 Hello and welcome to this next segment of CD spectroscopy and MOSBAR spectroscopy for chemist. My name is Arnab Datta and I am an associate professor in the department of chemistry IID Bombay. And today we are going to take a few more examples where we will look how MOSBAR spectroscopy can aid us to understand the mixed valency. So we are discussing this from the last segment. So we are going to take our example number 10 over here. So today we are going to take another trinuclear molecule but over here only 2 of them is actually our iron and there is a cobalt in between and there is another iron. So 2 iron centered by a cobalt. Now this is only the metal centers I am writing I also have to write the ligands around it. So let us take a look what are the ligands present in this particular molecule. So first of all we are having 3 nitrogens over here and these are all amine nitrogen which is coordinated to this and this one is coordinated. So each of the nitrogen you can see it is bind with the other nitrogen by a ethylene linker CH2CH2 linker. So each of them. So that is the interaction over here. And now there are other ligands on this particular side and they are all sulphurs and this sulphurs are coordinated with another chain. So the middle nitrogen capture the middle sulphur the top one captures the top nitrogen and same for the bottom one and they are connected with the linker chain over here. And these sulphurs coordinate with the cobalt and we expect the same thing on the other side is present and these sulphur atoms are present with the coordination site of the iron. And the rest of it it is again full fit by the 3 nitrogens. So quite the similar looking symmetric kind of ligands. And these 3 nitrogens over here it is going to be replicated on the other side of the terminal. And over here as we just discussed these nitrogens are connected with ethylene ring. So that is how it is connected and the sulphurs are also connected. So this is the structure of the full molecule. And over here what is the oxygen state we are talking about iron over here actually having oxygen state of 3 cobalt also 3 and 1 iron is in 2 state. So that is the thing we have to look into there is 2 iron 1 is plus 3 1 is plus 2 mi going to see the mixed valency or not. So that is the question we are trying to answer with this particular example and what is the role of this cobalt is playing in between. So when we first started looking into the Mosba spectroscopy, so I am drawing the Grabs problem it is the transmittance and it is the velocity which is actually coming for the how I am moving the Mosba source with respect to the sample with respect to the Doppler that is actually represented over here which is actually a representative of energy scale over here. So when you first do that at higher temperature by high temperature I mean 353 Kelvin, so it is almost 80 degree centigrade quite hot over there we are seeing only one set of iron centers. Then we go to little bit lower temperature we continue to see these 2 peaks but additionally we started seeing something else. So we started seeing other peak coming out another set of signals this is the red one I am also drawing how it looks like for the original signal stage there and this is we are seeing at 297 Kelvin, 298 Kelvin room temperature. And when we go further down what we are able to see is this this particular signals are actually started shrinking down this is happening at 80 Kelvin, so quite low temperature close to liquid nitrogen burning point. So this original black trace of the data it is now very small what happened to this red one and the green one. So they actually increase in their component size into the green one. So this one also start increasing this kind of very finely splitted doublet for the green one. This is at 80 Kelvin what happens if you go further down we go to 5 Kelvin and we saw nothing of this old peak over here they are almost gone what happens to this red and green. So the red one actually remain over there the red one remain over here while the green one also start dominating. So no stress of the original black trace no signal from there. How do we explain this result? So in the beginning I have only one set of data whereas later on it split in two different sets of data extra peaks coming out the red and green the black is still staying and slowly only the red and green remains the black is totally gone. So what is happening? So the red one and the green one what are those? So the red one is actually a iron 3 complex at low spin state and the green one is iron 2 in low spin state. So why there is a shift in the quadruple splitting because this is almost very low splitted this is the red one is quite highly splitted. So why it is happening? That is actually remaining on the splitting energy. So this one say it is quite close to an octahedral geometry and the same thing happening for the iron 3 and try to see what is actually happening between iron 2. So iron 3 versus iron 2. So over here we can say this is the EG level and this is the T2G level and over here what is actually happening? We have iron 3 means D5 system iron 2 means D6 system. So over here we have 1, 2, 3, 4, 5 because these are low spin systems present over here. Why low spin? That is we are coming into a little bit later because of the presence of the sulfides which is coordinated with this cobalt 3 plus system which is very strongly charged and that makes them very strongly charged system and over there the interaction is such that this become low spin system. The same thing happened over here D6 low spin system. So over here you can see how they are actually behaving over here. So that is how it is coming iron 2 and iron 3 and you can see iron 2 low spin system over here their valence contribution would be 0. So valence electric field gradient will be 0, lattice gradient will be there because there are 3 sulphurs 3 nitrogen's. So that remains same for both of them and in the case of AP3 plus in case of AP3 plus you can see there is an imbalance charge distribution. So valence EFG will be not equal to 0, lattice EFG is already not equal to 0. So altogether it is going to have a very asymmetric electric field gradient and that is going to show up over here and that is why iron 3 plus over you can see so widely distributed whereas in iron 2 plus only the electric field gradient of the lattice is coming plus valence is shut down. So that is why they