 Welcome back to the discussion of cytochrome P450. In the last class we have seen the reaction mechanism and this is really an exciting mechanism where you have seen that iron hydroperoxo is being formed and then oxygen-oxygen bond is cleaved in the process to give the high valent iron oxo species. Now, you must be wondering what is the difference between the let us say hemoglobin myoglobin and cytochrome P450. Of course, in case of hemoglobin myoglobin you have up to this iron superoxo species, but none of these steps are happening in hemoglobin and myoglobin. Now, what really differentiate hemoglobin myoglobin with and that of the cytochrome P450? Yes, you have got it right the cysteine this axial ligand that proximal ligand compared to the histidine that we have seen in case of hemoglobin let us say right. Another thing is of course, there are electron transfer processes and proton transfer processes which which can also be there, but in mainly in cytochrome P450 I think this axial ligand is varying and that makes a lot of difference. If this axial ligand was histidine this oxygen-oxygen bond cleavage may not have been that easy. Let us look at therefore, then what is the role of this cysteine ligand for this oxygen-oxygen bond cleavage ok. So, overall as you can see that this cysteine ligation to the iron III center actually helps in a multiple way. One of the thing that it definitely helps that for cytochrome C there are lot of side chain and this proton conduit that helps overall in protonating such a iron III peroxo intermediate right. So, this iron III peroxo intermediate is getting protonated with the help of of this proton conduit. So, this charge relay through this trionine 252 and aspartate 251 is suggested for the proton conduit to deliver proton to peroxo before oxygen-oxygen cleavage ok. So, this peroxo unit itself without this protonation that means, the peroxo unit itself that means, without this protonation it cannot undergo the oxygen-oxygen body. So, this peroxo unit itself without the protonation cannot undergo the oxygen-oxygen bond cleavage. So, the peroxo unit has to be protonated in the process and that protonation is helped by this side chains different side chain that is present over there. You see there is a big relay process that is happening by which this protonation is taking place right. So, the side chain definitely helps in the overall process of the cytochrome P450 mechanism and more importantly this cysteine can push electron through this S cell iron and through the iron-oxygen bond and therefore, the cleavage of the oxygen-oxygen bond becomes much more facile. Has it been the histidine then this push would have been missing and therefore, this oxygen-oxygen cleavage would have been much difficult. So, the histidine is not a negatively charged ligand. The negatively charged ligand cysteine thiolate helps overall in breaking the oxygen-oxygen bond. So, the cysteine thiolate provides strong axial donation to the iron center referred to as big push and therefore, facilitates the oxygen-oxygen cleavage and stabilizes high valent iron center. Overall, not only this cysteine big push cleaves the oxygen-oxygen bond it also helps in helps in stabilize the high valent iron-oxo intermediate that is going to be formed at this iron center. So, just to summarize this part therefore, the role of this cysteine thiolate versus hemoglobin myoglobin this histidine is quite crucial. Nature has utilized heme center in both the cases as you can see both in hemoglobin and myoglobin and cytochrome P450 there is heme center for both reversible oxygen binding and oxygen activation. If you are looking carefully for the hemoglobin myoglobin this is a reversible oxygen binding there is no substrate sitting close to this hemoglobin and myoglobin therefore, just very efficiently it can transport oxygen without doing any oxygenation chemistry with the any organic substrate. So, this is the same species the common intermediate for both hemoglobin myoglobin and cytochrome P450 despite having the common intermediate this case it is just transport oxygen or reversibly binds oxygen it does not do any oxygenation chemistry. On the other hand cytochrome P450 obviously, forms the same intermediate, but it engages the substrate as you have seen many different substrate in in taking taking the oxygenation reaction for forward right. So, this this is quite amazing by utilizing the same cofactor the iron for firing nature have decided to do completely different function in one case it is transporting oxygen in another case it is making the oxygen reactive to do the oxygenation chemistry. In other word by having or by changing the axial ligand from the histidine to cysteine it is possible to tune the heme function and therefore, both the oxygen oxygen cleavage and the stabilization of iron oxo species which are high valence species can be possible in cytochrome P450. As you have seen protein side change also helps overall in the process to provide to provide the required environment for the protonation and overall process of the oxygen-oxygen cleavage as well as the substrate