 Hi, welcome back. Today we will discuss methane monoxygenase. It is a fascinating enzyme and actually it is a class of enzyme. A series of enzyme are having similar activities and these enzymes are quite fascinating perhaps as fascinating in their activity as we have seen for cytochrome P450. These are non-heam iron enzyme unlike cytochrome P450. We will follow the books of principle of bioenorganic chemistry by Lippernenberg and also class notes and online available materials by Professor Lippern. So, methane monoxygenase has a diiron center. As you can see over here, two iron centers are bridged by two hydroxy moiety. Of course, they are also linked by this carboxylate group. Each of the iron center is having a histidine unit as you see on iron 1 and iron 2. In addition, there is also carboxylate linkages as you see over here. Clearly, these two iron sites are unsymmetrical. We have seen such unsymmetrical iron sites in hemeritrin. So, this is responsible for methane monoxygenase. That means, methane is converted to methanol at this site. As you know, methane to methanol transformation is the most difficult transformation one can perhaps think of and that is precisely due to the fact that methane is having 104 kcal per mole bond dissociation energy. This class of compounds or the class of enzymes are not limited for converting methane to methanol. You can have a series of other reaction even aromatic hydroxylation reaction. For instance, one can think of taking toluene and convert it at ortho toluene hydroxylate ok. So, ortho hydroxo toluene can be synthesized. Also metahydroxyl toluene can be synthesized. Parahydroxo toluene can also be synthesized. So, these sort of toluene or xylene as a substrate if the monoxygenase of toluene or xylene is thought of this is the similar active site where in these toluene or xylene will be hydroxylated ok. Of course, let us say for toluene you can have ortho hydroxylation, metahydroxylation or even parahydroxylation. So, this is the enzyme active site for the toluene parahydroxylation or toluene 4 monoxygenase. Essentially these hydroxylases a group of enzyme which are capable of hydroxylating the organic substrate. It could be aliphatic substrate, it could very well be the aromatic substrate. As you see here these are the crystal structure of different monoxygenases and very little differences are existing among these different crystal structures. There are also amylakene monoxygenase which can convert an olefin into epoxide. There are phenol hydroxylase which can convert phenol into catechol. But all of them once again will have a diiron active site and their active sites are quite similar with respect to each other. In all these cases oxygen is activated and they are often involved in these very difficult transformations. For the case of methane monoxygenase, these oxygen activation is coupled with the inactivated C-H bond activation as well as corresponding hydroxylation chemistry, right. Well, what is the source of this diiron center? What is all? We have soil methanotrops which is converting methane to biomass and that is one of the source for these methane monoxygenase. By utilizing such soil methanotrops there is a huge amount of methane, there is huge amount of methane that is converted to methanol. This is 5 to 50 Tg's methane per year which is overall let us say 1 to 10 percent of methane in air is converted to methanol by utilizing this method. There is methane to methanol formation at room temperature under you know under pH 7 let us say or under neutral condition. I think this is got to be one of the best reaction perhaps you can see in biological system or anywhere in synthetic chemistry setup. So, we are going to see how methane is converted into methanol. Methan would require oxygen NADH H plus to form the methanol. This is the reaction over here, this is a multi component enzyme, the components are hydroxylase MMOH, reductants MMOR, regularity protein MMOB. If you see at this protein, this is a gigantic protein, it is having many subunit alpha, beta, gamma subunits. In each of those alpha subunit there is this diiron center which is responsible for converting the methane into methanol that means, the hydroxylation chemistry is happening ok. Just to keep it in the perspective, you have seen previously the oxygen transport and oxygen activation for with iron porphyrin we have hemoglobin myoglobin which is responsible for reversible oxygen binding right, but utilizing the same iron porphyrin unit with cytochrome P450 we have seen the substrate activation as well as oxygenation chemistry. So, seemingly similar or almost similar active site, but doing completely two different jobs. We have seen the dinuclear site such as dinuclear di copper center in hemocyanin which can activate oxygen or bind it in a reversible fashion and deliver the oxygen in desirable position, but the same di copper center is also capable of converting your phenol into catechol or catechol into the quinone. So, those tyrosinase activity by utilizing exactly same species as in hemocyanin we can that dinuclear peroxo, sidon peroxo species forming in both the cases and in one case it is used for reversible oxygen binding in another case we have seen oxygen activation as well as substrate hydroxylation chemistry. For the hemorrhoid thrin case we have seen that two di iron through iron centers are involved for oxygen binding and over there in hydro peroxo species is formed, but most interestingly these are non-hem di iron center. Similar non-hem di iron center can be found as you see in case of methane monoxygenages, but structural similarities are also there these are all dinuclear or bis iron containing active site these are unsymmetrical iron centers, but in one case as you have seen hemorrhoid thrin is capable of converting oxygen into your oxygen into hydro peroxide during the process, but of this bindings are reversible in nature, but in case of di