 Hello, welcome to metals in biology. Today's topic is photosynthesis. You will be amazed in knowing how great this reaction could be where we will be converting water into oxygen. Now, overall if you see the all the transformation perhaps two reactions stands out of course, there are many reaction which stands out including the methane to methanol. Perhaps two reactions which are complementary with respect to each other and they are very much fascinating and that is oxygen to water formation as well as water to oxygen formation. Life comes to a circle I would say. So, reduction of molecular oxygen to give the water and water oxidation to give you the oxygen I think are the two most important processes that is keeping us alive. So, it is happening these oxygen to water is happening at mitochondria what where you have seen the cytochrome C oxidase, the heme copper oxidase and the intricate details of it that you have seen how oxygen is converted to water by the heme copper oxidase which is quite fascinating. This is the mitochondria where things are happening the cytochrome C oxidase heme copper oxidase is getting involved in there to convert oxygen into water. On the other hand in chloroplast ok in plants we have this photosynthesis where you have photosystem 2 the oxygen evolving complex we will see in a moment manganese 4 calcium compound or the structure cluster will be involved in the water oxidation. So, water will be converted to oxygen at chloropast over there. These are complementary reactions and these are right diagonally opposite reaction. These of course, requires 4 proton and 4 electron for converting oxygen to water and the reverse will be true for the water oxidation to make the oxygen molecule. These transformation require both these transformation require multiple metal center. So, that is quite fascinating you see the metal is everywhere of course, more than 50 percent of our body weight is metal, but more importantly wherever there is a difficult reaction or difficulty arising nature has always trusted on metal. Metal is the place to go when you have seemingly nowhere to go metal makes the reaction feasible. Metal play multiple roles in these enzymes both I mean in this today's case let us say heme copper oxidase and the oxygen evolving complex chloroplast. Metal plays multiple roles such as it binds with substrate. Substrate meaning in these cases let us say oxygen or water. Increase reactivity of substrate of course, increase the reactivity of oxygen or water. In this case it would be water activity, water binding and water activity will be improved. Prevent site reaction which is important and essential because these biological processes are completely bullet proof right. These are the reaction which happens no matter what. This is one dimensional single way one way one product formation or the desired product formation only. There is no detour almost no detour nowhere and then almost no other site product formation happening. Of course, depending on the concentration of the materials things can vary in few cases that we have seen. For example, peroxidase, catalase cases we have seen, but otherwise it always prevents site reactions. Provides electron quickly that is one of the great thing about the metal center. Metal centers are used very cautiously and very carefully particularly wherever required to provide the electron. It can couple a multi electron process that is also also also important of course, at a time only one electron transfer occur, but it can provide multi electron process to several single electrons process. It can have multi electron transfer at a time or several one electron transfer at a time or at times. So, you will see in a moment that this OEC oxygen evolving complex will have 4 manganese center. You have already seen in the previous classes where cytochrome C oxidase you have iron copper site along with a phenol or tyrosine cross linking over there. So, these manganese 4 manganese calcium 1 calcium structure we will see today for the oxygen evolving complex or for the photosynthesis where water is converted to oxygen. The diagonally opposite exactly opposite cytochrome C oxidase confirms transformation of oxygen to water. Of course, another very important enzyme is this nitrogenase which can convert nitrogen to ammonia. These are fascinating enzyme, these are fascinating time fascinating transformations. We will come back to that in moment. Let us look at the crystal structure of the photosynthetic oxygen evolving center or oxygen evolving complex. This is back in 2004, this is a crystal structure that you see is reported many structures are present as you can see non-hem iron center, heme center and cytochrome center many different centers are present, many heme and many non-hem centers are present. Many structures are present like this sort of different structure is as you see the one in the green here is the one which is the photosystem 2 and this is a great structure for the photosystem 2. As you see this is the oxygen evolving complex or oxygen evolving center this is the chloroplast structure in green over there. Of course, this photosystem 2 dimer structure is existing, but it does not still provide the definitive structure of OEC. So, what happens here is this crystal structure which is phenomenal and highly informative, but still it cannot give you the resolution at a level of what a inorganic chemist would like to have perhaps right. These crystal structure are fascinating many different iron center heme iron non-hem iron center many different metal centers are present. Despite the presence of these many different centers we see that still it cannot gives the give the clear idea about what is happening at these metal centers and what is the metal what are the exact association of these metal centers ok. So, we will see this OEC oxygen evolving complex over here in a moment we will be coming back, but what has been known or what has been understood so far is these four manganese one calcium structure which will start at S 0 we will come back to that in moment also it is photosensitized to give you S 1 then subsequently S 1 can form S 2 once again it these are the subsequent redox reaction each of the step involved one electron processes. The catalytic cycle of OEC is right over here it is going through S 0 to S 1 S 1 to S 2 and S 2 to S 3 and S 3 to S 4. So, all these steps are dependent on light and it is in turn oxidizing metal oxo cluster in each step metal is getting oxidized further ok. Finally, at S 3 S 4 the catalyst or 4 manganese center one calcium center is getting ready to react with water to give you the oxygen molecule. The S 4 to S 0 that means, the