 Welcome back, today we will continue discussing on the reaction mechanism of formation of methanol right. So, from methane we are talking about, methane is such a great starting material to convert or to be converted into biomass right. So, methane to methanol is hugely important and very difficult transformation. And we have seen in the last class that these di-iron bis-muoxos intermediate where iron is in plus 4 oxidation state, iron is in plus 4 oxidation state and oxide oxide is reacting with methane to give an intermediate or the in this case the first the transition state T S 1 C H 3 H O where both bond making and bond breaking is happening simultaneously. From there we have seen the intermediate where C H 3 and OH are interacting still with each other and subsequently a concerted pathway where C H 3 and OH is intimately involved in the C H 3 OH molecule formation. This pathway so called concerted pathway is little bit energy demanding compared to the one the bound radical intermediate pathway. So, in the bound radical intermediate pathway we see the exactly same transition state where bond breaking and making is happening simultaneously from there a distinct radical intermediate involving C H 3 dot radical and oxo hydroxy iron 4 iron 3 intermediate is forming. Now that I would say quite interesting. So, what has happened over here this one of the iron center is iron 4 plus another iron center is iron 3 plus now the oxo is no longer oxo it is hydroxide H O minus 1 minus this is oxide O 2 minus right. So, iron 4 iron 3 mixed valent intermediate is formed along with the formation of the C H 3 radical this is what is IBR intermediate of this which is really preferred over that you see for IC intermediate for concerted pathway intermediate for the radical pathway is this one. This radical then can react with this OH of course, the OH has to orient properly and of course, we will see in a moment that this rearrangement or the electronic reassignment has to be done. Some electron spin has to exchange overall we will see that this C H 3 and OH once the OH is properly oriented and ready to react with the radical H O is H O minus that means, two electrons are there. So, two electrons over here one electron over there is not going to be a radical mechanism. So, this double electron has to be a single electron by transferring one of them to the iron center and this is where this iron intermediate will come into the picture and this OH radical and this C H 3 radical will combine with each other right to give the C H 3 OH. Let us look at this pathway which is the preferred one in little bit more detail as you see the energy levels are not too high it is achievable and these reactions are achievable at 20 degrees C at nearly room temperature or less than room temperature at pH 7. So, to be able to convert methane to methanol at room temperature at pH 7 I think is phenomenal right. This is quite exciting such a transformation remained very difficult to do even for the synthetic setup enzyme can do it perfectly because everything all the active site is perfectly. We will look at these steps how methane is approaching this intermediate Q, how this transition state is forming, how these intermediates are and how the next transition is forming and subsequently the overall product formation is happening in the in the by the DFT studies in a little close manner ok. So, this is your methane is approaching the intermediate Q. So, you see these two intermediate these two iron centers are iron 4 plus 4 plus oxide oxide and rest of the cycle or rest of the ligands are as it is as in the active site. So, you have iron sites oxo oxo and the methane this approach or this is set at 0 because this is when it is going to come and start reacting. So, this is set at 0 Kcal and we are going to look at these overall hydroxylation of methane to methanol by this non-Himdi iron enzyme ok. Subsequently this is the transition state that we that we discussed where CH bond breaking and OH bond making is happening ok. The electron transfer in this state electron transfer from this CH bond CH bond electron transfer occurs and determines the barrier height here it is 17.9 Kcal per mole which is ok right which is achievable at 20 degree C. So, this is the highest energy demanding step and this is the step why we do see kinetic isotope effect when we displace CH4 with CD4. If these are deuterium this CH bond breaking becomes further slow because this is the one which is the most difficult step to carry out. CH bond breaking and OH bond formation this is the corresponding transition state. CH4 versus CD4 will give a kinetic isotope effect value of nearly 22 as you have seen earlier ok. During this process one of the two iron centers will be reduced to iron III as this OH bond is formed. This is the transition state by the way not the intermediate. So, this is the structure looks like in this transition state. If you move on from there on what will