iron center of methane monoxygenage as we will see it will be able to convert oxygen into an useful entity by converting methane to methanol. So, this is what I think are the similarities or the contrast between the reversible oxygen binding as well as oxygen activation. Let us look at the resources or source of methylococcus capsules which is responsible for these methane to methanol formation. These are these are beautiful location, these are the picture from internet from England bath. These are the places where people can go and take a bath and these waters are really holy and can convert and can be responsible for curing many skin diseases. So, this is one of the one picture. So, bath this is a small city in Somerset, England located on a bend of the river Avon about 185 kilometer west of London, but most importantly this is a very nice touristic place. Well, as you can read from the Wikipedia or any other sources, this is a beautiful place where many tourists visits and spends quality time with friends and family. The fundamental part of these Roman baths which are existing in England bath Somerset are the sacred springs. Hot water at a temperature of 460 degree C rises here in this sacred spring at the rate of this 24,000 gallons every day and has been doing so for thousands of years. To the ancients this remarkable phenomenon could only be the work of gods. So, if you are believing in holy man or sacred water, this is the sacred water you can think of and there is no wonder that these are the sources for methylococcus capsules which is responsible for the methane to methanol conversion. This holy water cleanses the body from all botches, suscarvical itchings and breaking out. So, any sort of skin disease can be recovered or can be cured by this holy water. So, if you see that ancient time or even present time the holy water sprinkle on your on someone's body and then miraculously the patients got cured or the skin disease or other problem got cured and this is due to the great water which can perhaps have this methylococcus capsules. There is just strong science behind it and we are going to see what is these active sites and how active they are and how they are functioning. Okay. Few more pictures from the mineral springs in Bath England. So, this is as you can see really, really you know wonderful atmosphere being created. It is great to take bath in these places and have nice time over there as well as it has extreme benefits of the and have you know medicinal value. So, if you are looking for a great time and wants to solve some of your skin diseases perhaps this is the place to visit and do enjoy your time, okay. All right. Another quite exciting stuff that is included or that we can discuss about this methanotrops bacteria is their ability for bio remediation. Let us look at this Wikipedia information on the on Prince William Sound which is of the Gulf of Gulf of Alaska on the south coast of the US state of Alaska. It is located on the east side of the Canine Peninsula. It is largest port of Valdes and you can read more from Wikipedia or from here. In 1989 the oil tanker Exxon Valdes ran aground on Blyreif after leaving Valdes causing a large oil spill in the Prince William Sound area which resulted in massive damage to the environment including the killing of around 250,000 seabirds, nearly 3000 sea otters, 300 harbor seals, 250 baldigals and up to 22 killer whales, right. That is quite amazing number I mean quite frustrating quite devastating number, right. If you are looking at this number the oil spill that has caused or that has happened in 1989 by or from this Exxon Valdes this was quite devastating and it has long reaching impact on environment. It has polluted the area quite naturally for long time to come but if you are looking to for bioremediation I think this is when methanotrophs comes into the picture and these bacteria are grown over there and overall it was possible over time to make this place a suitable place as it was before. Oil what was found that in the root zone was a rich reservoir of oil known oil eating microbes one family of which accounted for fully 95 percent recovery of the of this oil spill. So, therefore, natural disaster or unnatural disaster like this one as you have just seen little bit perhaps can be taken care of by this methanotrophs bacteria by converting the alkene or oils into the corresponding let us say alcohol products and setting of further degradation over long term. So, these methanotrophs bacteria as you have seen are found in one of the shores could be these you know Bath England, but they are having capability of bioremediation which is wonderful right. Now, let us look at the active site of these crystal structures ok. If you are looking at these active site you see that there are two iron center they are bridged by these two dihydroxo unit. Here is a water molecule as you have seen there is a glutamate unit it is a mono coordinated glutamate along with an histidine along with histidine coordinated with this iron center. This iron center is not exactly same as this iron center here you see the two terminal glutamate in this case it was just only one glutamate that was present as you see that there is histidine as well and both the iron centers are bridged by this glutamate 144. This is phenomenal and quite fantastic crystal structure right. In cases where it is also possible in the reduced form when one of the hydroxo is missing and then this glutamate one of this glutamate can have the bicoordinating nature and also can be relevant in the catalytic cycle. These are the resting state of the catalytic cycle. So, as you see over here clearly methane monoxygenase diming clear active site looking somewhat similar to what you have seen in hemiarithrine where these two iron centers were supported by the histidine side three on one side two on other side, but here you have you do not have all the histidine side, but