final step where water is converted to oxygen is light independent this step is light independent and releases oxygen. This overall transformation is very complicated and intermediates all have been postulated not direct evidence I mean some evidences are there that we will see in a moment, but these are not 100 percent clean and clear informations, but most importantly as you know by doing or by converting water into oxygen this whole process is also coupled with the carbon dioxide to form the glucose which is the pretty important molecule for our for our existence right. Water to oxygen conversion is also coupled with carbon dioxide that is I think you are perhaps more familiar with carbon dioxide to the glucose formation this whole process are coupled and we will see stepwise what perhaps is happening over there. I think one thing for sure unlike many other enzyme that we have seen in this class this enzyme is perhaps the most complex and very little understood. It is not at least I mean in some it is understood well, but it is not up to the mark what we would like to have because the thing is these are very sensitive metal cluster that is involved that we will see in a moment. There are many proposition that what would be the relative disposition of these different manganese center, but no direct or no clear indication exist till that ok. There are proposals there are argument in favor or disfavoring one of the structure, but nailing down one structure has still been inconclusive I would say still lot of studies are going on these are fascinating structure both in terms of the enzyme study as well as the as well as the synthetic studies has been done quite a lot. So, the possible arrangement of the 4 manganese ion that we are talking are many and some of them are shown over here as you can see many of them are having the butterfly like structure and these are the structure in the box which are getting support from the X-ups measurement. So, still from the X-ups also many structure are proposed it is not really one definitive structure or one clear structure that can be proposed, but for you know the course purpose or keeping it simple we will slowly evolve into one structure and try to say that let us try to follow that rather than trying to follow many structure let us keep one in mind knowing very well that that structure may or may not be the correct one because there are many structure as you see the different permutation and combination is possible between the 4 manganese and 1 calcium orientation as at least if you are just focusing between 4 manganese 1, 2, 3, 4 manganese you can have all these different structure orientation bridged between by the oxide right. So, manganese and calcium also comes into the picture how they are bridged how they are helping that can be debated it is still debated no clear solution a once again exist for these beautiful enzyme and most effective one of the most effective enzyme that is converting your water into oxygen right that is fantastic. We will try to see what is understood what is evolving and how we perhaps will be able to understand it better in future. So, let us get back to the crystal structure that OEC crystal structure 3 slides back we have seen this is the crystal structure if you zoom down over here further if you zoom down in this region you see that these are 1, 2, 3, 4 these are the 4 manganese clusters that we were talking about this is the calcium over there. So, what is the debate about? Well although this is looking like a definitive structure people do not believe that that is the original structure what has happened during the crystallographic characterization or crystal data collection people believe that this cluster is broken and rearranged. So, or I mean you know this is not actually is the real structure. So, extra damage to the manganese 4 calcium complexes has happened already although this is a decent 3.5 resolution structure which allows the density modeling, but it is still not definitive ok. The miwakso bridges that is present over there and the hydrogen bonding network are not really well resolved and this remains one of the most debated structure most debated active site so far. So, the structure perhaps that we can move along with is this one 1, 2, 3, 4 these are the 4 manganese bridged by these 4 oxo and also it is shared by the calcium. These are the 2 references you can study which are which are quite informative. In any case, but still the debate remain what would be these 4 manganese doing and of course, their role is little understand, but how they are relatively positioned you see these as if like these are dimanganese dioxo structure. There are different way you can look at this structure this is perhaps the one if you want to remember something perhaps this is the one from the crystallographic characterization. Let us look at little bit more into the electronic properties of these intermediate. So, you start with S 0 of course, all these intermediates are having 4 manganese 1 calcium structure. So, from the S 0 which is the fully reduced form it is getting oxidized at each step each of these 4 steps are dependent on light, but the final step is not dependent on the light that is converting S 4 into S 0. This light dependent step oxidize the manganese core to give you S 1 intermediate which is oxidized by 1 electron at every step 1 electron oxidation is happening. We just do not know or for sure what was the manganese 4 different manganese oxidation states are, but none the way none the least these will be oxidized by 1 electron this will be also oxidized by 1 electron S 1 to S 2 S 2 to S 3 another electron goes out in presence of light S 3 to S 4 another electron goes out this is the fully oxidized form, this is the fully reduced form. The fully oxidized form now will react with water to give you oxygen and reforming the fully reduced form S 0 and that is how the catalytic cycle would go like right. You have seen the cluster what perhaps it could be talk over here although knowing very well that is not the right representation of the structure that one would like to have. So, let us look at the same thing once again we are trying to understand where water is oxidized to oxygen and the structure of the photosynthetic 4 manganese 1 calcium cluster from X-ray spectroscopy that has been done and then what are the caveats remain this is a fantastic paper to read if you are trying to understand, but let me tell you that many things remain unclear these are some of the proposition still many evidences are required to nail down it further. So, the S 0 state as you can see over here these are the proposed structure or proposed oxidation