 and welcome to the final segment of CD and MOSBUS spectroscopy of chemist and over here we are going to discuss today the applications of MOSBUS spectroscopy where we are trying to find out and mixed valence state in a biological sample. So, let us start that. So, this is example number 12 that we are covering and today we are going to talk about a 4-iron 4-sulfur cluster. So, you can start from the beginning one more time. So, we are going to talk about this 4-iron 4-sulfur cluster today which is having a structure of a Cuban and the vertices we are having a iron and sulfur on the alternate spaces. So, that is how the structure looks like a 4-iron 4-sulfur cluster that is the core of it. So, 4 sulfides and 4 iron centers and now iron you can see it is coordinated with 3 sulfurs each of them, but iron need to have 4 coordination. So, that last one is actually coordinated with a cysteine. So, that is what is the overall structure of 4-iron 4-sulfur cluster. Now, once we have it the question is what is the oxygen state it actually varies and we found that there are actually different situation possible two different situation. So, there are 4 irons. So, I am just writing the oxygen state. So, one of the possibility is the most common one is 2 of them in plus 3 2 of them in plus 2 state that is the oxidized form and the reduced form is 1, 3 and 2 of them 3 of them in plus 2 state. That is the reduced state that is actually happening over here and this one is known as 4 iron 4-sulfur ferredoxin where we are seeing 2 of them in plus 3, 2 of them in plus 2 the oxidized state and 1 plus 3 and 3 of them plus 2 is the reduced state. And then there is another possibility that some of the ions of a cluster remain in 3 of them plus 3 state and 1 plus 2 that is the oxidized state and its corresponding reduced state is 2 plus 3 and 2 plus 2 and the protein which actually follows this particular set of redox system. So, these 2 it is known as high potential iron-sulfur proteins and if I take these letters it is sought from is HIPIP. So, this HIPIP proteins also use the same template of 4 and 4 sulfur cluster but it is using different set of oxidation states whereas, the common 4 and 4 sulfur cluster they use the different set of oxidation state this is the common one. So, it is oxidized state for the 4 and 4 sulfur cluster it is the reduced state for the HIPIPs. So, these are the different oxidation state possible what is the difference between them? Difference between them is the oxidation potential 4 and 4 sulfur cluster varies its potential from minus 415 to minus 280 millivolt versus standard hydrogen electrodes that is the potential it looks for whereas, the 4 and 4 sulfur cluster which is forming this high potential iron-sulfur proteins that actually varies from plus 90 to plus 460 millivolt versus SHE. So, you can see the difference it is more on the positive potential. So, that is why it is known as high potential iron-sulfur proteins and this is commonly figuring out a position on the negative side for the typical 4 and 4 sulfur cluster. So, that is the big difference. The question is why there is such difference in the potential? So, 4 and 4 sulfur clusters are majorly found in a hydrophobic media. So, this cluster you are saying it is coordinated to a hydrophobic motive. If it is in a hydrophobic motive, what is the overall change in the charge for this particular molecule? So, if it is a 4 iron-4 sulfur fadoxin protein, let us calculate the overall charge. So, in the oxidized state you can say it is 2, 3 plus and 2, 2 plus so total plus 8 charge from there and there are 4 sulfides. So, that will be minus 8 charge over here. And along with that we also have the charge from the cysteines. So, all of them if we combine together what is the overall charge we will see in the oxidized state and what is the charge we are going to see in the reduced state. So, that we are going to consider while we are calculating the overall charge of the 4 and 4 sulfur cluster. And if we did that for this particular system we see the reduced state and oxidized state we write typically write oxidized state versus reduced state. It varies from 2 minus to 3 minus charge that is how it is actually varying over here. And the same thing because over here it is actually 2, 3 plus and 2, 2 plus so total plus 10 charge and minus 8 charge for the 4 sulfides and minus 4 charge for the cysteines. This is for the iron, this is for the sulfide and this is for the cysteine. So, you can see the overall charge is minus 2. So, the reduced state obviously is going to be get 1 further negative so minus 3. So, minus 2 to minus 3 is the charge. So, over here it is going to be more negatively