 Hello, welcome back. Today we will discuss hemorrhoid thread. So, we are discussing metals in biology. The book to follow is the Principles of Bioinorganic Chemistry by Lippard and Berg. Well, it is a fascinating enzyme, hemorrhoid thread. It is a metalloenzyme that means metals are there. Well, in mammals for example, in human we have blood. Blood is read due to hemoglobin, but there are many other species in the world which does not have hemoglobin, but still they are aerobic species. They inhale air and they are air dependent. There must be some other enzyme which is responsible for carrying the oxygen in different parts of their body. The job this is done in the invertebrates or marine invertebrates by hemorrhoid thread. This hemorrhoid thread are having two iron centers. As you can see, this is a reversible dioxin binding in hemorrhoid thread. This is the reduced form of hemorrhoid thread also known as deoxy hemorrhoid thread. Iron centers are in plus 2 oxidation state. Each of the iron is having 3 histidine or sorry 3 histidine in one of them and on other one we are having 2 histidines. So, it is a diiron center that is responsible for oxygen carrying in those species where we do not have hemoglobin, not for all of them, but for mainly those which are marine invertebrates. Those are the species looks like this is something like the species which looks like these are having this hemorrhoid thread. Now, as you have seen 3 nitrogen or the histidine coordination are there on iron. 2 histidine coordination are there on this other iron. They are linked by aspartate and glutamate. These are bridging carboxylate linkage. In addition to aspartate and glutamate, they are also having hydroxy bridging between them. This is crystallographically characterized intermediate. So, it is very clear that this is what we have in the deoxy hemorrhoid thread. Upon binding with oxygen because they are oxygen carrier, a new species is formed where oxygen is reduced doubly. That means, each of the iron will provide one electron and will form a peroxo species. So, oxygen is 2 minus, 1 minus is bound over there and another minus is picking up the proton from this hydroxo. Overall, it is forming diiron 3 hydroperoxo species bridged by a new oxo species now. So, that is quite fascinating. While here during these oxygen binding and electron transfer process, a proton coupled electron transfer happened. So, this overall transfer of this proton happened in a PCET mechanism by a PCET mechanism. One can utilize the resonance Raman data to characterize these fully oxidized oxy hemorrhoid thin diiron species. Resonance Raman data shows that this oxygen oxygen stretch is around 844 wave number. We will see in the next slide what this mean? I can tell you in advance that this means that this is a hydroperoxo species. This is a peroxo oxygen oxygen stretch that is around this 844 wave number region. Resonance Raman also says that this iron oxo iron stretch, this iron oxo iron symmetrical stretch is around 486 wave number and the asymmetric stretch is around 757 wave number for this iron oxo iron. Well, as I mentioned these are found in marine invertebrates and this is the deoxy form of the hemorrhoid thin which is colorless. These are diiron 2 species bridged by glutamate and aspartate and hydroxy completely colorless species. Oxy hemorrhoid thin upon binding with oxygen the oxy hemorrhoid thin is forming. These are having both the iron in plus 3 oxidation state and this species is red in color. This was a colorless species turning into red upon oxygen binding. While one can assume that there is another intermediate that can be there or one can perhaps characterize them that could be a iron 2 iron 3 intermediate which is basically called the semi-met hemorrhoid thin which is inactive. I hope it is crystal clear for you guys that hemorrhoid thin is the oxygen transport protein for a number of species such as those marine vertebrates. They do not have hemoglobin like us that their blood is not really made up of hemoglobin ok. Now, these are the species which will carry oxygen and most importantly just like every oxygen transporting protein the oxygen binding has to be reversible. They should be this should be easily settling between deoxy form and the oxy form. This is quite intriguing that during oxygen binding it gets reduced doubly to paroxo not only that a proton coupled electron transfer process is involved. This such a compound is really fascinating as you may have noticed one side is having 3 histidine another side have 2 histidine. Therefore, the oxygen binding site is open or available only on this iron center not on the other iron center. This means that nature has really designed it perfectly show that everything remains as much constant as possible, but only this subtle oxygen transport or oxygen binding can happen at one of the iron center. Let us look at the resonance Raman data for the various