 Hello, welcome back we are nearing to the final few classes of this course and that is Betel-Sin Biology. We are trying to summarize little bit quickly so that you can study these for exam purpose relatively easily. What we have seen in case of the iron oxygen intermediates that they are very capable of doing oxygenation chemistry some of them are oxygenases some of them are oxidizes right. But more importantly almost every organic substrate can be functionalized by utilizing the iron oxygen chemistry. These chemistry can be between the two iron center between the oxo species is bridged in between or it could be as fascinating as those the terminal oxo species or these porphyrin oxo based species. There are non-him oxo based species as you have also seen, but these species are also known to be very very effective. So, you have seen these many different types of reaction hydroxylation oxidative ligand transfer desaturations cyclization electrophilic aromatic substitution and cis dihydroxylation reaction. All these reactions you are hopefully now familiar with now most of them are going via the radical mechanism as you can see in there. This one where arene activation is involved or arene oxygenation is involved it is going through an electrophilic aromatic substitution reaction. The cis dihydroxylation can be mediated by these oxo hydroxyl species to form this from this extremely exciting products. All these cases this is this oxo radical formation and corresponding the CH bond activation and the radical formation is key and that is what we have seen over the many many different high valent iron oxo intermediate cases. One of the exciting species you perhaps would remember as well that the alpha ketoglutarate dependent enzyme are capable of doing both the oxygenation chemistry as well as the halogenation chemistry depending on the slight changes they have in their coordination motive. But nonetheless they will be having this alpha ketoglutarate as an internal substrate for their activity. So, what you have seen earlier with the alpha ketoglutarate dependent enzyme is simply it binds with the non heme iron center and oxygen activation that takes place at the iron center before that the substrate will orient itself get it ready get set go and then oxygen will start against activated at the iron center it first form a iron 3 super oxo which immediately is trapped or attacked attacking the ketomoyety of the alpha ketoglutarate. The alpha ketocenter is getting reacted with forming an alkyl peroxo like intermediate which can then undergo oxygen-oxygen bond cleavage to give rise to these iron 4 oxo species. These are reactive intermediate and the substrate is sitting right close to it therefore, the oxo will obstruct hydrogen atom to form the hydroxo and the R radical right. This R radical and hydroxo radical can combine with each other to give the ROH. Once again this water molecule goes out alpha ketoglutarate comes in, RH texate position gets set perfectly oxygen comes in forms the iron 3 super oxo iron 2 plus was there it forms iron 3 plus it forms the super oxo this super oxo attack the alpha ketocenter to form the cyclic nice intermediate where it is now iron 4 thanks to the nucleophilic attack on this ketocenter which is then getting O minus by transferring one electron from the iron center oxidizing is to iron 4 plus from iron 3 plus right. Now, these iron 4 plus alkyl peroxo type of intermediate can undergo the oxygen-oxygen bond cleavage wherever whatever way you want to see it with the simultaneous expulsion of the CO2 moiety it gives rise to the succinate bind the iron 4 oxo intermediate which is the real active species for abstracting hydrogen atom from the SP3CH bond right over here this is nothing, but you can see that iron 3 O dot this O dot and this RH will form the bond or form this iron 3 OH bond and R R dot radical will be generated during the process. So, I am sure you are having clear understanding among these different OH and O bond for instance if you have seen earlier this cupril species for example. So, we are saying that cupril is nothing, but CO2 O dot this is exactly same as CO3 double bond O because this will give electron that will give electron to form that. Similarly, for example, if we are talking about iron 4 oxo species this is also nothing, but going to be iron 3 these are same species in different form one can write iron 3 O dot. Now, this as you have seen in the last slide these iron 4 oxo was reacting with RH to give you what to give you iron 3 OH how that is happening because these iron 4 oxo is nothing, but iron 3 O dot and if you are giving RH to this this is forming R dot this is forming H dot and these 2 bonds. So, overall you get iron 3 OH plus R dot hopefully this is clear. On the other hand over here if you want to draw it is nothing, but this bond is breaking and then that is giving and that is forming the R dot. So, that is R dot in any case whether you want to think that iron 4 oxo is a double bond O iron or it is a iron 3 O dot all leads to the exactly same product iron 3 hydroxo R dot iron 3 hydroxo R dot. So, this is the pattern of the radical formation you can you can say the same thing for a copper oxo intermediate or a iron 4 oxo intermediate although these species in a cupril species in the enzymatic setup although proposed are still doubtful in the synthetic setup. Recently some evidences are coming, but still one need to further develop this chemistry in the synthetic setup. So, that they are relevant can be understood in enzymatic setup ok. So, overall I hope you are able to understand this mechanism. So, you are forming the succinate from this alpha ketoglutarate you are getting rid of this carbon dioxide moiety from here and I hope you understood that how it is attacking over there and this is O minus is forming and then this O minus overall is get oxidizing or so, overall