 How are you all doing? Today we are going to discuss general mechanism and the some of the reactions of alpha Kg-dependent or alpha ketoglutarate dependent dioxygenases as well as halogenases. In the last class, we have seen the reaction mechanism for these dioxygenases right, where we have seen that how the enzyme is dependent on alpha ketoglutarate and then how the high valent iron oxo species is generated from this alpha ketoglutarate dependent enzyme. So, this is the alpha ketoglutarate and it is bound with the iron center, then oxygen activation takes place and it attacks on the keto center overall alkyl peroxide type of intermediate and subsequently oxygen-oxygen bond cleavage gives rise to the iron 4 oxo intermediate which then can react with organic substrate right. So, we have seen that organic substrate can be hydroxylated by using this mechanism or by following this mechanism where R dot and OH reacts with each other to give RoH right. Now, let us look at a little bit more on this, we will see one crystal structure how these sort of intermediate looks like. Let us say one of these we will see how it is looking like when alpha ketoglutarate is bound with iron and the organic substrate is also appended right next to or right at the active site ok. We will see tau D, this is active site of tau D with bound substrate. So, this is the tau ring that is the organic substrate and this is the aliphatic substrate that we wanted to get hydroxylated right. As you can see this is the iron center and two of the histidine and another aspartate is right over there right. This is called facial triad motif. So, three of these substituents are right next to it. This is the alpha ketoglutarate as you can see it is bound and overall the one coordination is vacant over here where tau ring is approaching of course, it is not coordinated. Now, the oxygen activation will takes place and the binding will takes place at this axial site right. Now, as you can see tau ring is right next to the iron center that means, it has the capability to reach out to the high valent iron oxo species that is getting generated right over there. One of the thing you must be noticing that in addition to tau ring here is a tyrosine which is not a you know desired substrate to get hydroxylated because its job is to hydroxylate tau ring, but when the tau ring is not present then what happens? We see quite a lot of reaction at tyrosine we will come back to that later. Let us see that what we can do or this sort of mechanism is quite familiar when the tau ring is not there actually tyrosine gets involved into the reaction right. So, that is quite unusual I would say, but what you can conclude is since the iron oxo species which is getting generated over there it is so reactive if the organic substrate that needs to be hydroxylated is not positioned perfectly or missing then tyrosine is going to be the substrate and tyrosine to catechol formation can be done. We will see that in a separate slide and the mechanistic study on this we will discuss. While this is also happening if these you know this sort of reaction also happens when you when you do not have alpha ketoglutarate present if you have this succinate which is originating from alpha ketoglutarate upon decarboxylation as you have seen in the last slide the succinate is there and then one can also get different type of mechanism ok. Let us look at this mechanism one more time little quickly. So, this is the alpha ketoglutarate and if the organic substrate this one is not there this tyrosine will come into picture in absence of the R H or in presence of R H how things are going to be let us quickly look at. So, this superoxo intermediate is generated iron 2 reacts with oxygen let us say this is a leveled oxygen iron 2 superoxo is getting generated then that attaches or attacks on the keto moiety right fantastic that attacks on the keto moiety and you have this you know beautiful ring compound from here on the rearrangement reaction gives rise to this intermediate where you have a iron 4 oxo species generation. Now, this iron 4 oxo species in absence of taurine or the suitable substrate it can abstract hydrogen atom from this tyrosine right tyrosine phenolic OH it becomes tyrosine radical and then hydroxylation can go on we will discuss this again in few slide. Now, in presence of substrate like this R prime H, R prime H if it is sitting right next to it ROH formation will be going on decarboxylation can lead to alpha ketoglutarate to succinate of course, carbon dioxide also will come out and the overall catalytic cycle can be completed by the regeneration of iron 4 to iron 2 right. So, so far so good. Now, let us look at a little bit related, but I mean much related lot different outcome reaction mechanism and the substrate halogenation chemistry right. So, there is a surge of enzyme or surge of reaction that can happen if you have little bit twist into your active site. What is that? Well, you we were having 2 histidine and 1 aspartate right previously with the alpha ketoglutarate dependent hydroxylases right oxygenases. So, we have 2 histidine and 1 aspartate if you remember 2 histidine and 1 aspartate was there. Now, what if this aspartate is removed and placed with another halide. So, this becomes a completely different enzyme now, this is called alpha Kg dependent halogenases. So, that means, a halogen is sitting over there instead of aspartate and of course, you still need alpha