 Hi, how are you doing? Today, we will discuss cytochrome P450, this is a jolly good enzyme, this is a very very important enzyme right. It can do many reactions, many different type of reaction almost any oxidation reaction that you can think of, I think it can be done by this enzyme. It is having a porphyrin center at the heart of it, you have seen the similar type of structure in let us say hemoglobin, myoglobin, it is a proto porphyrin 9 type of species ok, this is a porphyrin center. But one of the difference now you are seeing is there is a methionine unit right, cysteine sorry not methionine there is a cysteine right. And in front of it the active site structure shows that there is this organic substrate which is going to get hydroxylated. So, this is the CAM4 ok, while there are many different version of this enzyme, essentially the one great thing that it does is very simple hydroxylation chemistry, it can do effectively at the aliphatic center. Obviously, there are many other reaction that it can do very effectively, but other than hydroxylation reaction those reaction we will discuss in a moment. So, this is the organic substrate that is sitting right in front of the iron site, there is the cysteine binding and as you can imagine oxygen molecule will come and bind over here and then it will react because oxygen will get reduced by these iron species, those species are very reactive oxygen intermediate, those reactive oxygen intermediate will end up reacting with this organic substrate. So, what we are trying to tell you here is this metalloenzyme in presence of an organic substrate is capable of doing oxidation reaction right. So, if you have an aliphatic substrate in front of this metalloenzyme cytochrome P450 it will be oxidized and this is precisely the reason why lot of let us say drug medicine that we take prescribed medicine that we take. These enzymes are so good that they can react with those medicine and medicines normal function can be complicated or it does not allow the medicine to react at a desired location before medicine does its job it starts working on the medicine itself. So, this is a big problem for the pharma industry that presence of cytochrome P450 enzyme. It is such a universal reactor that almost every organic substrate perhaps can be reacted with it ok. It has many implications this enzyme has many implications in biosynthesis of many different molecule including those we will see biosynthesis of vancomycin which is a secondary metabolite also primary metabolite and different also different other materials if we accidentally inhale or accidentally consume in our body they can start reacting with them. And if sometime what happens if those material itself may not be that very problematic let us say not carcinogenic not having any bad effect, but upon reacting with cytochrome P450 it creates some intermediate which is becoming now carcinogenic in nature. So, this enzyme is really really important to understand how it is show reactive that it reacts virtually with let us say everything right that we take. And therefore, we need to understand the chemistry of the cytochrome P450 in greater detail it is simple chemistry, but very powerful and effective chemistry right. Just to tell you again that this is a porphyrin center there is a proximal site where the sulfur binding is there there is the distal site where oxygen binding will take place and along with the different protein residue there is the organic substrate that is hanging in front of the iron oxygen species as if like it is perfected to react with the iron oxygen species that will be getting generated at this site we will see that in a moment. Before that I think I we did not discuss this MO for oxygen molecular orbital diagram of oxygen as you have noticed previously that it is having these two unpaired electron at the anti-bonding orbital and this is the triplet ground state and with a bond order 2. So, as you can see over here there is 6 electron minus this 2 electron that is 4 electron divided by 2. So, that would be bond order 2 this is a triplet ground state for oxygen, but most organic compounds have singlet ground state that is quite phenomenal and that oxygen can have a triplet ground state and still it is so popular and so important and because of this triplet ground state I think a lot of reaction that can be operated while reacting with the metal center right. Let us look back at the processes of course, cytochrome P450 requires 2 electron to convert the second oxygen atom of the oxygen molecule into water where are those 2 electron coming from as we have discussed in the electron transfer cases these are coming these are coming through the chains of events that is happening over there. So, essentially iron sulfur cluster that we discussed in the electron transfer protein is the one which is delivering the electron to cytochrome P450, but before that these electrons are getting hopped in from NADPH from the flaboprotein and then to iron sulfur protein and this is what the flaboprotein looks like all of them are acting as electron transfer site ok. So, electron is hopping from this center to that one to that one and to that one right. Finally, to the cytochrome P450 so it is a chains of events that are happening to provide the desired electron to the cytochrome P450 sites ok. Let us look at some of the chemistry that can be done by utilizing this cytochrome P450 as we mentioned there are plenty of reaction that can be done by utilizing this chemistry. So, it is capable of doing substrate hydroxylation chemistry for instant if you are having a substrate like organic substrate it can go and go and give you