charged as it is getting reduced and if it is in hydrophobic media hydrophobic media does not really favor the stability of a negative recharge system. And if you are going more negative the delta G is going to be positive because it is unstable and that is going to show in the E value it will be more negative and that is what is shown over here. Now, what happens for this HIP IP? In HIP IP what is the oxidized and reduced state? In oxidized state you can say is plus 3 of 3 of them and 1 plus 2. So, it is plus 11 minus 8 charge for the sulfides and minus 4 charge for the 4 cysteines coordinating the iron centers from the protein. And all those things together you can see what will be the overall charge of this system. And you can see it is a charge of minus 1 and the reduced state is the plus 10 minus 8 minus 4 which is minus 2 charge. So, it is going from 1 minus to 2 minus charge. And this HIP is also found in a hydrophobic media HIP IP is also found in a hydrophobic media. But over here one important thing is that this background of this protein they have amide bond and this amide bond has a dipole delta plus on the N H and delta minus on the carbonyl and the delta plus is actually situated directly towards the ions of a cluster and a multiple of them. And those are actually positive charge and stabilizing this negatively charged system minus 1 versus minus 2. And over here minus 2 will be more stabilized because more negative charge and more ionic interaction dipole ion interaction coming from the amide backbone. And that actually brings the stability towards the more reduced state and that is why the delta G for this one will be negative e value to the positive direction and that is what we found over here. And that is what is happening in the HIP IP and the 4 and 4 sulfur cluster. Now, all these things now understood and we kind of know the overall oxygen state now take a look into the MOSBUS spectra of this particular system. So, first we will try with 4 iron 4 sulfur cluster what is actually happening in its oxidized state and as we said it is a charge of plus 3 plus 3 plus 2 plus 2. So, now let us say what is actually happening in the MOSBUS spectroscopy what is the isomeric shift what is the quadrupole splitting that we are observing over here. So, we found only one set of data over here which is actually one set of data like this and over here this is the value of isomer shift and the quadrupole splitting is 1.12. Now, previously you remember if it is a plus 3 state for the ions of a cluster the value of isomer shift is around 0.3 and if it is a plus 2 state around 0.6. So, where right now we are in the middle of somewhere because what we expect this is the experimental and what is theoretical if it is remaining plus 3 and plus 2 we expect 2 sets of data 1 for the plus 2 and 1 for the plus 3. So, plus 3 and plus 2. So, 2 sets of data we expect at the ratio of 1 is to 1, but what we are getting only one set of data and this is the value we are getting which is in between iron 3 and iron 2. So, this data suggests that I am actually seeing a state of iron 2.5 plus for all the iron. So, in the ions of a cluster in a state over here in this particular geometry where they are in a cubical form the iron centers are actually exchanging electrons through this sulphur and gaining a mixed valence state and that is what we are getting from this particular MOSBAR spectra it is clearly speculating that and this is also supported by the EPR study and magnetic study. But MOSBAR spectroscopy give us the most clear data that yes it is in a mixed valence state and only one kind of iron is present over here not two kind of iron center that we would get if it is delocalized sorry localized. So, if it is delocalized we are getting to see only one kind of iron center. Now what happens if we go to the reduced state so over there we are going to see plus 3 plus 2 plus 2 plus 2 what is the theoretical thing we are expecting if it is localized. So, it is also the theoretical one previously if it is a localized sample if we would be getting and that is not we got we got this one. So, over here let us say theoretically what we say we should see 1 3 plus and 3 2 plus. So, 3 plus we know it is going to show very narrow down signal and 2 plus we know a large signal like this and the ratio should be 3 is to 1 that we expect. Now let us see what are the values we get and what was their ratio between the different signature. So, we got two peaks 1 at 0.58 and 1 at 0.49 and the 0.58 signal has a quadruple splitting of 1.89 and this one has a quadruple splitting of 1.32 and very interesting the ratio was 1 is to 1 it is not 3 is to 1 that we are expecting. So, we