binding, substrate orientation everything helps to make cytochrome P450 and very effective and effective effective a very effective enzyme ok. So, I hope it is now clear that why nature has chosen the different axial ligand for different enzyme in case of cytochrome P450 it is one axial ligand everything else remains same in case of hemoglobin myoglobin it is another axial ligand ok and you have seen the reason behind it ok. Now, we are going to see the you know reactive intermediate in case of cytochrome P450 well as you have noticed that we have proposed a number of intermediates. Now, is there any support in favor of those proposed intermediate or these intermediates are going to be extremely difficult to crystallize only I think reliable data you can get perhaps through the spectroscopic technique. For instance resonance Raman, X-Aps, Jazz or even visible low temperature definitely is one of the most characteristics characteristic feature or characteristic thing you can get. But many attempt has been made to make these material crystalline and use different trapping techniques by which the intermediate perhaps can be trapped. Now, there is cryo crystallography lowering the temperature and doing the crystallography under super cool condition, but then these techniques has its own limitation and more importantly if the crystal structure or crystal quality is not great enough the conclusion that can be drawn from those study can be problematic ok. There are many issues controversies, controversies, caveats of these cryo crystallographic studies and the trapping technique that has been used for these for these for this cytochrome P450 intermediate studies, but nonetheless still it can shed light about the putative intermediates. Once again these intermediates are crystallographically although some of them are characterized, but still they are not free of debate. That is particularly because the the resolution that is the resolution that one can get and therefore the conclusion that can be drawn at a particular iron oxygen center or iron center regarding its regarding its existence I think can be questionable. Well, despite having that let us see this is a porphyrin iron center as you have seen this crystal structure before iron is sitting outside because this is this is not in the plane yet it gets inside the plane only upon oxygen binding and then reduction to the super oxygen ok. It is only gets into the plane only upon oxidation to iron 3 plus and binding with oxygen. So, this is the crystal structure we have seen before and here is the here is the organic substrate camphor that is placed right in front of the active site. Now it has been proposed that there is a iron 4 oxo intermediate and it has been crystallographically characterized. Now once again there is a debate, but that debate is whether this is really iron 4 oxo or an aqua molecule although the crystal structures can be informative in this case it cannot resolve beyond doubt that this is a high valent iron oxo intermediate, but it looks quite reasonable though as you can see the bond length and bond length is shorter, but perhaps not short enough and overall it seems like it is an it is the intermediate for what has been suggested for the cytochrome P450 compounds ok. There is yet another intermediate which shows that iron center is bound with oxygen. So, these are all remain quite interesting intermediate and some of them can be debated, but these studies are very difficult to do and therefore, it could be highly rewarding, but perhaps still it is not 100 percent clear how these things must be happening in the enzyme and this is where synthetic studies can be quite useful. If one can have the crystal structure of such compounds then the matching of the data with that of the cryocrystallographic technique of the enzyme can be extremely useful. So, let us look back the mechanism once again. So, what has been proposed that this iron 3 plus is getting reduced to iron 2 plus first till it is not part of the complete for firing plane upon oxygen binding and electron transfer it forms the iron 3 superoxo it gets into the cavity of the porphyrin and then electron transfer protonation gives the iron 3 hydro peroxo species. Now, this oxygen binding or superoxo intermediate structure or the peroxo intermediate structure these intermediates remain quite exciting to crystallographically characterize, but overall this intermediate is suggested to be crystallized, but again although crystal structure is quite definitive usually, but in these cases there is there is still debate whether this is really the intermediate that is been trapped, but nonetheless this mechanism has been supported by many different groups and evidences in support of these intermediates has been collected although crystallographic characterization remain bit controversial. We will come back some of these iron oxygen chemistry pretty iron hydrogen peroxos chemistry pretty soon just to remind you that these cytochrome P450 once again is quite exciting enzyme. In the next part of today's lecture we will discuss peroxidase and catalase ok. So, you