ketoglutarate this is alpha ketoglutarate dependent halogenase right. So, the substrate just like previously for the oxygenation chemistry or the oxidases you have seen previously this oxadioxygenases, you have seen that substrate is coming on to the organic moiety or onto the metal center. So, substrate is oriented similarly over here also the substrate is going to be oriented or fixed right next to the iron center right. It is the exactly same thing what is happening previously 2 histidine 1 halide you have instead of this aspartate. Reaction mechanism and things remain similar we have seen the next step would be after substrate binding next step would be the oxygen activation or dioxygen activation that is what happens first of course, has to undergo binding that is true in everything case you will have the bind bound oxygen intermediate iron oxygen bound intermediate this is fantastic 2 histidine 1 halide and alpha ketoglutarate then 1 electron transform from iron 2 to this superoxo occurs you get the iron 3 superoxo intermediate that attacks on this center not this center this is the carbon dioxide releasing part on this superoxo once it generate it will attack over there as you have seen previously further cleavage will gives rise to the iron 4 oxo intermediate. Now, up to here exactly same actually up to next step is also exactly same as you have seen in the dioxygen age, but over here what you see that of course, iron 4 oxo will abstract hydrogen atom from the aliphatic substrate that is all fantastic and will give RCH 2 dot or the substrate radical intermediate and you have the hydroxo intermediate right. If it abstract hydrogen atom you will get RCH 2 dot and OH. Now, what happens in the halogen ages enzyme the halogen is sitting very close and nicely placed with respect to this radical even compared to the hydroxo and therefore, quite excitingly for the halogen ages you get exclusively halogenated product formation of course, succinate comes out carbon dioxide comes out and then you deliver halide to regenerate the catalytic cycle. As you have seen in the earlier case when X is aspartate and that means, no halogenation business and hydroxo is close to that and then it can do the hydroxylation chemistry. Again succinate and a carbon dioxide comes out as the byproduct. So, what you have just seen right now by changing just one ligand of course, that is the iron halogen we are talking about iron X just one ligand nature can modify control absolute control I think that is that is what is really amazing that nature can completely control the reactivity everything else is remain same only the aspartate is replaced by halogen. Now, all of a sudden no hydroxylation products are forming, but exclusively exclusively halogenation products are forming I think that is quite phenomenal and that is why you see it is going to be so difficult to compete or try to mimic what we have in nature right these efforts are going to be very tedious and lengthy. Indeed you would notice that that there exist mimics for the alpha ketoglutarate dependent dioxygenases, but there is no mimic so far on the structural plus functional together these halogenages one. Well that is quite amazing how nature really does. Let us look at this one more time little bit. So, what we are saying that from the iron oxo this R H undergoes hydrogen atom abstraction to give you hydroxo and R dot. Now, this R dot is not going to rebound with the hydroxo instead this R dot is going to bind with or react with halogen or halide over here to give sorry halide over here to give the halogenated product. Well that is quite phenomenal as I would say because this hydroxylation is quite challenging or in these cases, but overall you have seen exclusive halogenation was happening in the other case right. Now, no halogenation only hydroxylation only halogenation is happening this has to do in two counts one this is due to the fact that this radical R dot radical is positioned really perfectly to and very close to the halide over hydroxy. Another thing is the you know that reduction potential for the halogen is suited to transfer over there compared to hydroxo. Hydroxo can transfer, but halogen transfer is much more facile. So, that is how nature has designed and decided to take advantage of this system right. So, one common intermediate almost throughout the catalytic cycle one saddle change complete different product distribution complete exclusive product distribution. So, in one case you have seen the hydroxylation in the first cases and these halogenation enzyme can exclusively give the halogenation product. Now, you can imagine when synthetic chemists are trying to promote some reaction it is actually always going to be the hydroxylated product that is going to be predominantly forming ok. Almost 95 percent efforts that has gone in mimicking the halogenage activity actually ends up failing to mimic it, but it just gives the hydroxylation product. Almost in most of the cases no halogenation product whatsoever can be formed. In some cases at best mixture of halogenation and hydroxylation product can be formed so far. Well there exist alternate mimic which we may not be discussing too much, but there it is possible to promote halogenation reaction by utilizing high valent iron oxo intermediate by taking a completely different route. In any case let us look at one of the practical examples. This is what the organic substrate you see over here and this is in