1 and then to S2, then to S3 and S4 in each step one electron one proton is involved and overall the fully oxidized S4 oxidation state will be converting water to molecule of water into oxygen and this is where mainly we were interested in learning what happens at this S4 so that water can be converted to oxygen right. If you look at this S0 to S4 overall catalytic cycle you will see that manganese oxidation states are varying and these are the two possible assignment for S0 this is the single possible assignment for S1, this is also the single possible assignment for S2 and then these are the two possible assignment for S3 and subsequently S4 which is the fully oxidized form. More importantly 1, 2, 3, 4 these four steps are light dependent or photo dependent the final step is photo independent. Many different studies or many different spectroscopic techniques such as EPR, FTIR, ZAS, UV visible studies are employed for characterizing these intermediate ok. So, today let us try to see what happens to these intermediates, what are the oxidation state, what is the proposed intermediate for these water to oxygen formation well. I think one of the key step that we are interested in is the oxygen-oxygen bond formation because if water has to give oxygen there would be a step where OO bond formation takes place oxygen-oxygen bond formation takes place. And it is believed and with some evidence that this is the calcium hydroxide which is the nucleophile here and the manganese oxo centers can be acting as the electrophile. For instance as you see in the right so the calcium hydroxide which is arising from water molecule and this oxo which is also arising from water molecule will see the mechanism of how water is giving rise to oxo in the next slide. So, this calcium hydroxo can attack on the manganese 5-oxo bond to form the oxygen-oxygen center or oxygen-oxygen bond of the dioxygen molecule ok. It could be in this form or it is a radical form as it is shown over there manganese 4-O dot. Alternatively, calcium hydroxide can be attacking a bridged oxo species as it is shown over here and then form the oxygen-oxygen bond. This rearrangement can be between the 3 manganese 1-oxo or 2 manganese 1-oxo. See the clarity at 100 percent level or for a synthetic chemist level for a bioorganic chemist level are not really there. So, therefore, we will discuss the possibilities and we will assume some of the possibilities to be actually happening and then move on from there. This is the model from the cyanobacterial photosystem to crystal structure, the crystal structure we have seen in the science paper well. Many different studies are known right this is one by Yachandra et al. The proposal simply is that part of the 4 manganese calcium structure remained intact throughout the catalytic cycle only 2 manganese center is varying the oxidation state and therefore, they are involved into the oxygen-oxygen bond formation process. The part which remained completely constant is shown in the bottom in here. If you look at S0 to S1 to S2, S3 and S4 this part remained constant. So, in all the cases it is a dimanganese 4 bis-meoxo intermediate, dimanganese 4 bis-meoxo intermediate see dimanganese 4 bis-meoxo intermediate. The part which is varying is this dimanganese with a lower oxidation state. To start with it is proposed by Yachandra et al along with the experimental evidences that it is S0 where we have a manganese 2 and manganese 3 center and this is the aqua molecule that we are talking about and this is another water molecule or aqua molecule which is with calcium. So, the oxygen-oxygen bond will form between these two oxygen center. This manganese 2 plus will be oxidized by photon to give you the oxidized manganese 3 plus a proton and electron release from the center will gives rise to the manganese 3 aqua complex. This OH bridging hydroxo can give rise to the H dot as is in here overall H plus and electron and then this electron that is getting generated O dot is getting generated that can be reduced further by the manganese center to form O 2 minus. So, that is going to be the oxo. So, manganese 3 manganese 2 oxo hydroxo species becomes manganese 3 manganese 3 dioxo intermediate. So, these are also dioxo the only difference is these are in manganese 4 plus oxidation state over here this is a dimanganese bismi oxo species. So, oxidation state is only varying, but remaining things are the same. At this position or at this point yet another electron oxidation of this score will happen or will take place where this manganese 3 will now be oxidized to manganese 4 plus. Now, this is a mixed valent center manganese 3 manganese 4 bismi oxo intermediate water remains as it is calcium hydroxy remains as it is. Consequently from this S 2 state as you see S 0, S 1 and S 2 all of them are spectroscopically characterized specifically if you are multi-line spectrum will be informative although very