happen once the OH bond formation is happening meaning we are looking at the second transition state this OH is now formed and the it has of course, of course, have undergone the electronic rearrangement. Overall now this OH is ready to bind with CH3 and the corresponding transition state will be looking like this. This transition state is very little up filled from the radical intermediate that we have discussed two slides back. So, these are the two transition state once again this is the first CH breaking OH bond formation step transition state which is the most demanding step and this transition state is not that very demanding it is rather easy to form rather less energy demanding where OH rebounding with the CH3 radical. So, this is of course, oxygen rebound or OH rebound step with the CH3 radical, but before that the rearrangement of electron has to happen. Let us look at those rearrangement of electron. So, this is once again from this book sorry from this reference from this research paper where this is the radical bound or bound methyl radical. So, CH3 dot H H H here is carbon this is the p orbital of carbon this is one electron over there CH3 dot donor orbital is there. Here is the acceptor orbital this is one way of looking at it one of the orbital we are looking at T x 2 y 2 of the iron center which is having beta spin lumo. So, we will see that in a moment if you are looking at hydroxide HO minus HO minus have two electrons and so, the two paired electron one up another down this doubly occupied intermediate or doubly occupied state then we will transfer one of the electron from here to the iron center right. Let us look at that. So, this is not really this OH minus and this double electron is not going to react with the with this CH3 radical that very easily this OH also has to form the radical that means, only one electron should be over here another electron should go somewhere most likely in this iron center we will see that in a moment. So, what you need to do you need to overlap. So, the rotate this p orbital or rotate overall over here to do the bonding between this d d d orbital of the iron center let us say iron one center and subsequently one of the electron from here will be transferred to the to the lumo metal based lumo and then you left up with one down spin beta spin one alpha spin on the OH and the beta spin on the lumo of the iron center. So, what has happened the alpha spin over here is now reacting with the beta spin of the radical to form the CH3 OH bond. So, these will form the new bond and then there is the one electron reduction center from the iron center making it from iron 4 to iron 3. So, this HO rotation this rotation promotes intramolecular beta electron transfer. So, intramolecular beta electron transfer from the oxalone pair orbital to the metal based lumo. So, this is the metal based lumo. So, oxalone pair to the metal based lumo this transfer is happening the remaining radical ad that is over here after one electron transfer the remaining alkaloid alpha electron on the bridging oxo group has the correct spin now to recombine with the beta electron of the of the sigma of the CH3 radical and these two now will form the sigma bond. So, what is essentially happening from here if you look this is the CH3 radical, this is the diiron center, this is the beta spin lumo of that means, dx2O2 orbital for the iron one center we are not looking at the iron 2 center. If you are looking at OH 2 electrons over there it will has to have to rotate in any case this is the 2 electron. So, this is single electron this is 2 electron it will have to rotate one of the electron has to the beta electron has to be transferred to the iron center and this is what you see over here the alpha spin sorry the beta spin and the alpha spin often rotation and transfer of one electron from this OH to the iron center gives rise to a situation where OH radical is there and the CH3 radical is there. Now, these two radical will combine to form the sigma bond. So, we have seen how CH4 to CH3 radical and CH3 OH bond is forming. So, overall this is this is the diagram where you started as we have seen with the bis mi works of species so called the Q intermediate and this is the Q star intermediate Q intermediate the acting or approaching methane is approaching towards is that is set to 0 and this CH bond breaking and OH bond formation is happening. So, CH bond breaking and OH bond formation is happening you are having a transition state right over here this is the most demanding transition state 17.9 kcal per mole where if you have the CD4 you will also get the same thing, but it would be significantly slower and then therefore, you will see that the CH3 radical and OH is now formed this hydroxide still has to form the OH radical and that is what is happening over here OH radical is formed and CH3 radical is over there. So, they will combine this is not that very energy demanding step and this is when