oxygen derived species. If it is oxygen O2 the from air this oxygen oxygen stretch would be at 1580 wave number. If it is oxidized form of oxygen where oxygen oxygen bond is stronger then we have a increase in the oxygen oxygen stretch from 1580 to 1905 wave number. Similarly, there is a change in the oxygen oxygen distance from 1.21 angstrom to 1.12 angstrom, but if oxygen is reduced by 1 electron which is known as superoxide this oxygen oxygen stretch goes down to around 100 1097 wave number and the oxygen oxygen distance increases because now it is reduced by 1 electron. If it is reduced by 2 electrons from this molecular oxygen if it is reduced by 2 electron this species is called peroxide or peroxide this is the superoxo. Now this peroxide species will have a further decrease in oxygen oxygen stretch around 800 wave number. Of course, these value may vary slightly, but as you can see nearly 300 wave number difference is there. Even if they are varying depending on the ligand on the metal center still it will be very characteristic to a particular species whether this is a superoxo, peroxo. These are almost a fingerprint spectral features that can be seen in these oxygen bound compounds. As you have noticed that the oxygen oxygen stretch goes up significantly to 1.49 angstrom ok. Remember what we have seen in the in the oxyhemerythrin case that oxygen oxygen stretch was 844 wave number ok. So, 844 wave number is the oxygen oxygen stretch. Now if you try to match that that is falling in this region that this is a peroxide in nature right. And more importantly as you will see that oxyhemioglobin has this oxygen bound in a superoxo format, but you see that superoxo stretching is matching quite well with that of those expected. So, 1097 and observed is 1105 that is fantastic I mean these are quite interesting. So, these can serve as a very readily available tool to confidently identify any reactive intermediate such as those we have seen in hemerythrin, oxyhemioglobin, hemoglobin and even hemocyanin and so on. Many different oxygen containing species can be characterized very easily by such resonance Raman spectral data. Moving on well there is earlier studies which shows that there is this crystal structure that is I believe somewhere in 1980s I forgot to put the reference here. So, this is azidomethymerythrin, erythrin so azidomethyrhythrin. Now in this case in the last case as you see that 2 histidine was there and the vacant coordination site was binding oxygen, but before those structure were known this structure was first reported with an azidobinding group and later on these sort of species or similar species with acetonitrile or OTF other species are also known. So, this vacant coordination site can be easily probed and these 3 histidine as you can see are definitely there on another one and these aspartate and glutamate binding as well as the hydroxy binding is there. This is an inactive form of the protein. So, this protein crystal structure this contains a mu oxo di iron III core. So, this is the mu oxo di iron III core and this is artificially of course oxidized. The azido ion this is the azido ion occupies the place of hydroperoxo anion in oxy hemrythrin as we have mentioned earlier that this is the site is now actually bound by this azido the hydroperoxo is not there that is why azido it is bound with azido anion and these are the early crystal structure which is clearly showing the characteristic core of this di iron center. There is antiferromagnetic spin exchange between the 2 iron center the 2 high spin iron III center in this case are exchanging their spin through these oxo bridge right. So, these are early studies, but quite informative about the relative orientation relative binding and the core of the hemrythrin ok. There are a number of studies that is done towards mimicking such azido met myohemrythrin structure. So, this is from this 1983 report as you can see clearly 3 histidine on 1 iron, 2 histidine on another iron as well as the azido group. Now, there are and then the bis meoxo bridging is between their meoxo bridging is they are not bis meoxo bridging single meoxo bridging is there between the 2 iron center right. Now, to mimic such species scientists or the synthetic chemist has worked long and quite successfully and now the model of this species has been reported. This is an earlier attempt where trispyrazole borate ligand system has been used for synthesizing this the iron based system. And as you can see these are the acetate bound intermediate bridge between the 2 iron center as well as the oxo bridge is there, but one thing that is missing is the vacant coordination site. Both the iron center are having 3 iron center once again this is a model study right, synthetic study. This is not really the enzyme this is a synthetic study which clearly shows that each irons are having 3 nitrogen and both of them, but as you remember in the enzyme