oxidizing iron 3 plus let me draw that step again. So, this is here a oxygen, oxygen radical is attacking a keto center. Now this keto center over there will form a radical overall it would be forming O O C O let us say these are oxygen level maybe it would be easier to understood understand. So, that is there these are there, this is iron center. Now this so, I am redrawing that over here. So, this is now attacking and this keto moieties are drawn over there this oxygen, oxygen iron is giving rise to a carbon center and O dot right. This is iron 3 plus, this is remain iron 3 plus and then this iron 3 plus transfer one electron from here to give to oxygen which becomes iron 4 and O minus that is what I am trying to explain. Hope the electron transfer does not throw you off too much and you can count these electrons stepwise without much of a problem. Let me draw that one more time very clearly for you and that is very simple process I hope it is not getting complicated for you. So you start with iron 3 super oxo iron 3 super oxo ok you have a keto center attached with it with you this is the one which is going to form the carbon dioxide in any case I am not drawing the rest of it. So, this intermediate goes on to attack this center right. So, this pi bond will cleave to give you this oxygen iron 3 remaining part remain constant right. So, that is what you see right. So, this is what you see along with the O bond O dot over there. So, this O dot over there is attacking and forming this radical and that radical forming in this bond right. This is the new bond form and this is the radical that is coming over there. This radical and then pick up one more electron from this iron 3 overall to give you this center as iron 4 and this oxygen becomes O minus along with formation of this alkyl peroxo intermediate right. I hope you got it correct without any trouble in understanding right. So, this radical attack at this carbon center and that oxygen radical form one electron transfer from that center to give rise to iron 4 and this carboxylate. Anyway, let us move on what we have seen that these alpha keto glutamate can form iron oxo intermediate very interestingly and very simply then that can go on to react with the substrate to give you the substrate hydroxylation product. The twist here is if you do not have that you know 2 histidine 1 carboxylate intermediate 2 histidine and 1 aspartate intermediate the facial triad the facial triad so called facial triad intermediate if you do not have 2 histidine and 1 aspartate these 3 moiety if you do not have then you are you are in for a quite exciting part and that is just 2 histidine just 2 histidine and 1x the halogen. So, this was aspartate in the last case now this is halogen this halogen will then be reactive with the radical center that is getting generated at iron. For instance, if you are bringing the alpha keto glutamate of course, at alpha keto glutamate in substrate is sitting right over there iron 2 center is there reacts with oxygen binds with it and then form the iron 3 super oxo intermediate iron 3 super oxo intermediate right iron 3 super oxo intermediate then that attack on the carbonyl center of this and subsequently it form the iron 4 oxo with the succinate this is upon decarboxylation of this alpha keto glutamate unit. Now, this iron 4 oxo unit as you can see over here iron 4 oxo unit over here then can abstract hydrogen atom just like what you have seen in the hydroxylation chemistry, but only differences you have the halogen over there. Now, it can abstract hydrogen atom from this substrate to give the substrate radical intermediate along with the formation of OH. Now, there is a competition between X and OH where OH loses because this radical is close to this X and more importantly the reduction potential is helping out as well overall this X selectively transfer over there no hydroxylated product is found. So, RCH2X is form in this case RCH2Cl is form if X equals chloride and if you have you know other things not present or you have difficulties then hydroxylation product may be formed. So, these alpha keto glutamate dependent enzymes are I guess quite exciting not only they are capable of doing the hydroxylation chemistry they can also do the halogenation chemistry right. We have seen that as well. So, what we have seen so far that not only reversible dioxygen binding, oxygen activation is also fascinating right and more fascinating I would say and more challenging more opportunities and more substrate scope can be involved. Iron for firing cytochrome P450 chemistry we have discussed already and summarized it di copper center tyrosinase also we have discussed. We have discussed in the last few classes about MMOs I will briefly mention these again. So, the main reaction we are trying to study is in case of MMO these are going to be a non heme iron center once again do not mix between the heme and non heme iron center. These are going to be the non heme iron center methane is going to be converted into methanol with the help of the oxygen if you are having the leveled oxygen this is going to be leveled over there right that is correct. So, these are fascinating enzyme as you have seen there are going to be two iron center and these both the iron centers are quite exciting over there. These are unsymmetrical as you see the coordination environment around this site is completely different compared to that one which is quite fascinating you have one glutamate here you have two glutamate over there right. Now, if you have seen the earlier mechanism once again the oxygen comes into the picture and earlier mechanism suggest that this oxygen will follow up all the way through to first of all transfer one electron one electron from here and one electron from there to form the superoxo intermediate. This superoxo intermediate can again transfer one electron from there to give you