 So, if not we are going to move toward the next example. The next example is a biological example of a protein called rubredoxin. Rubredoxin is a protein where there is a one iron center and it is kept a hedgerally ligated with four sulphurs coming from cysteine groups. So, that is actually a part of a protein where it has four sulphurs coming from cysteine site chains and this cysteine creates a binding pocket for this iron. And over there the iron can go between two oxidation state iron plus 3 to iron plus 2. It is one of the most basic iron sulphur cluster proteins which is the most simplest iron sulphur cluster proteins and it is widely used in biology for electron transfer. And generally these proteins transfer electron one electron at a time. That means this whole system can transfer one electron at a time. So some electron comes over here from outside it gives electron to someone else and it goes from iron 3 to 2 first step next step it goes to iron 2 to 3 back again. So it shuttles between iron 3 to 2. So it is very much important to understand us that what is the oxidation state of this rubredoxin proteins inside the biology so that we can understand in which direction the electron is moving and what are the different experiments or reactions these rubredoxin proteins are participating. So as we just said this iron can have two different oxidation state iron 3 oxidized state iron 2 the reduced state and our goal is to find out can we predict how the system will look like for the MOS perspective. Now what is the coordination geometry over here iron tetrahedral previously we were talking about only the octahedral so it is a tetrahedral system. So E at the bottom T2 at the top iron 3 is a D5 system and this is all bound with sulphurs and sulphurs are pi donors so they are all going to be high speed system it is very rarely you see a tetrahedral geometry a low speed system. So 5 electrons so 1 2 3 4 5. So now looking into the system D5 system are you going to expect any valence contribution because it is a E2 T2 3 system so it is not going to show any valence contribution and lattice contribution it is tetrahedral geometry tetrahedral geometry is not as symmetric as it is for an octahedral or square corner or spherical so lattice contribution will be there for tetrahedral coordination geometry. So lattice contribution will be present valence contribution will be absent. So what will be happening so let us write down the delta value of iron 3 and compare that with iron plus 2 system so iron 3 versus iron 2. So iron 3 has a delta value around 0.32 millimeter per second these are all in the unit of millimeter per second and delta EQ value around 0.5 millimeter per second. Now if I reduce this system to iron 2 what do I expect what I am going to expect is the following is a D6 high spin system 1 2 3 4 5 6. Now I have a asymmetry over here so I am going to see some valence contribution in this case lattice contribution is anyways present over here. So what do I expect if I go to iron plus 3 to plus 2 I am reducing the oxidation state that means I am increasing the D electron density that means I am going to increase the shielding effect that D orbitals is going to hamper the interaction between the 3s orbital and the nucleus the S electron density will be less inside the nucleus compared to the iron 3 psi 0 square will be less it is multiplied with the negative term of delta r by r so it is going to move towards the positive side and that exactly what happens it moves to 0.7 millimeter per second you can see the positive shield over here. What happens to delta EQ previously there is no valence contribution but only lattice now both valence and lattice contribution will be active so the splitting should be higher and that exactly what we found it goes to higher value 3.25 so it clearly shows that totally different system for rubydoxin between iron 3 and iron 2 so if I want to plot that together for rubydoxin what we are going to get so say I am putting iron 3 in red and iron 2 in blue what do I expect in the same graph if I want to put series percentage of transmittance it is going to be millimeter per second so for iron 3 what do you expect 0.32 low splitting so it is going to be somewhere the more negative side so first draw a line this should be 0.32 and what is the splitting 0.5 so either side 0.25 so I should see a signal like this so this will be 0.57 and this is another point where 25 so it will be 0.07 so you can see the difference is 0.5 delta EQ and the average value is 0.32. Now what happens to iron 2 iron 2 is going to show a value of 0.7 the delta value splitting is 3.25 so 0.7 is going to be the average somewhere around here and 3.25 let's say 3.2 so 1.6 on either side this is 0.7 so 1.6 on either side so what will be happening 1.6 plus 1.7 is how much 2.3 so 1 will be around 2.3 and the other is be 1.6 on the other side so it will be around minus 0.9 so it will be far negative something like this so I have not drawn it properly in the scale but if we draw it properly the average will be at 0.7 the difference will be 3.25 millimeter per second so you can clearly see how different the signal will be not only the average value will be shifting with respect to the reduction but also the splitting will also be increasing so by that you can easily see what is the oxidation state of the iron in the rubric reduction. Next we will take an example of another ions of a cluster where there