you have the glutamate replacing some of those histidine side, but quite naturally there is similarity between hemiarithrine and these methane monoxygenase active side. If you look at the overall activity by these active site you will find that this bridging a diiron center which is abbreviated over here in this form where this is the reduced form. You start with both the iron center in plus 2 oxidation state oxygen molecular oxygen is reacting with this diiron center to give the superoxo species where one electron reduction from one of these iron center is happening giving rise to the iron 3 iron 2 superoxo intermediate. This oxygen is singly reduced right now so this is MMOH reduced form this is MMOH superoxo form. Now from there on you one can think of transferring another electron to this oxygen moiety which is now superoxo upon upon getting reduced by one electron from the second iron center you will see that both the iron center are now in plus 3 oxidation state and the oxygen moiety is reduced by 2 electron to peroxo. Not too much characteristic spectroscopic character data are available for this intermediate nonetheless this peroxo intermediate is suitably characterized for example, MOSBOHR spectra or MOSBOHR data are quite quite definitive of these iron 3 species and UV visible spectra have the characteristic 725 and 410 nanometer once again indicating such a species is existing. Now this is the species one can think of introducing the substrate 2 and substrate can get hydroxylated by utilizing this peroxo species indeed many studies have been done often generation of the species and quick study shows that this is capable of converting RH substrate sp3 CH bond can be hydroxylated to corresponding the hydroxylated product right. Well from this side on peroxo intermediate one can think of or one can characterize this bis muoxo species which is a iron 4 iron 4 bis muoxo species here MOSBOHR spectroscopic data as well as the UV visible and XFs data are consistent with this intermediate which is known as intermediate Q MMOHQ. So, this intermediate quite interestingly both of the iron are having iron 4 as you have seen in this previous case this was iron 3 this is a mixed valence scenario and have not much proof in terms of the reaction mechanism or reaction intermediate study. Now this intermediate once it is formed can react undoubtedly with methane to methanol. So, this is the process we are interested in following up too much of course, it has to follow step wise this is the real active species which will convert methane to methanol and from there on the dihydroxo intermediate is generated MMOH ox which can then in presence of NADH and reducing equivalent can give gives rise to the original compound as we have seen over here. So, these dinuclear iron active sites are quite fascinating as you can see and can convert methane to methanol. If we are to follow up this procedure or these steps over here we will see that this di iron 4 plus oxidation state intermediate will be reacting with methane to form methanol, but there will be an intermediate getting generated into the process. Let us look at the modified mechanism which has been recent which has been corrected little bit wherein you see that almost everything remains similar where iron 2 reacting with oxygen to give you the iron 3 peroxo intermediate or a iron 3 iron 2 super oxo intermediate from there on it can undergo further reaction or electron transfer to give you the iron 3 iron 3 peroxo intermediate. So, this peroxo intermediate can directly give rise to the substrate reactivity from Rh to RoH. So, that could be one of the possibility, but likely another possibility that can be comes that can be coming into the picture is the oxygen oxygen bond cleavage of the peroxo to give the iron 4 oxo species. Now, these iron 4 oxo species can then react with substrate methane for example, to give an intermediate the characteristics of which is not let us say known so far subsequently it can it can go and form the di iron 3 di hydroxo bridge intermediate. So, some of these intermediate remain same. However, these new intermediate such as Q star is important as well as it is important to know that this H peroxo at this peroxo intermediate can directly react with Rh and to give you RoH without perhaps formation of these intermediate. So, so far we have seen that these methane monoxygenase enzymes are having the dinuclear iron center and it is supported by these side chain of glutamate and histidine and the bridging ligands such as dihydroxo. These units are capable of doing or converting methane into methanol with the help of the other subunits which are essential part of this catalytic cycle, but most importantly there is this reaction mechanism as you have seen in the previous slide wherein this di iron center is reacting with oxygen to give rise to the superoxo then peroxo. Now, the revised version of this mechanism says that this the peroxo can directly be linked with the with the with this MMOH ox intermediate where it is a dihydroxo intermediate from there on the rest of the catalytic cycle can be completed. Alternatively, this peroxo intermediate can gives rise to an intermediate di iron 4 mu oxo intermediate which we were seeing. This intermediate can further react to give a metastable intermediate which is once again a very reactive intermediate which can then go on to form the iron 3 dihydroxo intermediate. So, in the next class we will try to see how these studies are done to better understand these intermediate that is forming over here. These iron 4 dioxo intermediate is quite reactive we will see the similarity of the reaction of this di iron 4 mu oxo species with that of the copper 3 bis oxo or mu oxo intermediate or keep studying we will come back soon later on in the next class discussing the mechanism of this reaction and how people have studied this reaction mechanism in greater detail. Thank you very much.