get two signals actual experimentally one is pleated and the other one somewhere in between but it is also similar ratio. So, 1 is to 1 ratio now the question is why it is so. So, now again we look back to the value. So, 0.58 and this splitting up to 2 millimeter per second. So, that is kind of saying it is an iron 2 center but again 0.49 it is somewhere in between 0.3 and 0.6 that we expect for iron 3 and iron 2 respectively. So, this is actually showing a iron 2.5 plus charge system. So, again over here what happens between this 3 plus and 2 plus they become mixed valence and delocalized and form this 2.5 plus 2.5 plus where the other iron 2 remains iron 2 which is showcase over here the delocalized one is shown over here. So, we have a mixture of delocalized and localized system over here, but in reality we can say we are having a mixed valence system which is quite obvious from the MOSPA spectra of the reduced state of the 4 iron 4 sulfur ferred oxins. So, in the ferred oxins if we look into the experimental what we are seeing is not different sets of signature with different ratios in the reduced and similar ratio in the oxidized form what we are actually looking only one set of signal in the oxidized state and 2 set of signals in the reduced state but the ratio is 1 is to 1. So, it says only one kind of iron in the reduced oxidized form and 2 iron sites of 1 is to 1 ratio in the reduced state and by that we now know the over oxygen state that is really present on the state of ferred oxin is all 2.5 plus 2.5 plus 2.5 plus for the oxidized state and the reduced state is 2.5 plus 2.5 plus and the rest of them is 2 plus 2 plus that is how it is behaving for ferred oxin. Now, we will look the HIP IP which is in its oxidized state say here 3 of them are plus 3 and one of them is plus 2. So, what do we expect over here theoretically? Theoretically expect that there will be 3 of them 3 plus. So, as we know 3 plus is actually much more narrower splitted. So, that will be there and along with that we are going to see 1 2 plus. So, it will be largely splitted and the ratio would be 3 is to 1 this is the iron 3 plus and this is the iron 2 plus that we expect. Now, let us see what we actually observe in Mossberg spectroscopy of this HIP IP oxidized sample. So, over here we again got 2 signature 1 at 0.29, 1 at 0.40, 0.29 was splitted 0.88 millimeter per second and the 0.41 is a little bit large splitted. Very interestingly the ratio of the picture 1 is to 1. So, what we are actually seeing in experiment this is again not exactly what we are thinking theoretically if it is a localized 1. So, now what we are seeing is actually closely splitted thing and along with that largely splitted thing and the ratio is 1 is to 1. And over there we are seeing this largely splitted thing this is the largest bit is saying the delta Eq of 1.03 and their middle point the average value of the delta is 0.4. So, what we are seeing over here that is you can see again it is failing in between a typical iron plus 3 why do you see 0.3 which is actually shown over here. So, this is obviously iron plus 3 system, but this one we are seeing it is neither iron plus 3 because it is not 0.3 neither iron plus 2 which some where it comes around 0.6 it is on middle in between it is coming because it is again a mixed valent 2.5 plus system. So, what we are actually seeing over here we are seeing a ratio of 1 is to 1 because now over here it is a delocalization happens. So, 2 of them remain plus 3 plus 3 and the rest 2 of them do the quadrupolar splitting such a way and isomership such a way that it is actually doing a mixed valency and it is coming 2.5 plus and 2.5 plus and that is what we are seeing over here and it is a mixed valence system over here. So, that means in HIPIP oxidized state we are seeing mixed valence and we are seeing a set of 2 3 plus and a set of 2 2.5 plus systems 1 is to 1 ratio all of them and the reduced state it is going to be exactly the same of the oxidized state of ferred oxene. So, its reduced state is exactly the same for oxidized state of 4 iron 4 sulfur ferroxene. So, we have just discussed that few minutes back. So, we are not looking back into that but this is what is actually going to happen. So, by that you can see MOSBUS spectroscopy can give us very detailed analysis how it is actually interacting between the different oxidation states of iron in this ion sulfur clusters which are very crucial biological entity for electron transfer and we know exactly where the electrons are transferring and what is their particular oxidation state. So, with that we would like to conclude over here the applications of MOSBUS spectroscopy. Thank you very much.