are two irons which are breached by inorganic sulphides like this kind of diamond geometry so it is known as the Fe2S2 cluster and the rest of the iron coordination sites are filled up by protein bound cysteine sulphur so this is the core and that is why it is known as Fe2S2 cluster or Fe2S2 ferrous ferrodoxene in short from Eptius and over there there are two centers over there and we found in the oxidation side it is iron 3 iron 3 in reductive condition it is iron 3 iron 2 so it doesn't go to all the possible oxidation state this ferroxene can still translate on one at a time and the oxidation state it shuttles between is plus 3 plus 3 in the oxidized state to plus 3 plus 2 in the reduced state now the question is how the delta and delta eq value looks like you have already discussed how the values will differ it is very much similar to the rubrodoxene so iron plus 3 plus 3 it shows 0.27 and delta eq value 0.6 and over there both iron shows similar value nothing else so it clearly shows how good the interaction is happening between this iron through this bridging sulphur so they are quite well balanced with respect to the electronic distribution so that is why we are seeing only one kind of iron what happens when we reduce now it is 1 iron 3 1 iron 2 so we see two different signals iron 3 1 is coming at 0.35 iron 2 come around 0.65 so they are differing in the delta values 0.35 belongs to iron 3 0.65 belongs to iron 2 again higher oxidation state means to the negative side lower oxidation state means to the higher side delta eq value it remain 0.6 corresponding to the pit at 0.35 and this bit 0.65 it goes to 2.7 very similar to what we have discussed in the earlier system so iron 2 splitted further because it now have both valence and lattice contribution whereas in iron 3 case it is only the lattice contribution so that is why you can see the difference so what you expect in the preliminary system you expect only one kind of signal in oxidized state in the reduced state you will see two sets of signals like this which clearly distinguish between the oxidation states of the iron in this complex proteins which are very difficult understand in other experiments and Mosbier is very critical because in Mosbier only iron is active all the other systems are actually blank or doesn't contribute to the signal so it is very easy to do that with respect to the Mosbier spectroscopy okay that is how it is going to look like for this Fe2H2 so one more example we will take and that is the example of 3 iron 4 sulfur cluster or 3 iron 4 sulfur peroxide what is the structure of 3 and 4 sulfur pedesting it looks like a cube and alternatively you have iron and in organic sulfurs bound over there and one of them actually doesn't have an iron over here so over here the iron is actually missing otherwise it will be a complete 4 iron 4 sulfur cluster so over here it is a 3 iron 4 sulfur cluster one of the iron is missing and over there you can see the irons are still missing some of the coordination geometry which are actually filled up again by the cysteineal sulfurs and through this cysteineal sulfur bonding this cluster is covalently connected with the protein and this system also transfer electrons and what it has been found that the oxidation state it is iron 3 iron 3 iron 3 so what is the delta value expect very similar compared to the previous one 0.27 delta EQ value is going to be 0.63 right now what will be the reduction state? Reduction state is going to be one electron reduction so you expect it will be iron 3 iron 3 iron 2 now over here what do we expect in iron 3 iron 3 iron 3 it is very straightforward you see one signal at a time now what happens if it goes to iron 3 iron 3 iron 2 so you're going to see a signal belongs to iron 3 and one signal belongs to iron 2 and what should be the ratio the ratio should be 2 is to 1 2 signal belongs to iron 3 one signal belongs to iron 2 and iron 2 is going to be on the positive side and should be further splitted and iron 3 should be on the negative side and less pleated so the data we are expecting is the following that iron 2 will be most pleated and iron 3 will be less pleated but almost twice in size that is how the signal it should look like right with respect to the 2 is to 1 ratio we are expecting or now let me show you how the signal actually looks like let me do it over here how the signal actually looks like so that is how the iron 2 iron 3 system comes out the top one is actually the iron 3 iron 3 iron 3 one signal only once pleating so that is fine the rest of them should be two signals so we see two signals over here over here we see for this one two signals but in the opposite way we expected the middle one should be higher in intensity because that should belong to 3 and the side ones should be lower in intensity that belongs to iron 2 but over here see it is actually kind of opposite the outside one is actually higher intensity than the middle one so instead of 2 is to 1 it is showing something 1 is to 2 how I can explain but at the same time I am taking only one electron that can be explained because it is actually not forming iron 3 iron 3 iron 2 it is actually going through a mixed balance system one iron 3 and one iron 2 actually interacts between them and it forms iron 2.5 iron 2.5 by interaction between this iron 3 iron 2 it creates 2 iron 2.5 system the other one remains iron 3 now you can imagine a iron 3 will be remaining in 1 equivalent