a desired product that you would need if you have an aliphatic substrate for example, there are different sites of which can be reacted with these iron oxygen species. So, substrate aliphatic substrate hydroxylation is possible if you have the olefin, olefin can give you the olefin epoxidation product. If you have N alkylated product substituent such as let us say N N dimethyl substituent it can react from the N formyl species and then undergo a further cleavage to let us say give you NH and methyl. So, many if you have the sulfur containing compound you can get the sulfoxide species many different reaction that can be happening over here. So, most importantly you can also get these reaction quite going with these places for example, S D alkylation chemistry is also possible where this D alkylation of this moiety will be possible O D alkylation is possible this whole unit can be D alkylated to give you first of all hydroxylation and then this alcohol formation. So, the alkyl part is forming the formaldehyde in these cases you can have the N hydroxylation chemistry as you can see here N hydroxylation chemistry can happen, N oxidation can happen as you can see over here sulfur oxidation can gives you the sulfoxidation product and overall your aliphatic substrate C H bond be primary secondary or tertiary all can be hydroxylated to give the C O H product, olefin as we mentioned can give you the olefin epoxidation product. The alkyne can form into the carboxylic acid terminal carboxylic acid even a benzene ring can form an epoxide to give you finally, phenol when with the transfer or the rearrangement of the phenol. MD alkylation reaction can be also possible where this methyl group is turning into the formaldehyde and the R N H part is becoming R N H 2. So, it is a flavor of reaction it is a series of reaction that it can be done that cytochrome P 450 can do as you can imagine all these reaction put together if all these things are happening let me go back if all these things are happening these reactions are going to complicate the whole spectrum of the reaction that can happen in our body for example, right. So, cytochrome P 450 is an enzyme not to be messed with too much. So, any drug molecule that any pharmaceutical industry has to design they have to take care that these many different varieties of reactions are not easily happening or even if they are happening the molecule still remain active even after these reactions. So, that is a big challenge because cytochrome P 450 can do many, many reaction that can be unheard of that can be unthinkable by the chemist perhaps they are involved in designing the drug molecules, right. So, this is a very important enzyme once again and can give rise to the many important factors that can be happening in the enzyme, right. So, we will keep this in mind many different reaction system many different possibilities that we is coming into the picture. Well, this is the vancomycin the you know antibiotic of the last resort even in antibiotic in these cases we will see that these important carbon oxygen bond formation and carbon carbon bond formations are taking place by the cytochrome P 450. So, the biosynthesis of vancomycin is controlled by the cytochrome P 450. In fact, 3 units of 3 cytochrome P 450 comes into the picture one is for there another is for there and another is for here. So, cytochrome P 450s are involved in biosynthesis of different primary metabolites, bolides for example, steroid hormone secondary metabolites such as vancomycin and of course, it is also involved in metabolism as you are mentioning of different drugs of different medicine and it can involve in toxification or detoxification of external molecule, right. So, if any external molecule is coming into our body that can be detrimental for our body because or sometime it can convert into something good even if bad things are coming in. So, of course, it can do a lot of bad things because unknowingly since it is cannot discriminate too much it is so reactive, but of course, it is involved in many other important enzyme synthesis or you know important compound synthesis such as even this vancomycin synthesis biosynthesis. So, we will see in a moment also so these sort of CCO bond formation and CCO bond CCO bond formation CCO and CCO bond formations are taking place with the help of cytochrome P 450. So, you can imagine this porphyrin oxygen chemistry is extremely effective and that is why we need to understand this chemistry little bit in molecular level so that we know what exactly is going on in these cases. For instance if by accident this by benzo pyrin is getting incorporated in our body this benzo pyrin can be you know can be involved in oxidation reaction by utilizing cytochrome P 450. So, that therefore, molecules can be converted sometime into mutagenins and carcinogens. This is not by design of cytochrome P 450, but cytochrome P 450 once again it is so reactive if any foreign molecules are getting in it can start acting on those molecule for example, it can it can epox form the epoxide as it shown over there cis hydroxylation species it can form subsequently further epoxylation chemistry and overall once these species are forming these are not really great species right. So, these are the molecule which are carcinogen and it can intercalate it can interact with DNA it can end up damaging the DNA and can give extremely dangerous intermediate it can lead to different diseases and deadly diseases right. So, this compound itself is not carcinogenic or not too much harmful for our body, but in presence of cytochrome P 450 which is there anyway and therefore, this will start reacting with those