they will combine with each other and will give you the CH3 OH bond intermediate that we have seen earlier. So, to summarize simply in this case what we have seen that a diiron mu oxo intermediate is approached by CH4 radical obstruction or you see that CH3 radical formation or OH formation is going on between the iron 2 centers. From there on electronic rearrangement has to happen to give you the hydroxy radical and then you also have the CH3 radical ready they combine to give you the product that is all nothing else is there ok. So, if you are looking at CH4 by versus CD4 that we have seen that this is having a high kinetic isotope effect value nearly 22. Quite surprisingly if you are changing the methane substrate from methane to ethane just change of substrate methane to ethane and if you are studying this enzyme you see that there is no kinetic isotope effect value between C2H6 and C2D6. Here CH4 versus CD4 you have a huge difference in the reaction right right. Over here C2H6 versus C2D6 which is ethane versus deuterated ethane there is no difference in the kinetics data or k-obs data. So, this is quite amazing right for ethane the rate determining step therefore, is not the CH bond activation step. So, what we are trying to tell here is the scientist has now figured out quite phenomenal observation and that is methane and ethane reacts completely differently or at least the intricacies of the reaction mechanism are different. In case of methane CH bond activation is the rate determining step can you imagine that and just you change from methane to ethane CH bond formation is no longer the rate determining step. You do not have a kinetic isotope effect value at all its kind of KiE is 1 although the k-obs is same which is fascinating I would say. So, for methane hydrogen atom abstraction is rate determining for C2H6 the binding the approach the C2H6 is approaching the Q the diffusion control is happening. So, the binding of the ethane with respect to the Q intermediate Q is important. See this is how nature has evolved in such a way the enzyme is designed in such a overall enzyme pocket the approach road is such that it just fits methane perfectly ok. If you want to fit in ethane that is also not possible and this is why one should realize that although nature is really great, but nature is only designed for one substrate and this is also true why we have such difficulty in synthetic setup in mimicking these chemistry and more importantly having a broad substrate scope for this sort of active site if we are able to mimic in synthetic setup. Because nature can do it for one substrate but synthetic chemist want every substrate over there that is not even nature has done it right that is going to be even more challenging that is why synthetic methodology development or catalyst development for a wide variety of substrate is always going to be challenging. One thing perhaps would be better for synthetic chemist to do is to try identify one great reaction and try to do these transformation effectively just what perhaps nature is doing. But yeah synthetic chemist's demands are little different in any case C286 as we are discussing the binding is the rate determining step. The bond in C286 is weaker of course, this is weaker significantly I would say by nearly 5 to 6 kcal compared to CH4. CH4 was 104 kcal per mole and C284 or ethane CH bond breaking would be something like you know 104 minus 6 let us say 98 or 98 99 kcal per mole. So, this is also saying that CH bond dissociation is not the rate determining step that means, CH bond dissociation is relatively easy in directly saying this is also can be proved by experiment and theory. In any case the CH bond dissociation is not the rate determining step, but the approach of ethane or diffusion of ethane to the intermediate Q is the rate determining step. But for methane diffusion was not of a problem, but CH bond breaking was the problem right. For ethane you can see that the approach of ethane towards the Q is problematic or the most demanding ok. So, a number of substrate one can study one such substrate that has been studied again this is a different paper. So, that is the nitromethane and in this case actually react IR can be used to monitor these by IR spectroscopy the kind of the dissociation or the CH bond breaking or the new product formation can be followed by the react IR. So, CH3 NO2 versus CD3 NO2 can be studied by react IR of course, it can one can still think of doing the UV visible spectroscopy, but this is a fantastic way of studying the reaction mechanism once again. The CH3 NO2 and CD3 NO2 will will will give this this sort of plot chaos versus nitromethane at pH 7 and 20 degree C this also gives rise to a very high kinetic isotope effect value. As you see between CH3 NO2 which is faster CD3 NO2 is significantly slower ok. So, this is not by UV visible spectra study, but by IR study you can follow the kinetics of this reaction with respect