 to the discussion of cytochrome P450. Today, we will discuss the substrate CAM4 which is right over here this is a crystal structure of P450 CAM, this is one of the really cool crystal structure you can ever come across that mainly due to the fact that the organic substrate is crystallized with the heme iron center. As you can see heme iron center was over there, this is the iron side oil, this is still outside the plane right. So, this is not really in the plane yet, it has to bind with oxygen get ready or get the oxygen reduction going and then only this iron will come inside the plane ok. But the fascinating factor is substrate is right over there of course, there are protein side chain which are omitted for here for clarity. But you note also these binding of the axial ligand or the proximal ligand this is the proximal side this is the distal side once again. So, the proximal side of the that is the axial center we have this sulfur coordination from the cysteine which is fantastic see that also interaction of the other side chain with this sulfur or S minus right. So, this is from journal of inorganic biochemistry and the references therein and you see that this CAM4 substrate overall which is over here it is redrawn over here and selectively this CH which is the XO1, XOCH is getting abstracted and then subsequently hydroxylated. So, CH at bond abstraction or H dot abstraction and then hydroxylation occurs to gives rise to the 5 XO hydroxy CAM4 unit. So, which is very very fascinating right CAM4 as a substrate for cytochrome P450 is quite exciting as you can as you can see over there you need still the overall process with need 2 electron because we are starting from the resting state of the enzyme which is iron 3 plus 2 electron each of those are supplied by the NADH in that pool of electron pushing or gushing towards the towards the cytochrome P450 sites you still need also 2 proton and nothing will happen with within absence of oxygen. So, oxygen 2 proton 2 electron gives rise to this water and the substrate hydroxylated product as we have seen the mechanism before we will discuss one more time very very quickly here. So, this is where we start this is the iron 3 aqua complex right this iron 3 aqua complex also known as the resting state of the enzyme it reacts with the organic substrate I would not say it is a reaction it is just a displacement of water molecule in presence of the substrate when the substrate comes a come seen water molecule goes out. So, substrate sits very close to the active site and then this iron 3 sites gets reduced to iron 2 plus in presence of the substrate over there still I would say these centers are outside the porphyrin ring now once this iron 2 is formed still although outside the porphyrin ring oxygen will then react with it react with this iron 2 which is fully capable of doing the reaction with oxygen to form the iron 3 superoxo iron 2 note here is getting oxidized to iron 3 plus the electron from this is reducing the oxygen. This electron is coming from NADPH as you have seen through the iron sulfur cluster and all the way to the iron center. So, these extra electrons are coming from outside, but as you have seen these oxygen is forming superoxo by taking the electron from the iron center now the superoxide species this is iron 3 superoxide species can then also be further reduced by another electron once again from the NADPH or that electron transfer conduit overall channel of the electron transfer. If this electron transfer gives rise to the iron 3 peroxo intermediate this iron 3 peroxo intermediate if you look at the oxygen with respect to this oxygen this is doubly reduced it is a peroxo species now this peroxo species is ready to accept another proton or one proton. So, this proton gives rise to the intermediate compound 0 which is nothing, but porphyrin iron 3 hydro peroxo species which is I think is quite exciting intermediate because once this species is formed it is kind of getting ready to react with the substrate. See throughout this cycle you see the RH is sitting pretty and nice over there as you have seen the crystal structure that this is sitting very nicely in front of the active site. Now, we see that this RH is getting ready and of course, RH is ready, but the species was not ready now this active site is getting ready and another proton is again required to cleave the oxygen-oxygen bond between the iron 3 hydro peroxo intermediate. This oxygen-oxygen bond cleavage would give rise to the iron 4 oxo intermediate iron 4 oxo intermediate by removal of water from this equivalent right. So, water goes out and so, proton comes in water goes out iron 4 oxo species is formed and a radical cation is also generated during the process. So, overall it is a 2 electron process. So, one electron comes from this porphyrin another electron from iron. So, iron 4 so, that is where the double bond is formed. So, this double bond is formed by taking one electron from the porphyrin and taking one electron from the iron 3. So, this double bond is formed and this hydroxo goes out as hydroxide HO minus. So, HO minus is getting protonated to give the water molecule and this iron 4 high valent oxo intermediate with radical cation on the porphyrin ring is getting generated. Now, at this situation this is a really really reactive intermediate. This is a super reactive intermediate abstract hydrogen atom from the RH to give you the iron 4 hydroxo species iron 4 hydroxo species. And during this process you can see that RH can be homolithically cleaved R dot gets generated that H dot electron can be quenching this porphyrin radical and during