complicated. Now, this manganese 4 manganese 3 center as you can see over here can undergo further one electron oxidation reaction in presence of light to give the manganese 4 hydroxo center. So, this H dot from this aqua molecule can come out H plus an electron in the form of H dot or overall can come out to give rise to the HO dot which can be converted to HO minus by transferring one electron from the manganese side to give manganese 4 HO minus. So, this is where manganese 4 HO minus is there and therefore, overall you see we started with manganese 3 manganese 2 then 3 3 then 3 4 then 4 4 overall manganese di or bismi oxo species with di manganese center is forming along with that there is a hydroxo which is essentially is ready now to form perhaps the oxygen-oxygen bond not yet not yet right now it has to be oxidized further. So, it can gives rise to again an H dot H dot will leave MN4 O dot which is nothing, but oxidized form would be manganese 4 is giving one electron to O dot to make it O 2 minus. So, oxo 2 minus and manganese 5 oxo that is forming. So, this manganese oxygen S double bond can be formed right over here right. So, at this stage it is believed that this hydroxo which is sitting very close to this manganese oxo now can attack this nucleophile can attack on this nucleophile to form the oxygen-oxygen bond and therefore, overall 2 water molecule is giving rise to the oxygen and proton and this overall process will convert S4 to S1. So, what you have seen right now is quite fascinating reaction where these manganese centers are involved into the oxygen-oxygen bond formation reaction. Not all of the centers apparently are involved in this process, it is the terminal manganese oxo bond formation which is the key and subsequently calcium hydroxide or calcium aqua complex will be giving rise to the desired oxygen-oxygen bond formation to give you the molecular oxygen from water. What an effective reaction it is, you see it is fascinating as I said this core remained completely constant throughout the catalytic cycle only this is the core or only this is the center which is getting oxidized manganese 2, manganese 3 we have started with in the final oxidized form it is becoming manganese 4, manganese 5 oxo right. So, this is the change only change that you see is this manganese 4, manganese 5 oxo and that is happening quite easily right. So, what you have seen over here is manganese 3 and manganese 2 over here you end up having manganese 4 and manganese 5 center along with the oxo and this oxo is the one which is responsible for formation of the oxygen-oxygen bond. As you have noticed this manganese 2 first goes to manganese 3, this manganese 3 remain constant and then these manganese 4 center is happening. So, and finally, and then another water H atom transfer gives rise to the change in this manganese center finally, that is the again the manganese center which is undergoing. So, overall in this step if you see this is the center terminal center which is responsible for the oxidation almost all at all the step except in one step the second manganese is involved. So, if you are looking at the overall crystal overall structure or overall chem draw of this proposed mechanism then you will immediately realize that it is this center which remained completely constant or almost constant except one time oxidation from manganese 3 to manganese 4, but otherwise this is the center which is the one responsible for chemistry. So, manganese aqua complex is converted to manganese oxo complex. Now, I would say that is a really fascinating example what we are overall saying then is well it is transformation into manganese oxo. So, we are starting from manganese aqua complex overall with steps manganese 2 aqua complex this is now forming manganese 2 oxo well this is quite a phenomenon right. So, you can break this OH bond you can break this OH bond overall you can make it let us say manganese OH minus or dot. So, each step these manganese 2 let us say was there and then it can keep on getting giving one electron to form a bond or one electron to make it hydroxide overall it will reach to manganese 5 oxo when we are trying to trying to form these manganese 2 to manganese 5 oxo bond formation this proton and electron transfer will be the key transformation in these cases right. So, let us look at some other proposal that is existing over here let us look at in this you know quite interesting another yet another proposal where it has been discussed by Blondin et al that this is actually the tyrosine radical which is from tyrosine phenol unit is involved into the overall manganese oxo species formation. For instance these manganese 4 hydroxo that is generated into the process. So, we are looking at manganese manganese 4 4 oxidation state oxo bridge this as I said this remain constant and the terminal case is over here this is where manganese hydroxo is forming and then of course, there