to the concentration different concentration of CH3 NO2 the study is done similarly as we were seeing for intermediate Q and then change the CH3 NO2 to CD3 NO2 and vary the concentration of CD3 NO2 you will get the plot as it is over here. Overall many different substrates are studied ok and this remains quite interesting for methane as you see nearly 23, 20 to 23, 23.1 plus minus 1.1 this is the kinetic isotope effect value as you see that is for CH4 and CD4 the CH bond breaking is the red limiting and therefore, a huge kinetic isotope effect. For CH3 NO2 and CD3 NO2 once again there is a large kinetic isotope effect not as large as CH4 CD4, but CH3 NO2 and CD3 NO2 is still high kinetic isotope effect this is saying that once again both for methane and nitromethane CH bond breaking is the red limiting step. One can even study the nitrile let us say acetonitrile for its hydroxylation chemistry. Acetonitrile versus CD3 CN is giving perhaps the highest kinetic isotope effect value in this series which is once again saying that the CH bond dissociation is problematic step rather than the diffusion which is happening in the other case this is the diffusion controlled process. Now both for all for methane, acetonitrile and nitromethane it is a significant kinetic isotope effect value indicating that these are the substrate where in CH bond dissociation will be the red limiting. I think it is also quite fascinating as we have discussed that ethane, methane to ethane you change the change the dimension completely. Methane CH bond was the difficult step ethane of course, CH bond is little weaker nearly 98, 99 kcal per mole bond dissociation energy, but this is no longer CH bond dissociation is no longer the red determining step. The red determining step is going to be the one wherein it is diffusing or approaching the intermediate Q and that is the difficult step rather than the CH bond breaking and such a similar case will be happening in case of methanol. Methanol versus deuterated methanol is also having kinetic isotope effect value of 1. So, methane to sorry ethane and deuterated ethane this kinetic isotope effect value of 1 indicates that the CH bond dissociation energy is not the red determining step, but the same thing is also true for methanol versus deuterated methanol where once again the CH bond breaking is not the red determining step. So, based on these studies one can think of having 2 different classes of substrate class 1 substrate where hydrogen atom abstraction is red determining as you see in these 3 substrate let us say those are studies methane, acetonitrile and nitromethane. For class 2 substrate binding rate is the red determining step substrate binding with respect to the active site or active species that is the intermediate Q that di iron di iron 4 plus with the bis meoxo intermediate that is the red determining step right. That is quite fascinating I would say and this is true for C2H6 and methanol ok. One can perhaps think of studying many other substrates as well but therefore, overall the whole spectrum can be split into 2. One is substrate 1, category 1 and the category 2, but I think you have seen the power of these reactions or power of these active site and that is this bis meoxo species are capable of forming the great methane to methanol ok. I think you have seen that there is similarity between the iron chemistry and copper chemistry I will take that reaction mechanism in a moment where you will see that iron chemistry and copper chemistry are similar. So, let me divide. So, let me try to draw the copper chemistry what you have seen in case of copper chemistry you have seen that copper oxygen or super oxo species is forming from copper 1 right. So, this is I am drawing copper 2 ok. So, you have taken copper 1 reacted it with oxygen right. So, one electron transfer happens to give you that and from there on you have seen another copper can come in copper 1 can come in to give you copper 2 O O let us say copper 2. I am deliberately drawing that is as an end on that will be easier to follow and from there on you see the one electron breaking from there and one electron from there another electron from there will give rise to the copper 3 intermediate right copper 3 bis meoxo intermediate. So, this is oxygen 2 minus this is oxygen 2 minus right. So, similar thing you have seen in case of iron iron 2 plus let us say let me draw the similar intermediate iron 3 would be there similar looking intermediate if we are trying to draw iron 2 is reacting with oxygen right. So, if we start from copper 1 we start from iron 2 it is a iron 3 super oxo and from there on another iron 2 can come in although this could be a side on geometry just I am drawing as an end on just to keep it clarity iron 3 can form. Now, this iron 3 intermediate can then once again give one electron break hom analytically and this can give one electron this one electron and this can give one electron overall you can form