are very splitted in very small amount and that is how it is happening and that is what we are seeing. So now we know exactly what is iron 3 plus and iron 2 plus look like. What is the original splitting over here that we are seeing what is that is representing and that we found that it is somewhere in between of them. So it is iron 2.5 plus so it is the delocalized system or mixed valence state. So that is what we are showing over here and what we found is iron 3 and iron 2 is coordinating in between them and how they are coordinating. So let us draw me the structure. So there is one iron center first over here and that is interacting with the sulfur. So I am just drawing in such a way that I am going through this iron sulfur cobalt sulfur iron. So this is over here now this is interacting with this cobalt this is another sulfur. So I am just simply matching the phase of the orbitals and then the last iron is there and now you can see how the iron from one side can connect to the iron of the other side through this sulfurs and cobalt system and we have a cobalt 3 plus system which is also low spin in nature. That means all its EG system is vacant and that is can be come into handy over here say if they are using dx comma y square kind of orbital it is going to come in handy and for exchanging electrons. So this is actually say one electron that I am going to exchange to the sulfur which is filled this is vacant this is again sulfur filled and this is where I want to put my electron. So that can travel through that and reach the iron very easily and that is what is actually happening and over here the orientation of the cobalt sulfur and iron is very important so that the there is a proper matching of the orbitals and that is happening when we actually heat the system and enough dynamical flexibility is there so that they can achieve the orientation they want to have and they can exchange electron very fast and that is actually what is happening and we are seeing this mixed valence system. But as we start lowering the temperature the iron centers stop interacting or exchanging because at lower temperature the sulfur and cobalt are not that much flexible so that you can attain all the particular orientation you want to have for this electron exchange. And as we go further lower temperature at 5 Kelvin where there is almost no interaction at all and you can say this mixed valence system is almost gone however it stays quite visibly even up till 80 Kelvin temperature. So it shows that only a little bit of option you give to the iron to bridge through the sulfur cobalt sulfur to the other iron it will do that so if you want to totally stop it you have to go to very low temperature only then you stop otherwise you are going to see this interaction. So over here we can say we are seeing a delocalized mixed system which comes to a localized system only at very low temperature so it is temperature playing an important role in delocalization of electron and which in turn is connected to the formation of mixed valence component. So that is one of the other examples we would like to cover in this particular segment. And now we move to our next example example number 11 where we will cover an example from biology. So previously we have discussed the iron sulfur clusters and over there we have discussed about rubidoxin and two iron two sulfur cluster which is known as the two iron two sulfur ferredoxin in short form we write them FDX. And we have shown them how it looks like so just to refresh your memory this is actually coming in the following way. So it is a two iron centers bridged with two sulfides and form this very nice diamond core but the iron is coordinatively unsaturated so it have to cover this tetral geometry around it and all the terminal ones are actually bound by sulfur system which if you remember has the side chain CH2SH which actually very readily form S- and that is actually binding to this metal so it is a monodentate ligand and that is what we are saying and we found this ferredoxins are actually electron transfer proteins so that is why they exchange electron and they can be in oxidized state and in the reduced state and we found in oxidized state it remain in iron III iron III and these are the proteins which typically exchange only one electron so the reduced state will be iron III iron II now the question is over here iron III and iron II I have two sulfides bridging ligand am I going to see a mixed valence system or they will be localized so for that we looked into the Mosba spectra for this system and what we found in the oxidized state value of isomer shift is 0.27 and quadrupolar splitting value is 0.6 whereas when you are looking into the reduced system we actually get 2 signals 1 at 0.35, 1 at 0.65 and let me draw it in this particular way and the 0.35 signature has the quadrupolar splitting of 0.6 so it is kind of remaining on its original position whereas the 0.651 that goes to 2.7 so that is the quadrupolar splitting we are finding over here so that means what we are saying that when you are doing through this particular experiment we are getting a system as such that in the beginning it has a very narrowed quadruplet splitting over here narrow quadrupolar splitting system but when you go to the reduced form now we are seeing two different signals one is remaining almost similar just move a touch on the positive side in addition to that it has an another signal whose midpoint is much more further down so somewhere close to this and then there were huge quadrupolar splitting and all these bonds are in the ratio of 1 is to 1 and it is predicted that whatever we are seeing over here is different from this signature that we are seeing over here so this is for iron 3 plus system and the broadly splitted one is the iron 2 plus system. Now the question comes why do we see this particular shift and for that we will just go a little bit back on this iron centers and try to find what we are seeing so if you look over here in the iron centers you will see there are two different iron centers possible one is iron 2 and iron 3 and they are in a tetrahedral geometry in tetrahedral geometry signature is the following T2 and E say it is the first time looking into iron 3 system they are always high spin in tetrahedral geometry mostly and there this is the iron 3 plus D5 system would look like and how it would look like for iron 2 D6 system everything safe except this one