iron 2.5 is 2 equivalent iron 3 is higher oxidation state so it is going to have a value delta value to the negative side 2.5 is going to have a value on the positive side 0.46 and also look into the value 0.46 previously we found the values around 0.65 or 0.7 for the iron 2 and for iron 3 it is around 0.27 or 0.3 and this is giving me a value in between around 0.45 so it is also showing from the value it is not 3 not 2 it is somewhere in between so it is iron 2.5 and it also affects a similar way to the delta aq it goes to 0.52 and 1.47 and that is why instead of this particular signal we saw that signal in the different way so the outside ones are actually higher in number middle one is actually lower in intensity and we get a 2 is to 1 value like that okay so this one equivalent is belongs to iron 3 and this 2 equivalent belongs to iron 2.5 so this is actually a very unique example of Mosbauer spectroscopy so it not only give you the exact oxidation state it also shows that mixed valency which is very difficult to understand from other spectroscopic in general but it clearly and significantly differentiate with respect to the other states and give you an idea what is actually happening over here okay now the next example next example is an example where most of you have gone through it is about the hemoglobin we already know in a hemoglobin there is an iron system binds with a porphyrin gene in plus two oxidation state connected with a histidine like this very loosely connected to a water molecule which is we know as a deoxy form before it binds to the oxygen and once the oxygen comes over here it binds to that oxygen and it comes to this porphyrin ring plane before that it lies a little bit lower side than the porphyrin and this one it is suggested it is an iron 2 high spin system and there's a lot of controversy what is the oxidation state what is the spin state of this oxy form now let's take a look if Mosbauer spectroscopy can resolve what is happening here so we take the Mosbauer spectroscopy of the deoxy and oxy form the delta value is found for the deoxy form is 0.89 millimeter per second the delta eq it is fine around 2.23 millimeter per second so that is fine it belongs to a iron 2 high spin system so that is what this value is so that is why it is so much highly shift positively positively shifted delta isomer shift value because of oxidation state also the spin state in the oxy state once they measure it they found the delta value shifted to very low value 0.23 millimeter per second and delta eq value is 2.12 so what is happening so first of all what is this huge shift positive to negative side so it shifts is actually belongs to both the change in the oxidation state and the spinning state so over here the suggestion was that it is actually going from iron 2 high spin in the ox deoxy form to iron 3 low spin in the oxy form so the low spin if it is happening it will shrink down a little bit the overall iron center and it will fit in the profile ring so it is actually favoring that and not only that it is going to iron 3 state and that is why the change of oxidation state change of spin state and that is why that much of shift because it is removing the delectron density now whether it is a low spin or iron plus 3 it is also supported by this delta eq value you can see the delta eq value is not shifted that much why because previously we are saying it is actually a high spin system d6 high spin system and that is why we are creating not only lattice contribution but also valence contribution over there it is going to give you valence contribution and also lattice contribution appears to because of a asymmetric coordination and if it also goes to a low spin system and iron plus 3 state then it is also going to contribute valence contribution and also lattice contribution and that is why both of them are going to have very similar delta eq value so the delta value changes isomership but the delta eq value remains almost same and it can be explained if it remains in iron 3 low spin value so if it is becoming iron 3 low spin from iron to low speed that means someone has to take one electron out who is taking that electron so the only possibility is that this oxygen is taking one electron and becoming a super oxide so if it forms a super oxide iron or not how we can prove so people have drawn resonance Raman spectroscopy and try to find out what is the splitting or the stretching frequency of the oxygen oxygen bond in this system and they found it is around 1100 centimeter inverse which actually falls in the range of super oxide if it is peroxide it is around 850 centimeter inverse if it is oxygen it is 1500 so it is 1100 which shows it is super oxide so with the help of the MOSFER and resonance Raman's it has been almost univocally find out that in the hemoglobin when the oxy form is formed it is actually an iron 3 low spin system and oxygen is remaining in the super oxide so there is an electron exchange between them however the electron exchange is quite reversible that's why once the hemoglobin comes to the cell it interacts with the myoglobin the myoglobin binds the oxygen and the hemoglobin of the iron readily gets the electron back from the oxygen the super oxide and release the oxygen and it goes back to iron 2 state so MOSFER spectroscopy was a huge help to find out that really you can figure it out what is actually happening in the hemoglobin during the oxygen interaction