know these are once again the him iron center and these makes quite an exciting story for themselves because they can convert also intermediates into quite fascinating examples ok. So, for instance you can start just like cytochrome P450 peroxidase are starting with iron 3 aqua complex it is in ferric resting state ok. As you can see of course, it would be it would be out of the plane and aqua molecule is over there. So, this is in resting state in presence of hydrogen peroxide just to remind you as we have discussed iron 3 can react with hydrogen peroxide to give the iron 3 hydro peroxo species right that is the compound 0. So, we have discussed it in the peroxide shunt mechanism. So, this is the peroxidase chemistry one equivalent of hydrogen peroxide gives rise to of course, displaces one water molecule and gives rise to the iron 3 hydro peroxo species. This hydro peroxo species upon protonation of this hydroxide unit terminal hydroxide unit gives another equivalent of water and it forms the iron 4 radical cation also intermediate ok. So, this is the same chemistry as you have seen in case of cytochrome P450, but these are completely different enzyme or in other words cytochrome P450 in absence of suitable oxygen and the reductant these species can be formed. So, these are a completely different class of enzyme which are fascinating which converts hydrogen peroxide into corresponding water right. It can be instead of hydrogen peroxide it can take an alkyl peroxide in presence of the hydrogen atom donor it can convert into the alcohol if it is let us say alkyl peroxide corresponding alkyl alcohol alcohol will be formed along with the formation of water molecule. So, once again we start from ferric resting state react with we will we have a water binding this hydrogen peroxo reaction with iron 3 gives rise to the iron 3 hydro peroxo intermediate. This displaced water is one of these water and then the protonation on this hydroxo terminal hydroxo gives rise to the gives rise to the another water molecule. That proton source can be one of the terminal proton for from the hydrogen peroxide right. Overall it forms these iron 4 oxo radical cation intermediate which is nothing, but as we discussed iron 5 oxo as you know that these iron 3 hydro peroxo it can be called as compound 0 this iron high valent oxo intermediate can be called or is usually called in the literature as compound 1. So, radical as we have discussed in the last place and seen that radical will be on the heme site ok on the heme usually or it could be a tryptophan or in trirosine. Overall it would require still one electron and one proton that means hydrogen atom to convert these high valent iron oxo into the iron 4 hydroxo intermediate ok. We have we have seen this before in the same way in cytochrome P450 this compound iron 4 hydroxo without the radical cation on the porphyrin is known as the compound 2 ok. So, compound 0, compound 1 and compound 2 from there on another equivalent of the hydrogen atom which is the combination of electron and proton will gives rise to the generation of the ferric resting step. So, overall as we discussed that if it is hydrogen peroxide then it will end up giving 2 equivalent of water. If it is alkyl hydro peroxide it will end up giving one equivalent of alcohol and another equivalent of the water molecule. So, this peroxidase chemistry comes into the picture when there is let us say even for cytochrome P450 when there is no oxygen available or reducton available these sort of chemistry start kicks in, but this peroxidase enzyme is quite effective in converting the hydrogen peroxo into water and this is useful because hydrogen peroxide can do quite a lot of other side reaction in absence of such mechanism of the peroxidase chemistry. Next, let us look at the catalase chemistry. Catalase this is the catalytic cycle of catalase once again we start from the ferric resting state. This is the iron 3 plus water molecule the ferric resting state and in this particular case we will convert once again similar to what we have seen we have taken a hydrogen peroxide and converted into 2 equivalent of water just via this iron 3 hydro peroxo intermediate as we have discussed in the peroxidase cases. It forms this high valent iron oxo intermediate iron 4 oxo with the radical cation intermediate. Now this radical cation iron 4 oxo intermediate can then go back to the resting state directly without having those 2 hydrogen atom if enough hydrogen peroxide is present right. If enough hydrogen peroxide is present then this hydrogen peroxide can give another equivalent can give water and oxygen to regenerate the iron 3 hydroxyl aqua species. So, catalase catalyzes the composition of hydrogen peroxide. The second H 2 O 2 is responsible for the 2 electron reduction of iron 4. So, this is of course, this is essentially iron 5 because this is a radical cation is there. So, the second H 2 O 2 that means, this one is responsible for reduction of 2 reduction by 2 electron to form this high valent iron intermediate to iron 3 intermediate. The iron 4 hydroxo that is getting generated in the