halogenase cytochrome C3 and you have a iron for oxo chloro intermediate. Now in these cases this CH2 and Cl dot transfers and you see exclusively CH2Cl product along with the iron to hydroxy product formation. This is quite great all we will not be discussing wait too much into detail these are very very fascinating enzyme and lot is known lot of studies has been done on the enzyme if you are further interested feel free to study. Now we will try to see one of the other aspect which we are mentioning earlier and that is in the absence of organic substrate for example, that is in tau D tau ring is absent what happens to the tyrosine because tyrosine as you have seen in the crystal structure is appended right close to the active site. Is the tyrosine going to be participating in the reaction in absence of the natural substrate that is a tau ring for tau D right. Well the answer as we have briefly mentioned yes phenol is going to be participating into the reaction, but what happens to phenol ok. Before that let us look at once again the same reaction mechanism that we are discussing it forms a iron 2 oxo iron 2 oxygen intermediate subsequently with the help of alpha ketoglutarate. We do see that this iron 4 oxo intermediate is forming all this iron 4 oxo intermediate can then react with tyrosine if the organic aliphatic substrate that is required to be present is not there right well. So, you get a phenoxy radical intermediate tyrosine radical intermediate along with iron 3 hydroxo formation right. Now you can imagine if this is the case now this can go on and form a oxygenation or undergo oxygenation at the orthophogism and that is what is exactly happening this phenoxy radical then undergo oxygenation reaction to give you the catechol intermediate. Well as you have perhaps noticed if you are using leveled water let us say wet in leveled water in these cases when tyrosine is reacting that means, the organic substrate is absent then this water molecule oxygen. So, oxygen derived or oxygen of the water molecule can be incorporated in the phenol equivalent or phenol molecule right. So, this is going to be catechol now and you know that is fantastic if you under oxygen it can further get oxidized to iron 3. So, what you have just seen right now in the absence of the natural substrate organic substrate that is supposed to be hydroxylated by alpha keto dependent oxygenage you do not if you do not have such organic substrate you end up getting a completely different reaction and that is the phenoxy radical generation because iron 4 oxo is show reactive it is not going to be sitting ideal just and you know just be sad that it does not have the organic substrate it is not going to be sit down over there it is going to react with anything that is available to it in this case phenol is a easy substrate to react and it ends up reacting and giving us phenoxy radical subsequently the catechol moiety which is alright right, but I think the most interesting part in this case is if you add wet in leveled water as you have seen wet in leveled water is getting incorporated the wet in level oxygen is getting incorporated in the product. So, what water oxygen atom is incorporated into the product this is a clear cut evidence that something must be going on and that something is this iron 3 hydroxo and this water can exchange with each other. So, this hydroxo can become water and this water becomes hydroxo and that hydroxo can be incorporated into hair right. Well, that also would mean that this stability of the phenoxy radical is quite high and unless until this is stable or this has some lifetime you would not be able to see this sort of exchange right. So, the reason or the moment you know that this exchange is happening then you are certain that the phenoxy radical is long lived enough. So, that it allows the exchange and then the reaction can also happen of course, it is not going to be exclusive formation of the O 18 level water you still can get O 16 water O 16 oxygenation here, but the fact that the leveling can be found is indicative of the two things as I said one thing is it is exchanging this hydroxo and this water molecule are exchanging with each other second thing is this phenoxy radical is long lived right. So, that is what we have written over here and well this is the self-hydroxylation mechanism right. So, we see that self-hydroxylation of the enzyme is happening in absence of organic substrate, but this mechanism will vary based on the presence of what you have if you have alpha ketoglutarate this is the pathway, but it is not necessarily you have always the alpha ketoglutarate if you run out of alpha ketoglutarate you can essentially can have let us say succinate because succinate is the product derived from alpha ketoglutarate and as you have seen alpha ketoglutarate undergoing the reaction overall in these enzymes to give you succinate. So, succinate is going to replace alpha ketoglutarate if it is not present in enough amount right. So, in those cases succinate is definitely not alpha ketoglutarate it will act just as a monodentate ligand as you have seen in the last slide alpha ketoglutarate is a bidentate ligand right. So, succinate that is getting produced over here is going to be likely the monodentate ligand and of course, it has also an probability of bringing these two acid together, but in any case this is almost going to be similar to let us say aspartate type of substrate and sorry aspartate type of