this process we see that iron 4 hydroxo is getting generated. Now, at this point R dot sitting with iron 4 hydroxo this hydroxo can be transferred to the R dot and then this 2 electron from this bond 1 electron goes to the OH if you are thinking homolithically and another electron comes to the iron. So, iron becomes iron 3 and this becomes hydroxy radical this hydroxy radical binds or combines with R dot to give you the ROH molecule right that is fantastic. Now, this water molecule displaces the ROH to slowly but steadily form this iron 3 aqua complex which is nothing, but the resting state of the enzyme. Note that this is where the radical is getting generated and that is the origin of the radical mechanism overall that we see over here. Now, in case of the peroxide shunt where mechanism where iron 3 is getting generated you do not need all the way travelling by reduction and oxygen activation and one can directly react with this iron 3 to form the iron 3 hydro peroxo intermediate. In other word this hydrogen peroxide reacts with iron 3 to gives rise to the iron 3 hydro peroxo intermediate let us discuss that intermediate quickly. So, we will discuss how these species are forming in presence of in presence of this iron iron 3 species. So, if you have iron 3 and if you are reacting with hydrogen peroxide this is your hydrogen peroxide right. So, if you are bringing a base in the medium so, in presence of base deprotonation will be possible now this is just like a acid base reaction. So, it can form the iron 3 hydro peroxo species. If you have a base available hydrogen peroxide this sort of reaction is quite done in in terms of iron also and also in terms of copper. In case of copper it would be copper 2 plus hydrogen peroxide 2 gives rise to the in presence of base it can be this sort of reaction can be done even in synthetic setup. So, that can form the copper 2 OOH. In these cases you do not need to reduce the iron 3 to iron 2 and react with oxygen it has to have the hydrogen peroxide present. So, and essentially the same intermediate which we were seeing earlier same intermediate can be generated by these processes and therefore, these processes are quite effective in in their nature. Overall you form the iron 3 hydro peroxo alternatively you are seeing that electron is coming getting reduced to iron 2 it is getting or it is reducing iron 3 to iron 2 and then oxygen activation was happening. So, that happens when oxygen molecule is present in absence of oxygen or in absence of the electron which is reducing the iron 3 if hydrogen peroxide is available right over there into into the mix it will go on to form the same iron 3 hydro peroxo species and once iron 3 hydro peroxo species is generated one can then go on to the normal catalytic reactivity of cytochrome P450 right. So, that is a peroxide shunt mechanism or the alternate mechanism. In synthetic setup also one can start with a ligand iron 2 and iron 3 and ligand copper 2 complexes and can react with hydrogen peroxide to form such species. Alternatively it is also possible to react with ligand iron 2 and hydrogen peroxide to form a quite interesting intermediate. So, you can have a homolytic cleavage over there. So, 2 equivalent of this compound can gives rise to the 2 equivalent of ligand iron 2 and the hydroxy moiety right. So, OH radical here OH radical there iron 2 gives rise to another electron to form the iron 2 hydroxy bond. Now, once these iron 2 hydroxo species is formed they can act as a base the base we were talking over here similar type of base it can gives rise to the water molecule or H dot iron 2 dot overall it can then give you the this is hydroxide H plus then overall you can again get the iron hydro peroxo species formation. That is quite amazing I would say and this is where I think this is this is going to be of course, this is going to be iron 3 hydroxy this is going to be iron 3. So, iron 3 OH this gets oxidized in the process iron 3 OH plus hydrogen peroxide overall gives rise to the iron 3 hydro peroxo species. Similarly, if one is reacting or let me take another phase here of course, that is not really relevant over here just for discussion I would like to quickly mention here that if you are starting if you are starting with copper it is also possible to do such reaction right. So, where you can take copper 1 complex just like iron 2 complex and react it with HOOH this reaction need not be very clean sometimes many side reaction also occur in synthetic chemistry of course, you have ligand copper 1 there no metal ions are completely free if no no ligand such as bidentate tridentate tetradentate ligands are there it could be a monodentate ligand if nothing is available water is there water can water can form complex with the metal ions. So, it is always coordinated with something made no metal ions are free completely free in the biological condition right. So, this ligand copper 1 complexes can react once again similar to the iron species in an equivalent manner. So, this copper 1 gives 1 electron hydroxo homolytic cleavage homolytic cleavage gives you hydroxy radical and hydroxy radical 2 of them can give rise to 2 L copper 2 hydroxo species right sorry this is hydroxo this is hydroxo this hydroxo this hydroxo this hydroxo goes over there. Now, once you take 2 equivalent this copper 2 hydroxo species and then reacting with hydrogen peroxide then this water molecule goes out and it is just a ligand exchange you can say that hydro peroxo copper 2 hydro peroxo is getting generated copper 2 OH is getting generated. This is similar to what we have seen in the iron cases also of course, these