cytochrome P 450 and then will deliver a material which in turn can damage our DNA which is not that great news for our body. So, cytochrome P 450 is a great enzyme once again, but it does perhaps more than what we would imagine and that is where it is so dangerous and we need to understand and design things according to the cytochrome P 450's need. Well, this is that camphor structure we were talking earlier. So, this is the camphor which is bound right in front of the porphyrin iron center as you can see the iron center is little bit outside oxygen is not bound with it yet as you have seen in case of hemoglobin and myoglobin upon oxygen binding these iron 2 plus center will move inside the cavity of this porphyrin and that species which is generated upon oxygen binding and the electron transfer superoxo and the subsequent reduction will see that it can affect on even on camphor to give the camphor hydroxylated product right. So, these are quite important reactions and we will be seeing that these reactions will have a overall say in how these processes are going on. So, today one more slide we will discuss here which will be discussing how these reaction mechanisms are happening ok. So, to summarize over here that we have seen many different reactions are happening. Why so many different reactions are happening? What is the mechanism of these reactions? That is what we are now trying to see. Well, the resting state of the enzyme is this iron 3 aqua species the organic substrate that we were discussing in the last slide let us say this is the organic substrate it can then come in and resides in the substrate binding pocket right in front of the digital site or right at the digital site where iron oxygen binding will take place. So, this is what is happening iron aqua molecule let us say just for drawing it is Rh over here which is sitting at the digital side and you still have a iron 3 center iron 3 center will not be reacting with the oxygen iron 3 water molecule has to go out once this organic substrate is coming into the picture ok. This organic substrate comes into the picture and resides in front of the of the of the of the digital site of the iron site. So, Rh comes in water goes out one electron as we were discussing there are overall two electrons involved into this process. Now one electron in this whole process comes in and this iron 3 as you have seen iron this electron is coming through hopping from different centers. This electron is now reducing this iron 3 center to make it iron 2 plus this reduced structure is now ready to react with oxygen ok. The substrate is sitting right in front of this iron center oxygen it reacts as you have seen in case of hemoglobin and myoglobin also this iron 2 plus reacts with oxygen to give you iron 3 plus and superoxide intermediate. So, one of the electron from the iron 2 plus is now getting transferred on the oxygen molecule to give you iron 3 plus superoxide species. Well in case of hemoglobin and myoglobin this was a completely reversible process and that is how it transports and delivers oxygen in different part of the body in the in case of let us say hemoglobin, but in this case we do see that there is a organic substrate sitting very close to this ok. It is quite interesting and this is the step why then subsequent reaction goes on because these are reversible step this is a reversible step, but from there on and another electron transfer occurs. So, this is once again it is from one iron center gives only one electron the second iron center as you have seen from the iron sulfur cluster this electron is coming through a hopping of electron from different sites including those other oxygen other organic molecule containing site which are good for the electron transfer. The second electron first electron reduce it make it active second electron reduce the superoxo species to make it iron 3 peroxo species. This is iron 3 plus this is peroxo species it got reduced to peroxo oxygen if you look at from the beginning oxygen itself is one electron reduced at this space and at this space second electron reduced, but more importantly iron in this step remain iron 3 plus. Now a proton can transfer or proton can add with these iron 3 superoxo to form these iron 3 peroxo intermediate this is also known as compound 0 in the literature compound 0. This iron 3 hydro peroxo species is quite interesting because this is the one which will be responsible for chemistry that will be happening next. So, R H is still there substrate is still sitting right over there and another proton that comes in that converts this OH into water. So, what you have seen this oxygen atom two oxygen atoms are there and from this oxygen molecule two oxygen atom out of the out of the two oxygen atom one oxygen atom is converted into water. So, now this is the water molecule this part goes out and this iron 3 now is forming iron 5. Essentially this is iron 5 it is a porphyrin radical cation with iron 4 plus that goes on in there I will come back to that in moment and the water molecules forms over there right. So, from there on it is the substrate that reacts R H reacts with iron 4 peroxo to give iron 4 hydroxo and ROH, RO water replaces ROH to gives back the general species. Let me discuss how this compound 0 to compound 1 is forming this is compound 1 and then this is known as iron 4 hydroxo porphyrin species is known as compound 2 right. So, we will see that in a moment ok, what we are trying to see how these reactions are happening. So, what you have seen over there that you have a iron 3 hydro peroxo intermediate now this hydro peroxo intermediate with a porphyrin center this is compound 0 