ligand in presence of such ligand or in absence of alpha ketoglutarate you end up providing one electron and one proton in the system. So, in the enzyme it ends up forming iron III hydroperoxo. Now, it is not that something new in this yes iron III hydroperoxo species can form essentially what is happening here iron II is reducing oxygen to give you iron III superoxo. One electron transform from iron II into oxygen gives you iron III plus and oxygen 1 minus that is superoxo iron III superoxo is formed you give one electron and one proton that superoxo radical then it becomes iron III hydroperoxo. This is what you are seeing over here right iron III hydroperoxo you are getting and this iron III hydroperoxo can undergo further cleavage of the oxygen-oxygen bond to form to form iron V oxo hydroxy species. If you break the oxygen-oxygen bond and then that radical will give you that iron V hydroxyl. Let me see if we can if you can draw little bit over here and well I would need a new page perhaps white screen ok. So, what we are trying to say is essentially if you have iron II right and reacting it with oxygen ok. Now, this is going to form iron I electron gives I electron give I electron gives ok. So, you will get iron III superoxo radical right that is fine and then you can give a hydrogen atom ok. It could be 1 proton plus 1 electron 1 electron overall a hydrogen atom if you are giving then you are going to get iron III hydroperoxo species right. Now, if you have seen if it is breaking oxygen-oxygen bond that means, it go if one electron here one electron there. Now, iron III to form a iron IV it will it will end up giving one other electron. So, double bond O will be forming right over here and this hydroxo radical if it has to bind with iron iron will end up giving another electron. So, iron is overall producing 2 electrons or giving 2 electron. So, iron III becomes iron IV and iron IV becomes iron V. So, overall you have iron V oxo hydroxy right. So, iron V oxo hydroxy intermediate is happening and I hope you understand how it is happening iron III gets oxidized once iron III get oxidized another time. So, this iron hydroxy and iron V oxo is forming in these cases. So, that is what as you have seen that is what is happening over here in the in the right hand side ok. So, that is where you see in the right hand side in here and iron V oxo is fascinating and it is happening right over there and you can you can get a clear idea from what we have drawn earlier and from there on as you can see this iron V oxo hydroxo can also give this you know catechol type of intermediate formation ok. So, the conclusion from this reaction is tyrosine radical is long lived and water exchange occurs at iron III hydroxo as you you are mentioning over here ok and iron IV of course, iron IV oxo forms and attacks tyrosine 73. So, these are all all we have discussed so far. So, what we have seen so far then in this class two things I would say first thing is the alpha-ketoglutarate dependent halogenases which is a great enchantment right. You get exclusive halogenation it is not that great, aliphatic substrate halogenation it has to be one of those best reaction ever I would say right or if you do not have the halogen you have seen that it could be hydroxylated that is the that is the normal enchantment or most of the things time that is what happens, but special enzymes such as alpha-keg halogenases it can do halogenate the organic substrate that you have seen. And the mechanism you have seen it is nothing different it is almost exactly same as the oxygenases, but only varies in one of the ligand which is on the iron center. On the other hand the last part what we are discussing today contains what? Contains yes thus does not contains the does not contain the substrate. So, it does not have the substrate the desired aliphatic substrate and therefore, therefore, we see that the reactive iron oxo intermediate is not going to see it ideal it is going to react with itself means its own environment and that is where tyrosine 73 which is right next to the active site will come into the picture and it gets hydroxylated right. Tyrosine getting hydroxylated means phenoxy radical phenoxy radical leading to the catechol moiety ok. Now this mechanism or the you know the intermediates can vary it could undergo a traditional oxo mechanism that we have seen in the oxygenase and halogenases, but that is when we have the alpha-ketoglutarate present. In absence of alpha ketoglutarate essentially you need to have something and that something is succinate because succinate is getting generated from alpha-ketoglutarate in this enzyme show when alpha-ketoglutarate is running out or we ran out or of alpha-ketoglutarate and that is when succinate comes into the picture for the tyrosine in absence of tau D or organic substrate we are going to get once again the same product in terms of tyrosine because tyrosine is the substrate that is going to tyrosine will act as a substrate and will give you catechol, but more importantly the reaction mechanism is completely different here right. As you have seen that that reaction mechanism would require a iron oxo hydroxy intermediate formation right this iron oxo hydroxy intermediate formation ok. And I hope how it is forming you have seen it just practice it a little bit this is very simple and we will come back soon in the next class. Till then keep studying thank you very much for listening we will see you soon.