the iron 3 2 minus this is the peroxo intermediate from there on this oxygen oxygen bond can be kept to give you the this meoxo where oxides are 2 O O 2 minus O 2 minus iron 4 and iron 4 it was iron 3 this oxygen oxygen bond cleavage gives you iron 4 iron 4 2 minus 2 minus. Now, the methanol comes and react with this Q intermediate this is the intermediate Q which is responsible for the methane activation to give you the methanol product. There is a slight change or the revised mechanism subsequently this revised mechanism tells that this iron 4 oxo or iron 4 dioxo intermediate will be capable of forming a new intermediate perhaps as you have seen this new intermediate is going to be iron 4 hydroxo oxo iron 3 intermediate although further characterizing this intermediate is not that very easy, but more importantly I believe that that this peroxo intermediate can also be reacting with the methane substrate to give you the methanol along with the formation of di iron 3 di hydroxo intermediate. This peroxo and this bis meoxo this is bis meoxo meoxo intermediates are quite exciting intermediate for this species as you have as we have noticed there that these intermediates are quite exciting if you are following overall if you are overall following if you are overall following up to here this is a 30 milliseconds right 30 millisecond time frame. If you are following overall from here to here this is a 500 millisecond time frame right. So, this is forming this intermediate is forming if you are following for 30 millisecond if you are mixing oxygen with this reduced species within 30 millisecond or right at 30 millisecond you can follow up this one what happens to this what is the decomposition product and this has a characteristic band around 720 nanometer. If you are mixing these species with oxygen and waiting for 500 millisecond this one you form and this is having a very characteristic band at 420 nanometer right. These species are the one we can follow and then react it with methanol a methane to give the methanol product right. So, overall so, overall what you have seen previously is the decay of these say 420 peak or 720 peak can be rationalized can be followed quite easily to give the give the detailed kinetic studies. The detailed studies shows that it is actually more exciting than what we thought Q the intermediate Q as you have seen the bismeoxo intermediate can react with methane to give you a bond making and bond forming step over here where we have seen a oxo hydroxo species is generated. This oxo hydroxo intermediate can either go through a radical intermediate or through a concerted intermediate concerted intermediate is more challenging or more energy demanding therefore, we end up getting a more preferred pathway where this radical species are formed upon this hydroxo electron rearrangement we get hydroxo radical this hydroxo radical and the CH3 radical combines to give you the product. Of course, the alternative pathway is little bit more energy demanding therefore, you do not perhaps follow this intermediate that very clearly right. So, this is a quick summary for these methane monoxygenases you have seen also two types of substrate over there one substrate is the methane and corresponding other substrate which will have the very high kinetic isotope effect right. So, these are having very high kinetic isotope effect as you can see over here these are these highlighted ones right now are known as the class one substrate where methane approach could be could be a could be interesting and simple, but more importantly the CH activation is the rate limiting step. So, the diffusion of methane towards this Q intermediate. So, if you are looking a looking at the Q intermediate which is nothing, but iron 4 oxo iron 4 intermediate this intermediate is is approach of this with respect to this CH4 is very simple nothing, no problem happens over there and this approach overall approach of CH4 with respect to bis meoxo is not the rate limiting, but this activation of this bond is rate limiting for this case of CH4 and CD4. And similarly as you have seen in case of acetonitrile and in case of nitromethane these approach is not problematic, but CH activation is the most damaging or most difficult step. On the other hand for the substrate like substrate like this ethane and the methanol there this approach of this substrate towards this active site is the one which is critical or most challenging and therefore, we will end up getting the diffusion control process. If you are remembering the previous drawing carefully you will note that the for methane this is the black curve this path is the diffusion of methane towards. So, towards the iron 4 oxo species is not really problematic. So, this diffusion or pre orientation of the methane is happening quite easily this can happen quite easily, but this is not so easy if you have approach of ethane and that is how the substrate specificity perhaps is obtained. See this is the same intermediate as this, but if you have H CH2 CH2 that means, CH2 H that means, the ethane this approach of this into towards this is not that very easy and this diffusion control is becoming or diffusion step is becoming the red determining step. As you can see from the methane case this is not very high or alternatively, but the other step that is the CH activation step is much bigger and much stronger. More importantly for methane bond dissociation energy is around 105 kcal per mole for example, for ethane it would be 98 to 99 kcal per mole which makes a huge difference that CH bond dissociation becomes easier and this is also perhaps one of the reason why ethane CH activation will not be problematic one, but reorientation or approach of the substrate diffusing the substrate with respect to the iron 4 oxo intermediate will be critical as you see there. So, for the methane case this is shorter this is taller