are other ligand involved histidine is also involved this manganese hydroxo is the focus this manganese hydroxo that H dot formation is facilitated by or O dot formation is also is facilitated by this tyrosine radical. This proposal is I think quite interesting in a sense that this gives rise to a direction from to where the H dot will be transferred this phenoxy radical formation is quite seemingly quite exciting for the generation of manganese oxo radical. Overall this process helps you in forming the manganese 5 oxo intermediate from where calcium hydroxo will be able to attack electrophilically on this oxygen atom ok. And therefore, we will see that the oxygen-oxygen bond forming process will take place to give rise to the dioxygen molecule right. So, that is quite fascinating and we will be keep seeing these mechanistic details or change of the proposal from different groups and all these studies are mainly based on the enzyme. The synthetic studies are also done quite a few quite a lot, but the problem is without knowing the enzyme structure properly or without having a clear cut manganese for calcium structure. I think it is getting very difficult to assign what type of chemistry is happening in the enzyme and therefore, trying to mimic this enzyme is always much more challenging. But once again these has to be understood at a molecular level if the mankind has to understood the biological processes and our very existence I think these processes at a molecular level should be and must be addressed. Let us look at some more proposal. So, once again these are many different proposals exist in the literature for which we do not have a crystal clear idea although the crystal structure exist, but the reliability of those crystal data can be questioned because the extra damage of those crystal structure. Now, more importantly these studies are mechanistic proposal are based on some of the experimental observation in terms of x-ups, in terms of UV visible, EPR and FTIR studies and therefore, one can think perhaps the all or you know all of these mechanism might have relevance in the case of the ever important these oxygen oxygen bond formation reactions ok. The fact that even the tyrosine radical is involved or believed to be involved in such process makes it even more exciting ok. Let us look at a proposal which is based on the x-up study. Here again we are going to look at S0, S1, S2, S3 and S4 without detail characterization. These data are based on or these sort of proposal are based on the x-ups measurement on manganese complex of photosystem 2 in 4 estates ok. So, these are the 4 estates that is referred over here. Now, if you look at the manganese manganese distance between these sites are 2.8 angstrom right. So, and the second manganese to third manganese distance is 3.0 angstrom ok. These are part of the cluster we are looking at part of the 4 manganese 1 calcium cluster we are looking at and that is the site where water binding water activation and the oxygen-oxygen bond formation will take place as we have previously said this score remained somewhat constant throughout this process. Now, this overall S0 state can then be oxidized or hydrogen atom leaves this center overall to make it a S1 center where once again 2.8 angstrom distance is only changing up to 2.7 angstrom these x-ups measurements are can be quite accurate. The distance between these 2 manganese center can be very critical and as you see only very slight change in the manganese manganese distance take place. Subsequently, this is the second and third manganese distance is turning out to be 3 angstrom which is remaining constant even after this electron transfer and proton transfer processes. This is quite phenomenal because one should understand that this sort of electron and proton transfer does not make much of the change in the core structure and that is what is observed by the x-up study. We can further oxidize this center by one more electron wherein you see that dimanganese center is isolated by 2.7 angstrom and the third center is separated only by 3 angstrom as you see in all other cases. From this S2 state it is also possible to remove yet another electron as you have seen earlier, but in this case you will see the manganese manganese this center and then second and the manganese third center distance between them decrease significantly from 3 angstrom to 2.7 angstrom. These are little greater than 3 angstrom or greater than 3 angstrom and then these are going to be 2.7 angstrom. As you see now these manganese distance and these manganese distance become equal. So, this is the S3 species from which the fully oxidized S4 oxidation state will be created and then water will be converted to the oxygen molecule ok. So, in the next slide as you will see that there is other proposal by which people believe that these oxygen-oxygen bond formation is taking place. So, the mechanistic proposal by Dow