are the mechanism that by which these iron 3 hydro peroxy or copper 2 hydro peroxo species can be generated. Now, that can only happen when this sort of hydrogen peroxide reaction mechanism can only happen when we have the problem and that problem is either the electron is not available or oxygen is not available. So, there in absence of these if hydrogen peroxide is existing that can gives rise to the peroxide shunt mechanism nonetheless this compound 0 is going to be very reactive under that condition. Once it is formed it can go on to form compound 1 which is nothing, but iron 5 oxo iron 4 oxo radical cation and then compound 2 which is the iron 4 hydroxy compound which are again very great compound to have. We will see the same catalytic cycle one more time in little bit different context from another review. So, this is by NAM and co-workers where we see that the same reaction mechanism essentially, but this is depicting the iron center outside this is depicting the iron center outside the box right outside the porphyrin moiety which is the case actually this really nicely depicts how the iron center is. So, this is the porphyrin sites it is abbreviated as a flat line over here iron 3 plus it is it is really outside the outside the cavity of the porphyrin it is bound with a cysteine moiety and the R H at the at the distal site right over there sitting and sitting idle this is the resting state of the enzyme this is the resting state of the enzyme another electron comes in and you get it reduced, but still iron is outside the porphyrin ring R H is sitting pretty right over there and then we have the oxygen coming in first of all of course, oxygen first will bind with this center binding will not cause too much of a difference, but the electron transfer once the electron transfer is occurring. So, the sixth ligand has come because this is a 4 ligand system the sixth ligand system has come this is the 5th 1 4 from here 5th and 6th here and that oxygen is now reduced by iron to give the iron 3 superoxide this is the same mechanism what we have seen in the last slide as well right. So, from there on another electron transfer gives rise to the iron 3 peroxo as we have discussed in the last slide and the protonation gives rise to the iron 3 hydro peroxo species with the cysteine bound and further protonation double protonation you see electron transfer electron transfer protonation protonation all these things are happening in between only oxygen activation has happened. So, it gives rise to the water molecule and these iron 4 oxo radical cation. Now, this is quite interesting over here as you can see the radical cation will be we will be right over here into this porphyrin moiety right it is delocalized into this porphyrin moiety and this porphyrin moiety is quite interesting that it does not really allow a iron 5 oxo formation right away. So, it helps this overall process because iron 5 oxo formation could be little bit more energy demanding and that is why the porphyrin is participating. You see that is how beautiful the nature's strategy is when the reactive intermediate is needed to be really high valent oxidized intermediate then nature decides to oxidize even the porphyrin ring which is feasible and it ends up doing it instead of forming iron 5 oxo it decided to keep it still at iron 4 while another center like another porphyrin center can be oxidized to a radical cation. I think that is amazing to be able to control these things at a level where enzyme once perfectly is quite phenomenal and we will see such beautiful chemistry once again or again and again in different enzymatic setup. Subsequently these iron 4 hydrox oxo species can then pick up the hydrogen atom from the some from the RA substrate and at this is the this is when we get the ROH formation. So, all these steps where ROH is required it is just acting as a substrate without much of the action this is where it gets into action and gives rise to the product formation ok. So, I hope the mechanism of cytochrome P450 is quite clear right and we have seen that how beautifully these reactions can be can be propagated and can can can can be summarized very very nicely right alright. Let us let us get out of the reaction mechanism let us see some of the subtle things in here ok. We have seen these reactions earlier in few slides back of course, this that is not an exhaustive list neither is this one, but this slide gives an overview of various other reaction that you may not have seen earlier can do or can be can be transformed or can be catalyzed by cytochrome P450 enzyme. This is once again from this review 1996 you can look it up and you can see that there are various different type of reaction that is possible. So, far we have discussed the first few reactions ok. So, we have not discussed about other reaction it is actually gets similar. So, oxidative deamination reaction is also possible when you have an amine the next alpha carbon center is getting hydroxylated and subsequent rearrangement at this center can give you the ketone formation and the ammonia generation which is fantastic. If you have similarly and halogenated compound alpha position alpha to the carbon center alpha to the halogen, halogen can be also hydroxylated and subsequent rearrangement can give you the ketone product as well. You can take an alcohol or aldehyde you can hydroxylate the alpha position of the alcohol and can give the dihydroxy compound alpha alpha dihydroxy compound which can which can which can then dehydrogenate to give you the ketone compound. See all these cases all these cases you end up getting the ketone compound if