for ethane case this is taller this is shorter that is the summary from the MMO. So, the substrate dependent chemistry we have seen another exciting part we have we were seeing the 4 manganese 1 calcium cluster. I think the first thing that should come into mind that these are microscopic rivers of the cytochrome cytochrome C oxidase which is hemp copper oxidase which is responsible for converting oxygen into water, but in the photo system you are converting water into oxygen. And the crystal structure as you have seen the gigantic crystal structure have many caveats in it many problem in it, but nonetheless this is the 4 calcium 1 2 3 4 4 calcium 4 manganese center and 1 calcium center that is over there this can be redrawn like that, but although this crystal structure is known, but still this is completely questionable the x-ray damage is the major reason why people do not believe that this is the structure for the oxygen evolving complex. So, what is the structure? Well nobody knows still now these are the these are the major structure that is proposed and believed to be still active, but nonetheless although some except data are there, but still people are debating on these some of these structure perhaps can be ruled out, but perhaps can still be existing in the in the in the discussion, but I think for time being we can or for since this debate is still on we cannot really rule out any of these structure completely, but these are the structure people believe are mainly happening. So, we have seen S 0 oxidation state we have seen S 0 oxidation state S 0, S 1, S 2, S 3 and S 4 oxidation state in each of the steps is oxidation happening oxidation happening oxidation happening and in the final step oxygen oxygen bond formation is taking place. You have seen the oxidation state dilemma between these two these species are still debatable this is quite fixed this is quite fixed this is still debatable and finally, this is the fully oxidized form. There are multiple line spectrum in the EPR which is helping us in assigning some of those spectra always these simulation of this band as well as the experimental observation has to be quite critically done otherwise the conclusion can be problematic different experimental studies are done to understand these intermediate in better better better respect or better clarity, but still their problem exist in these cases. The oxygen oxygen bond forming process as you would imagine that is the most important step and that is that is that is that is the key point of discussion over here. Some believe that it is the calcium hydroxo which is I think is quite reasonable because calcium is the Lewis acid it activates the water molecule to makes its hydroxide and this hydroxide and then can attack on this oxy center, but in other believes that calcium is not really part of, but anyway that debate for another day this again calcium hydroxide either this hydroxo can attack on these bridged manganese oxo or the term terminal manganese oxo still that is questionable. I think we can we can we can we can perhaps live as it is these questions are very difficult to solve at this point and we can move on because in the literature so far no clear understanding is obtained ok. So, at the end what we have in the picture is I think if anything you would like to remember I think this is the case you should remember these next two slides you should remember although this is questionable, but still this is the thing you should remember. What is happening over here? The core over the air these center remain constant ok. This is one of the proposal and this is something I believe is most likely closer to be the truth, but again nobody has seen the real active site. So, what is happening over here is manganese aqua molecule is right over there ok and it is in manganese 2 plus this is center is getting oxidized to manganese 2 to manganese 3 and then this manganese 3 is oxidized to manganese 4. Subsequently this manganese 3 aqua complex further gets oxidized to manganese 4 hydroxo and finally, to manganese 5 oxo which is the real electrophile we are looking at where calcium hydroxo can come and attack. If calcium hydroxo is attacking on these manganese 5 oxo the oxygen-oxygen bond bondage form I think job is done. Well again this is remaining a question mark over there, but this is the one if you have to remember anything for the manganese 4 calcium structure because it is so complicated you have to you have to remember something to better understand it. There are there are caveats there are problems, but I think this is something you should try to remember if the manganese 4 calcium oxo structure is drawn for the oxygen-oxygen bond formation I think this is the structure to be drawn ok. Let us moving on there is another exciting twist in this business and that is the formation of the oxo radical from the tyrosine unit which is proposed in some of the cases in case of blondine that this is the this is the oxy radical which is responsible for hydrogen atom abstraction for this manganese hydroxo species in S3. I think this is this is quite fascinating most likely to be the truth or true, but who knows what is happening, but overall this phenol is forming as you can seen over here and manganese 5 oxo is formed and calcium hydroxo is rightly coupled with the manganese center where this attack can takes place and oxygen-oxygen bond can be formed. Well I hope you are able to make some sort of sense about this complex structure, but you can read always more there are many references given a great chemical reviews and other reviews are available and you are feel you are free to study all those please do understand that these remains still complicated and many question are still there which need to be addressed over the decades to come ok. With this we will see you soon in the final few classes very very soon ok. Thank you.