state assignment by the EPR multiline spectrum, but it fits with both one manganese being 2 1 is 3 and another being plus 4 or another 2 being plus 4 2 plus 2 plus 3 and plus 4 2 of them is proposed or alternatively it could be 3 of them is in plus 3 oxidation state and 1 of them in plus 4 oxidation state this is the fully reduced form. From there on one electron oxidation occurs and this is quite definitive and that is that upon oxidation of one of these 2 intermediate perhaps one of them these 2 are correct which have spectroscopic evidence we see that it would be manganese 3 2 of the manganese will be in plus 3 oxidation state and 2 of the manganese will be in plus 4 oxidation state ok. So, these gives the EPR and EPR very characteristic for these species. Further oxidation of this intermediate gives rise to a situation where once again one of the manganese remain in plus 3 oxidation state another manganese which was in plus 3 oxidation state now oxidized to 4 plus 4 oxidation state. So, 3 of them 3 of the manganese is in plus 4 oxidation state one of the manganese is in plus oxidation state. Once again this will give rise to the multiple EPR multiple line spectrum with 2 different G values one for the manganese 3 plus and another for manganese 4 plus these are these are quite significant understanding what we have from S 2 to S 3 then it is further oxidation that means you know we have a all manganese 4 plus oxidation state or a radical intermediate where manganese 3 and manganese 1 manganese 3 plus and 3 of the manganese is in plus 4 state as well as a radical formation ok. So, these are the EPR spectra study. Now the assignment of oxygen evolving complex or center electronic structure is complicated and controversial ok. Let me get to that once again that these are putative assignment perhaps some of them are correct, but definitely not all of them are correct and there are open discussions on this there are open debate on these still ongoing in the literature. These different structure or the different oxidation state assignment was possible by various EPR study. These are complex EPR studies one has to be really expert in EPR to study and conclude or try to propose some of the valid oxidation state from these EPR studies. FTIR studies, XS, ZAS studies and UV visible studies were conducted for these two gives rise to the to the original proposal that we have seen over here. So, finally, once you have these S 3 state one more electron transfer one more electron oxidation and subsequent water to oxygen formation is going on. We will see these in a chem draw format real show ok. So, what we have seen so far then then that is saying that perhaps there is a manganese 5 oxo formation as you see if you just for simplicity pick up only one of them let us say you pick up this one. So, 4 manganese 4 plus all of them are 4 plus. So, further one electron oxidation has to have at least one electron oxidized to manganese 5 plus. So, this perhaps will have one manganese 5 plus other 3 manganese could be in manganese 4 plus. So, you can you can remember one of these oxidation state in each of them. So, although these two are possibilities these two are possibilities, but S 4 will definitely have a manganese 5 plus. So, that manganese 5 plus oxo would be would be quite electrophilic in nature and quite naturally calcium hydroxide which is generated from Wacoa or water molecule can attack on this as a nucleophile is attacking on the electrophile to form to form the oxygen oxygen bond. So, this is the most critical bond we are looking for because water to oxygen formation would require not the breaking of the bond, but the formation of the bond and this is where perhaps that oxygen oxygen bond formation is happening. This is the terminal manganese 5 oxo species we are talking about we will come back that to in a moment, but it could be also the bridging oxo that bridged between the 3 manganese center can also be possibly forming the oxygen oxygen bond. This these are the structure or of course, different orientation would be possible instead of manganese 5 oxo in this format one can have manganese 4 O dot radical where OH will be attacking the oxygen calcium hydroxide is also will be attacking the oxygen in these cases ok. So, this is what we have come to this is again a simplified one of the cluster we are picking up not every cluster we are talking about one of the cluster which is proposed one, but it could be very well be wrong, but for simplicity let us try to remember this where we are saying that 2 of the manganese center are always is in class 4 oxidation state that although we did not say like that over here let us say we are ruling out this as a possibility we are just for you know these are still argumentative. We are just for clarity just taking this one 2 manganese is in class 4 oxidation state and another 2 is in manganese 2 and manganese 3 oxidation state. What we see over here these center will remain constant nothing will happen to this structure throughout the catalytic cycle or this is again a presumption not direct evidence are existing, but it is becoming simpler if we are trying to think like that. 2 manganese center is in plus 4 oxidation state bridge by oxo just like bis me oxo intermediate. What you have seen in the last class in case of let us say intermediate Q in that your methane monoxygen as cases right. This was iron 4 iron 4 oxo oxo this is now a constant site or the site which is not involved into the oxygen-oxygen bond formation. It is linked with another manganese center which is of course, it will be setting the redox potential really correctly in any case this is bridged by another oxo molecule and from there you have a one manganese 2 and one manganese 3 center. It is proposed that in this case you will see one of the water molecule will be bound with the manganese and therefore, this calcium hydroxide which is right over there will be then subsequently interacting in the future cases. So, in the next class we will be discussing the details of these steps how these step wise mechanism is giving you water to oxidize oxygen, but it is important to understand that this once again the mechanism are not mechanisms are not really crystal clear. We really need to do much more studies to better understand these OEC or the photo system too. One thing is for sure these are 4 manganese 1 calcium cluster and these are going to be the most fascinating and perhaps the longest unsolved enzyme so far where we despite having the crystal structure still we are not sure what is going on. We will see how the oxygen-oxygen bond formation is happening in the next class. Keep studying photo system we will be back soon.