you have alpha carbon center alpha to amine alpha carbon center alpha to halogen alpha carbon center alpha to hydroxy all these cases will end up hydroxylating the enzyme perfectly and that is what the beauty of these reactions. Overall these can gives rise to the ketone product formation in all of those cases, but fantastically all these reaction can be done again these over reactive cytochrome few before as if it is really really anergyzed. So, that can sometime be a disguise sometime can be a sometime can be a very useful, but more often it is it can also act as a nuisance anyway. So, you can also it can take the aldehyde as a substrate aldehyde substrate this alpha CaH bond can be also hydroxylated to give the acid compound that is once again quite interesting if you have even an aliphatic substrate in addition to the hydroxylation reaction it is also feasible to of course, abstract hydrogen atom that anyway happen during the hydroxylation and then with a suitable substrate it is possible to give the give the unsaturation that means, a double bond is forming and alkene can give rise to the alkene by utilizing this method. If you have the parahydroxy any salt acetyl light substrate type of substrate with 2 electron oxidation and 2 proton transfer you can also get these semiquinone type of or quinone type of intermediate iminoquinone type of intermediate. It is also possible to undergo dehydration such as as you see over here dehydration reaction on this one will in hydroxy imine will will give rise to the cyanocompound or acetonitrile type of compound. If you have the alpha position hydroperoxylated next to the olefin you can also react it with to form a nice epoxide alpha to the olefin center which is quite exciting. You can have a series of other reaction as you can see you see all those reaction we will not be discussing here today, but that that gives rise to a number of reaction formation which is which is fascinating I would say. So, this the horizon the spectra by which this reactions are happening I think are I think this is this is quite remarkable none no other enzyme can be this effective as the cytochrome P450 is. So, no other enzyme can do the better synthetic chemistry perhaps than the cytochrome P450, but the challenges remain the over reactivity getting these reaction very selectively and of course, efficiently without forming other side reactions is always challenging and it remains a challenge. And by looking at these a synthetic chemist has tried to develop synthetic methodology that really can mimic these reaction these are by far the most difficult and most encouraging chemistry that one can have one can find from the enzymatic setup and again this is really by utilizing oxygen molecule as a reactive species or part of the part of the reactive species ok. So, we will see few more a few more cytochrome P450 there are many different variants of it depending on the substrate, depending on the subtle variation one can think of utilizing different substrate. For example, over here you see this is an aldehyde substrate aliphatic aldehyde you can define it now this iron III species iron III species can react with it to give rise to a. So, this is the iron III peroxo species once again this is from this review you can look at original preparation from there the references cited there in we you can see that this peroxo species can react with aldehyde to give the iron III hydro iron III alkyl peroxo intermediate this is a peroxo iron III peroxo forming an iron III alkyl peroxo intermediate which can then rebound to give you or then can react to give you the formic acid this is you know overall you can see that deformylation reaction is happening. This is once again a very very fascinating enzyme which or variation of cytochrome P450 which gives the even the deformylation reaction if you have an aldehyde molecule or aldehyde moiety can be completely removed from the organic molecule to give rise to the give rise to the simple organic molecule or deformylated or deformylated organic molecule. There is another example right over here in the previous example as you can see the estron can be generated by such process of starting with 19 oxoendrostendion by the effect of cytochrome P450 aromatics one or and then you can have another organic substrate which is similar but it is little different this is 32 oxo lanosterol you can get really beautiful reaction similar to the deformylation reaction above you have seen. Here once again the iron III peroxo attacks on the aldehyde moiety to give rise to the deformylation reaction once again forming the formic acid. We will we will just keep on discussing on cytochrome P450 in the next class. I hope you are able to see that these reactions of cytochrome P450 are very very simple yet very effective. They are forming a nice high valent iron oxo intermediate which is capable of doing any kind of oxidation chemistry that you can think of right. These chemistry are so powerful that once again pharmaceutical industry has to be worried extremely about this reaction because any drug molecule any organic molecule they want to put in our body to solve our problem it could be metabolized it could be degraded it could be you know taken to the task by the cytochrome P450. Cytochrome P450 can be seen as a as a as a as terror at some point to the to the pharmaceutical industry. These are so good enzyme I mean so versatile at such a great synthetic chemistry component can be there in cytochrome P450 that it is unbelievably clean unbelievably great chemistry and we will keep on seeing this in the next class. Thank you very much see you soon.