iron 4 bis meoxo species. Now, you do the see the similarity. So, this is a copper 3 copper 3 bis meoxo intermediate over here you have a iron 4 iron 4 bis meoxo intermediate only difference is one electron ok we start with copper 1 we start with iron 2. So, we go to copper 3 we go to iron 4 right that is very simple. This is why you can see that these species are very reactive and can effectively do a very great chemistry and these are of course, can do many chemistry as you have seen as you have seen over here here you can react methane to give methanol. There are copper enzyme also which is responsible for these methane monoxygenases these are the chemistry and these are the studies which are ongoing the detail understanding are still not 100 percent clear ok. I will not divert too much right now we will come back later with the summary between the copper and the oxygen similarities and summaries between them in some classes later. Let me get back to where we were discussing and that is these are the class 1 substrate and the class 2 substrate we have seen and the these are the 2 different types of substrate that we have seen over there right that is fantastic. Now, let us look at the overall program then. So, this is once again from from from professional leaf arts note where we see that that the first case if you are looking at methane that is in the that is in that is in the black hair. So, relative free energy if you are if you are looking at the black one where it is showing that this is not really the high energy this is diffusion is not the rate limiting of course, this is energy demanding, but not to energy demanding, but if you follow the black line is having the CH bond dissociation is as the rate limiting space and then the hydroxylation product is forming. In case of ethane that is in red you follow the red line here red line is very up. So, this transition state where the diffusion barrier is quite high on the other hand CH bond dissociation energy of course, is energy demanding, but not too very high. So, this energy is much higher than this one. So, that is why this diffusion is the rate limiting the CH bond activation is not the rate limiting. In case of methanol the blue line over here once again you see the in case of methanol which is the class 2 substrate if you have forgotten methanol is the class 2 substrate here you see that diffusion is far more energy demanding it is very high energy. Of course, CH bond activation is also demanding, but compared to the diffusion it is less energy demanding. So, diffusion is the controlling factor over there. In case of in case of nitromethane as you have seen CH this nitromethane diffusion is not too much of higher energy, but this CH bond dissociation is high energy. So, as you can see over here. So, this relative energy plot. So, let us say just for methane if you are looking at that is the black line over there. So, this is the diffusion energy compared to methane this CH bond activation energy is quite high as you see over here. That that is indicating that this is this is not going to be too much troublesome, but doing this CH bond activation the black line if you are following is going to be the most determining factor or the difficult factor. So, to conclude for this methane monoxygenase I think you know we are very lucky to have such a great enzyme where you have non-heam diiron center in the very high oxidation state iron 4 iron 4 in the bismeoxo bound intermediate which has similarity between the copper 3 di copper 3 bismeoxo intermediate, but more importantly it can convert methane even methane to methanol. All other substrates are I would say relatively easier all not all, but most of them are relatively easier there are 2 class of substrate that you can study with respect to these are methane monoxygenase 1 is class 1 or substrate class 1 another is substrate class 2. Substrate class 1 is the one where we see that kinetic isotope effect value is more than 7 or you know significant kinetic isotope effect value is there. If kinetic isotope effect value is equal to 1 then that is the substrates class 2 those are the one let us say for example, ethane that is what we have seen and in case of also methanol we see the similar cases. So, the class 2 substrates are also can be it can be utilized for its hydroxylation chemistry, but the diffusion is controlling you have seen stepwise how these bismeoxo species is abstracting hydrogen atom from methane and then how little rearrangement is happening making the hydroxo in C2 form ready for the rebound and then subsequent chemistry is following that is fascinating. We will come back with a much more chemistry in the subsequent classes. I hope you are studying these in detail and also we will summarize them to make it a little bit easy and compare contrast among the different enzymes so that you can you can follow well in the in the exam. Thank you very much see you in the next class. Bye bye.