on one side there is only 2 histidine or the 2 nitrogen ok. These sort of mimicking studies are quite exciting ok. Because although in this case it is not the correct mimic, but these initial attempts lays the pathway for the future generation studies which takes quite a lot of inspiration and then subsequently we are able to exactly match this sort of structure or the enzyme deoxy even the oxy hemorrhoithrin structure we will see those should. This is a X-ray structure of oxy hemorrhoithrin back in 1991. This is clearly shows that these iron 2 iron center in the deoxy form the hydroxo bridging is there during oxygen reaction or oxygenation leads to this hydroperoxo intermediate and this as you can see the plot clearly shows that this hydroperoxo is bound over there. And I must tell you that these hemorrhoithrin or hemorrhoithrin is readily crystallizable almost easily it is one of those enzyme which can be crystallized quite easily and it was quite amazing to get these crystal structure even with the oxygen bound form right. While a number of studies has been done this is one study by professor Steebley part group himself and in these cases this dioxygen binding chemistry of hemorrhoithrin modelling is done. One of the thing that we mentioned in the last case that although this trispirozolyl borate mimicking was quite good and the early stage of those mimicking those are effective mimic, but one too many ligand were there instead of 3 histidine 1 and 2 histidine motif it was 3 histidine 3 histidine motif or 3 nitrogen and 3 nitrogen motif. You must know that it is not essential to mimic exactly the histidine you can perhaps play with pyridine you can add pyridine or imidazole I mean you know all these mimicking studies with essentially deal with the same heteroatom. So, histidine has nitrogen coordination with iron synthetic chemist will take any ligand that has nitrogen coordination it could be aromatic I mean it could be pyridine nitrogen, it could be aliphatic nitrogen, it could be any let us say imidazole nitrogen which mimics quite well the histidine. It is not limited to just the histidine any nitrogen containing ligand is fine. In these cases a series of such compound been synthesized and it is quite interesting to note that it is instead of 2 3 histidine now it has taken the researcher have taken 2 nitrogen on each of the iron and one of them is having OTF. And subsequently this OTF coordination can be let us say let us say displaced by another nitrogen coordination such as imidazole or suitable pyridine coordination or even azido coordination overall any solvent or an extra monodentate ligand can be ligated while displacing this OTF. But quite interestingly the oxygen chemistry done at low temperature shows that the hydroperoxo species can form in the synthetic complex ok. So, synthetic modeling gives an opportunity to better understand the enzyme itself what happens to the synthetic chemistry modeling if it is perfect then it is going to perfectly match those data of the natural enzyme. So, that is to be able to mimic and to be able to perform the chemistry in greater detail and in absolutely complete detail is quite exciting for synthetic chemistry. Because this sort of it is sort of understanding and ability can lead to a great catalyst synthesis synthesis and it can have implication in understanding the process as well as if there is any deficiency that can be deficiency of the enzyme study that can be kind of overcome by these synthetic mimic studies. In these cases although enzyme studies are quite good and known, but in number of cases enzyme studies will not be known a lot of metal or enzyme studies are not that very easy therefore, these sort of synthetic studies are quite exciting ok. You must be very excited to know that humans afford to understand and mimic these hemorrhoid thing was quite successful ok. If you look at the data of this compound which is a iron hydroperoxo species all the spectroscopic data including Mokhba, Resonance Raman UVV spectra matches almost as close as one can get perhaps can think of getting right. So, that is fascinating and these are some of those examples that we were talking that these are the bidentate ligand system different easily prepareable bidentate ligand system can be employed these are some of the representative example. And these were done quite beautifully as you have seen the oxygen-oxygen stretch remember it was 842 is I believe now it is 844 wave number or exactly 844 wave number previously. And not only that it is also possible to level the oxygen. So, it was normal oxygen and then 18 leveled oxygen this 6th Resonance Raman spectra data 6th from 844 wave number to 798 wave number that is quite fascinating. UV visible spectra is quite characteristic of what has been seen in case of the enzyme itself. So, this is one of one such compound where these these these sort of ligand and then a N-methyl imidazole is