peroxidase chemistry that can still form in here if insufficient amount of hydrogen peroxide is available. So, the amount of hydrogen peroxide will determine whether this species is directly going back to there or this iron hydro peroxo species is getting formed into the process. So, this settling between these 2 intermediate will be clean if enough hydrogen peroxide is there. If enough hydrogen peroxide is not there still this intermediate can start kick in ok. And these are in lot of cases these catalase activity are dependent on NADPH those are called NADPH dependent catalyzes which actually essentially ensures that the electron reduction or electron transfer to this site occurs without any trouble even if there is a deficiency in hydrogen peroxide concentration or amount of hydrogen peroxide. So, what we have seen so far in the peroxidase and catalase catalase cycle is essentially it is a page out of the cytochrome P450 chemistry. It has in particular for peroxidase chemistry it has compound 0, compound 1 and compound 2 which is exactly same as what we have seen in cytochrome P450. But for peroxidase chemistry it is essential to have 2 hydrogen atom donation from a source, but in presence of such 2 hydrogen atom donation hydrogen peroxide can be converted to 2 equivalent of water. Not only hydrogen peroxide it can take care of any alkyl hydro peroxo in the process. As you can see compound 0, compound 1 and compound 2 all are part of the peroxidase cycle just like what you have seen in case of cytochrome P450. The source of hydrogen peroxide or alkyl hydro peroxo will depend what type of chemistry or what type of product is forming over there, but for catalase chemistry it is only usage of or use of hydrogen peroxide no alkyl hydro peroxide will be involved. Now these hydrogen peroxide will cleanly form just the oxygen and water molecule 2 equivalent of hydrogen peroxide will be converted to oxygen and water molecule ok. So, this is quite fascinating provided there is enough hydrogen peroxide is involved or enough hydrogen peroxide is available this catalase cycle is quite exciting and we will be able to degrade hydrogen peroxide quite efficiently to oxygen and water. In absence of enough hydrogen peroxide there is always a possibility of forming this high valent iron 4 hydroxo intermediate, but this sort of intermediate prevents formation in the in the catalase activity can still be still be shut down if these are NADPH dependent hydro peroxidases right or sorry catalases if these are NADPH dependent catalases then it is still ensures that this is the catalytic cycle that is being formed. So, to summarize what we have seen today that catalase is the easiest enzyme to kind of follow it settles between the iron 3 aqua molecule of course, going by iron 3 hydro peroxo to form a high valent iron 4 oxoradical cation and these two intermediate resting state and the active substrate settles among each other very quickly, but for that to happen you need to have enough hydrogen peroxide present and NADPH can be also be of help. In the case of the peroxidase it is just the peroxide is important in addition there is also this hydrogen donor hydrogen atom donor right that comes into handy. So, as you have seen these all these chemistry has relevance with respect to cytochrome P450 as well. Cytochrome P450 mechanism let me take you there once more very briefly we can we can have over here as you can see for peroxidase chemistry it is settling here. This is the chemistry we are seeing for cytochrome P450 we can go through all these way for in case of catalase chemistry it is the settling between this state and that state ok and of course, it goes via the hydro peroxo, but so it can be this intermediate to that intermediate to that back to that ok. So, this settling is quite interesting see it is part of the bigger cycle only, but it is still still still settles. So, cytochrome P450 mechanism is exactly the circular one peroxide shunt or the peroxidase mechanism would be over here this is the peroxidase mechanism for the catalase mechanism it is going to be from here to there to there and back there. So, this 3 intermediate iron 3 compound 0 although it is transient and then compound 1 back to the iron 3 resting state right. So, you see the bigger picture it is very simple and clear once again cytochrome P450 goes by this mechanism peroxidase goes by this mechanism hydrogen peroxide is converted into water completely to equivalent of water by this process and catalase mechanism will be from here to there to there and back in there ok 1, 2, 3 only these 3 are involved ok. Well, as you can see there is always a possibility of drifting little bit, but the NADPH and the concentration of hydrogen peroxide ensures that none of these are happening way too much ok. With this I would like to conclude today our discussion cytochrome P450 and peroxidase and catalysis keep studying I hope it is getting clear and these are really interesting enzyme and they as you have seen can really do quite a lot of beautiful chemistry. So, we will be back soon keep studying cytochrome P450 and other enzymes.