one extra electron would come over here and this is actually bringing a valence contribution to the EFG whereas the same one for iron 3 plus is 0 because it is all symmetrically oriented and that is what you are seeing the iron 3 plus is so narrowly splitted iron 2 plus is quite widely splitted. So this is something we have to look into we have to find out what is oxygen state and how we can distinguish that to find out how much quadruple splitting I am going to have and that is how it looks like. Now there is another version of answer for cluster is known as a rescape protein and this is the 2 iron 2 sulfur FDX. Now we are talking about something called rescape protein and how it is different it is having a similar structure of the iron we start with a bridging 2 iron 2 sulfur system and then over here it is bridged with cysteines and over here instead of cysteine now you have 2 histidines so that is the huge difference over here that over here you have 2 histidines over here instead of cysteine in the previous case and that is known as the rescape protein. Now the question is if this is actually happening how the rescape protein signals will differ in the oxidized and the reduced case and what we found that iron centers are remaining in the same oxidation states the strategy remains same iron 3 3 to iron 3 2 and previously we have found that these signals are not showing any mixed balance systems so they are all localized up so over here we can say it is mostly localized there is no delocalization possible or seen in this molecule but what is happening in the rescape protein that we are trying to find out over here and that is the systems we are seeing we are seeing 2 different signals for oxidized 1.24 and 0.32 and 2 4 and 0.32 and each of them have their own quadruple splitting 1 is 0.32 1 is 0.91 sorry this is 0.6 and 0.6 and then 0.91 so that is what we are seeing so why we are seeing 2 different isomer shift now because now we have 2 different iron centers one over here other over here and one of them is actually FeS4 coordination whereas the other one over here is FeS2 N2 coordination and this is going to showcase different electronic distribution on the iron centers and that is reflected on the MOSBUS spectroscopy of the oxidized states so they are already separated now which one is the histidine bound and which one of them is the cysteine bound so over here we can see that it is actually bound so let me I am just drawing this scaffold of histidine and cysteine let me draw it in the way I have drawn it over here so this versus these are the centers I am talking about now over here which one of them will be different and why so what is the difference between over here S-cysteine versus N-cysteine now N-cysteine is actually the imidazole ring that is come over here wrong place so through this it is probably going to bind or this one after the deprotonation so either of these two natures are binding but over here you can see typically it binds in this neutral stage so this N-cysteine is a neutral ligand whereas S-cysteine we have already shown there it is a charge system so now the charge system versus neutral which one of them is going to stabilize a oxidized iron system the charged one so this interaction of iron and interaction that is over here the iron 3 plus will be more prominent compared to this iron 3 plus over histidine that will be much more charged and the slight difference in the charge will showcase over here so one of them is 0.24 one of them is 0.32 so 0.24 is the iron S-4 system because it is on the much more charged side so more charged as you know it will move towards the negative side and 0.32 is a histidine side so it is the Fe2 S2 N2 coordination side so that is what is happening now what happens when you go to the reduced system in the reduced system also we are seeing two signals 0.31 and 0.74 0.31 signature has a splitting of 0.63 and this one has a splitting of 3.05 so previously also we can see the quadrupole splitting you can see this is much more higher 0.91 this is smaller 0.62 so why this is higher because we are talking about a Fe S2 N2 system much more asymmetric compared to Fe S4 relatively symmetric so as you induce more symmetry in the coordination geometry this is actually getting further split and that is going to continue even in the reduced state where it is C2 signature now which one I can actually confer like it is actually the N2 S2 1 or S4 1 so you can see the splitting and the value is remaining almost same for this iron III for this Fe S4 system but this one 0.32 is pleated to 3.05 much for wider even wider than what we expect for the normal 2 iron II sulfur ferroxene where all them are sulfur even then it is going beyond that so that is actually coming because of the asymmetry related to Fe S2 N2 so over here it is already large because we have already discussed Fe2 has the valence contribution from the EFG and not only that this Fe S2 N2 bringing more lattice electric field gradient over here which is actually shown the splitting on this particular side and that is what we are getting for this 2 iron II sulfur cluster and the resk protein and this is actually showing us a very unique example how MOSBA spectroscopy can be found even with this particular molecule and we can say like which one of them is actually exchanging the electron histidine side or the cysteine side so MOSBA spectroscopy without any doubt showing us that it is on the histidine side which is actually exchanging the electron and not only that we are also gaining an ideology how this change in the oxidation state and change in the coordination side can affect the overall isomer shift and quadrupole splitting in MOSBA spectroscopy which is showcased in the spectra shown over here and over here this is a very nice example how a biological sample can be assessed with MOSBA spectroscopy and with that we will try to close over here for this particular segment and we will come with another and final example of a biological sample of iron sulfur cluster and how we can distinguish about the different oxidation state and its orientation and probable mix valency or not through MOSBA spectroscopy and over here what we found these are all remain localized there is no mix valency at all in 2 iron II sulfur cluster or resk protein but is it going to remain same when I move to 4 and 4 sulfur cluster that we will take a look into the next segment thank you thank you very much