and co-workers shows that a complete or little bit different structure as you can see these we are looking at here the manganese S4 oxidation state or S4 state where manganese 4 and 1 calcium structure is there. Now, they according to this proposal this is going to be the reaction between these manganese 5-oxo that the one we were discussing two slide back manganese 5-oxo and bridged by these dimanganese di-oxo intermediate. These manganese di-oxo intermediate actually is participating in activating the OH bond unlike what we were saying that this is the calcium structure that is involved, but this is the aqua molecule which is getting involved not the calcium hydroxo, but the aqua molecule is getting involved it in this stage it abstract hydrogen atom from this water molecule slowly, but steadily ok deprotonation and electron transfer overall occurs to gives rise to the manganese manganese manganese this dimanganese center with an HO dot radical right. So, this manganese center will first then create a manganese 4 and O dot which is nothing, but manganese 5-oxo no problem in there, but most importantly this aqua molecule is interacting with this bridged meoxo species. Now, this bridged meoxo species will subsequently give the hydroxo radical this hydroxo radical upon removing one H dot. So, this hydroxo radical and manganese oxo radical will then combine to each other to form the oxygen-oxygen bond. As you can see this is a manganese 5-oxo which is nothing, but manganese 4 O dot that is fine, but these H dot transfer will reduce the manganese 4 center to manganese 3 and then hydroxo intermediate these electron transfer from H dot from this OH bond to manganese center will make it manganese 3 plus O dot and this O dot and this OH can then combine to give the O OH which is then can undergo an OH bond cleavage or activation to give the O O dot. This O O dot then can then can then can form the pi bond as is shown over here and then gives rise to the S 0 step. So, overall in this slide we were looking at the details of the oxygen-oxygen bond formation step. Here how S 4 is forming S 0 is considered and it is quite amazing that that step wise mechanism can be suggested so nicely, but without involving the calcium intermediate. Once again these proposals although remain quite exciting all these proposal, but there is a missing consensus in the community that which is the real you know mechanistic understanding for these oxygen-oxygen bond formation process that one still really still need to discover and convey to the scientific community. So, overall what we have seen so far are little variation of the reaction mechanism, but nonetheless it is the manganese terminal oxo center which is involved into the oxygen-oxygen bond formation or the bridge manganese oxo species involved in the oxygen-oxygen bond formation which is the critical step in the in the in the formation of the oxygen molecule from the water molecule right. So, water is forming the oxygen dioxygen molecule with the help of this manganese oxo the details of this procedure still remain unclear. These these sort of studies or these studies both in the enzyme as well as the DFT studies and in conjunction with the the the mechanistic studies as well as modeling studies from bioinorganic chemists are quite crucial in setting light into this highly complex intermediate and I think the study has must be go must be going on to better understanding procedure. I will not discuss this one this will be there for you to further diverse into it. So, this is another study based on that crystallographically model of Freira that science paper it is little more complicated let us not get into that. So, let me then once again summarize by saying that there are many different cluster that is proposed still some of them perhaps can be ruled out, but many of them cannot be ruled out and so are the many proposals that are existing in the literature still cannot be ruled out there exist little bit variation among these proposal, but nonetheless it helps us understand how perhaps the oxygen-oxygen bond is forming in the experimental studies or in research you do not have to understand everything right on day 1. Sometime it takes decades sometime centuries to completely understand a mechanistic proposal sometime perhaps it would never be understood because these reactions are so fast understanding each and every intermediate becomes next to impossible, but efforts are on many synthetic studies many experimental studies both in enzyme agile and synthetic setup are on the computational studies are paramount effect having great contribution in this field to better understand this oxygen-oxygen complexes or oxygen evolving complexes and the oxygen-oxygen bond formation reaction. So, we will come back with more discussion on these and mainly we will be next summarizing what we have learned so far in this topic. Thank you very much see you soon.