right. So, from there what will happen that it can take one proton to give you water molecule right. So, this goes on as water molecule. So, this part gives you the water molecule. So, essentially 2 electron from iron is going and kicking out the water molecule the hydroxide comes out hydroxide binds with proton to give you the water molecule. So, this should be then iron 5 double bond O right. In these cases what happens the reduction potential is such that it is not really iron 5 oxo it is actually iron 4 and the porphyrin unit is giving out one electron iron 4 radical catalyst is forming and then oxo species is forming ok. This is why this is called this species not this is why this species is called compound 1. So, this is compound 0 and this is compound 1 as you have seen that 2 electrons are coming overall for this process one electron from the porphyrin ring another electron from the iron center that is why this species is not the right representation this is the right representation where you see this is a iron 4 oxo species, but it is a radical cation on the porphyrin moiety right. So, this is compound 1 and from this compound 1 as you can see well for chemistry purpose it is perhaps easier if you think that this is iron 5 oxo although it is not iron 5 oxo. Subsequently what will happen the organic substrate Rh will come out and then let us say this oxo will attack on it as a radical mechanism and this iron these 4 oxo species will give them iron 4 hydroxo with a radical cation is there this would be called compound 2 ok. So, once again let me clarify it it is easier for chemists to think that this is the species although this is exactly the species if you just for a moment think that this is the species it gets little easier this is little easier to understand for us let me get back once again ok. So, what we are saying that if you have iron 5 oxo and then you have let us say Rh it is easier to think that this is going to iron 4 and this is how it is that iron 4 hydroxo is form along with Rh of course, in this case that is what is happening, but during these cases you still have this porphyrin unit. Now in these cases in this compound 2 this is not the radical cation this is actually the iron 4 hydroxo that is over there originally this was iron 4 oxo with radical cation this species this is not the species, but that is what we are drawing from this species that is the species that is forming the porphyrin is no longer actually radical cation. So, this is the species known as that compound 2 right. So, let us go back once again what we have seen then we have seen that this iron 3 over here it is getting reduced by one electron to give the iron 2 species these iron 2 species reacts with oxygen to and so, one of the electron comes in into the oxygen it reduced to the super oxo. So, this is the super oxo species super oxo intermediate and iron becomes iron 3 because it gives up one electron another electron comes in this is the peroxo intermediate that is getting generated over there this is now remained iron 3. So, this electron is coming from outside. So, gives the iron electron Rh is sitting over there nothing has happened like a good guy sitting over there nothing happened the protonation on this peroxo gives you the hydro peroxo species. So, this is the hydro peroxo species this hydro peroxo peroxo species this hydro peroxo iron 3 hydro peroxo known as compound 0 once again Rh nothing happening here one proton comes in this goes out at OH in this case as I was saying 2 electron is pumping in one from iron 3 plus one from the porphyrin ring. So, it is iron 4 oxo iron 4 oxo with a radical cation on the porphyrin right. So, this intermediate which is nothing, but iron 5 oxo species this intermediate is actually responsible for reacting with Rh. So, it will take up one H dot. So, to make it iron OH and then it quenches one of the electron this quenching happens on the porphyrin. So, the R dot sits over there and porphyrin now radical cation gets quenched and it is just porphyrin. So, it is essentially iron 4 oxo in other word you can think of that that iron 5 oxo upon reacting with Rh giving you iron 4 hydroxy and R dot that is what is easy to think, but anyway you should not draw this as a iron 5 oxo because this is iron 4 plus with a radical cation on the porphyrin. So, overall this is how the radical mechanism is getting initiated this is where you see that a radical is being formed and that is quite far full radical. This is the radical which will then can let us say for hydroxylation reaction a rebound of hydroxo will happen on the R dot to give you the Rh intermediate and we will get the product formation. Well, we will come back to that in the next class what we have seen in briefly then in cytochrome T450 we have beautiful chemistry, beautiful hydroxylation chemistry, Ndalkylation chemistry, sulphoxidation chemistry, olefinipoxide chemistry, cyclization chemistry if required in hydroxylation chemistry what not I mean we have seen quite a lot of chemistry that can be done by this enzyme and this is not only a great enzyme sometime it is too great and can create problem for us even for the medicine that we take can become or can be very problematic in presence of these enzymes. So, we need to learn the better designing of the drugs because cytochrome P450 is always there it is going to be always reactive we need to learn how to prevent our medicine to get affected by this enzyme. We will come back to cytochrome P450 once again in the next class keep studying the book to follow as I said lipid and bugs bio inorganic chemistry principles of bio inorganic chemistry keep studying. Thank you very much.