attached and the hydroperoxo species is also formed. This is once again the report by Professor Stibliper's group. So, I hope you are able to get some sense that these mimic synthetic mimic are going to be quite useful in understanding these enzymes and the greater details and the ability to understand almost every aspects of a metalloenzyme which has been perfected by nature I think is quite remarkable ok. So, let us let us try to overview this class what we have discussed so far. We have seen we have seen that this hemerythrin is part of a marine invertebrates and these are the diiron center completely bridged, bridged species with bridged by multiple ligand as you can see one aspartate one glutamate and one hydroxide this is an unsymmetrical diiron core one side is 3 histidine another side is 2 histidine it reacts with oxygen and binds oxygen at this vacant side of the second iron center and form a peroxo, but not just any peroxo it is a hydro peroxo while formation it is undergoing a proton coupled electron transfer right. So, this is really clear I hope and this oxygen-oxygen stretch is 844 OM number exactly the number also matched by the synthetic analogs or synthetic mimic where 3 nitrogen not need not be necessarily histidine, but 3 nitrogen from the ligand is mimicking not only on this side of the enzyme core, but also the left hand side with 2 nitrogen and 1 hydro peroxo along with this oxo bridge. This sort of mimic this sort of structure is completely mimic of course, it there has been a continuous effort in understanding this enzyme over decades. Now, we are in a position when we believe that we understood in really great detail, but initial studies back in 70s and 80s were quite preliminary I would say which has been mature over the years and decades. Quite interestingly I hope you also noted that this resonance Raman is quite exciting tool for this sort of species or this sort of intermediate characterization because you must be understanding that these intermediates are very very sensitive these are often most often these are only low temperature stable intermediate you will not be able to see these intermediate at room temperature and that is quite phenomenal to mimic I would say. And then thanks to these spectroscopic technique which is now taken as a standard in characterizing these species with or without the crystal structure. Now, these oxygen-oxygen stretch are invariably going to dictate the assignment of the right species. For example, as you have seen if it is getting reduced the stretching goes down as well as the length of the oxygen-oxygen bond goes up, but for that you have to have the crystal structure right that is quite not feasible in a lot of cases. And therefore, therefore, this sort of this sort of studies are quite important just to have a resonance Raman structure or resonance Raman assign spectra will tell you what is there in reality. Of course, getting a crystal would be perfect, but life is not that not that rosy all the time most often for these sort of species you have to rely on spectroscopic technique such as low temperature UV visible spectra resonance Raman and if possible let us say low temperature EPR and NMR if it is feasible or MOSBORES or in addition to that for iron for example, iron centers this is the MOSBORES spectra will be quite amazing to characterize specifically to tell what sort of spin state is there, what is the electronic configuration and most importantly there are other spectroscopic technique in addition to the X-ray crystallography if X-ray is there that is fantastic if it is not available also the XAS, JAS, XAPS these sort of different spectroscopic technique can be used to further characterize this intermediate with almost 100 percent confidence for these sort of species. And those are very sensitive studies and of course, mind you that these are also very expensive study, but nonetheless those can be now routinely done thanks to the development over the last few decades which has made these possible, made possible characterization of these species in greater detail. We have seen the azido bound hemorrhoithrin and the mimicking aspect of these how and what people have done some of it there are many studies that have been done we did not get into the detail to keep it simple. So, there were initial attempt which were almost as good, but then that was not mimicking the vacant coordination site iterative designing then has addressed that issue this is the crystal structure showing exactly what we have seen in the earlier drawing. This is the phenomenal studies by Leopard Group which exactly mimic what we have seen in the enzyme. With this let me conclude then that we are able to able to see these spectral features as well as the human efforts to understand these enzymes in greater detail. So, keep studying hemorrhoithrin and other related topics and we will get back to you shown in the next class. Thank you.