 Now, this is the one example I am trying to tell you, again you do not have to worry about few things, let me get it very clear. You should not be worrying about these drawing, these are the protein backbone, protein backbone means amino acid put together it is forming, it is a huge polymer. Polymer means let us say it is a huge length, it could be a lot of things together a lot of amino acid together and that is how this sort of different orientations are given, because that is how they look like different, different you know way of their structural aspects that is going on over here structure are different. So, you do not have to worry about all these protein backbone or the peptide backbone, if you zoom on over here you see that amino acid backbone, these are from different amino acid, all those amino acid if you look at you can see their structure, they are nothing but organic compound ok. For example, over here tyrosine, tyrosine is nothing but phenol containing amino acid which has a phenol let us say phenol. Tyrosine oxygen can bind with the iron center, another tyrosine oxygen phenol phenol oxygen can bind with the iron center, this is histidine and this is aspartate, aspartate is having carboxylic acid CO2H, carboxylic acid that can bind with the iron center, this is having histidine unit ok, just nothing but imidazole, imidazole you have heard of imidazole phenol carboxylic acid these are the side chain of amino acid, all those amino acid we know which it is having some side chain which can bind with the iron center or any other metal center. So, this is the one let us say it is transport iron that is what I was telling this is a PIDJA delivery guy, it is a delivery guy iron delivery guy right. Now, it picks up iron from different side and it essentially see it is not like they have to move they can just pass on also, you can I can take I can give it is a it can be a relay process as well right. Overall this is where it picks up or that is how one of the example we are showing how the metal center is going to be picked up. It binds with the amino acid backbone and it gets picked up and then delivered at a certain point where it can be stored pick up iron from storage iron protein ok, you can read, but usually what we see is iron centers during this movement when it is moving this is pick up and delivery time it is going to be plus 2 during transport and storage it is stabilized in plus 3 state. Because plus 3 are the one which is oxidized state little bit stable state during this storage it has to be stable. So, their redox chemistry is used for their transport as well right. Storing is in plus 3 form and during transport also it is in plus 3 form, during picking up and during delivery it is in plus 2 form ok. So, what essentially we are trying to tell you is a metal ion gets delivered by different approach. One of the main approach is simply different protein backbone that is nothing, but amino acid backbone protein backbone is nothing, but amino acid backbone. Amino acid has this phenol unit this imidazole unit carboxylic acid unit these are ligand these usually acts as a mono dented ligand and thereby can bind with either let us say iron 2 plus or iron 3 plus or zinc 2 plus or copper 1 plus copper 2 plus of course, you have to also understand that hard soft I told you hard soft acid base when you have a harder one for example. So, harder anion will come right when you have let us say you have copper 1 plus versus copper 2 plus copper 2 plus is harder one. So, if copper 2 plus has to be delivered it and let us say the ligands are very hard copper 2 plus is hard. So, the ligands associated with it during the delivery time it is let us say very hard right hard anions are bound. Now, the place where it is going to get delivered it is not having the harder anion what copper has to do or copper has to undergo is reduction copper 2 plus will undergo reduction to come to copper 1 plus since copper 1 plus is soft that side previously which was having hard ligand it will not bind it very tightly. Now, that softer copper 1 can be exchanged from one place to another let us say storage place if you say or that or the delivery place wherever it is delivering. So, that is how things gets also delivered you have very hard situation very strong situation hard anion all the oxides let us say are bound let us say carboxylate O minus or phenolate pH O minus differ whatever you know as hard, hard ligands are bound with a hard cation. Now, then you want to deliver it to a softer place. So, the metal center has to undergo a reduction and thereby softer center will pick it up and the reverse is true also the other case soft 2 going to hard. So, all these simple principle actually matters what ligands is there what ligand environment is there what oxidation state is there and thereby you can deliver in a particular manner. So, this is another enzyme I am just giving again you do not try to memorize too much because you may not be able to get into too much detail we do not want you also at this point these are protein backbone it looks quite funky and big, but if you nail it down if you zoom it down what you see is this. So, different protein backbone is coordinated with metal center and another different protein backbone is coordinated with another center. Essentially once again hard soft acid base principle will be followed of course, also another thing you have to see what is available sometime whatever is available they can still go with it if there is no competition between hard and soft center. Even if it is in a soft center hard if metal center is soft, but hard anions are the one which is available it is ok it will go with it it is not it is not like they cannot bind binding efficiency may not be great that is it. Overall I think the what we are trying to say is this enzyme is able to function since copper can undergo oxidation state blah blah blah you read it I think I explained it copper 1 plus and copper 2 plus iron 2 plus and iron 3 plus these oxidation state change help you to do a lot of chemistry 1 or 2 chemistry we will discuss today that is number 1. Now, zinc plus does not undergo usually these oxidation state change they can give the structural aspect they hold things together and thereby provide some chemistry to be done at that center let us move on. Now, I think the major part of the syllabus is on hemoglobin and myoglobin or so called the porphyrin centers right. I do not know whether you have studied little bit before fine. So, porphyrin center is something this is one thing I would ask you to remember and draw it again and again. Usually what people do is they cannot provide this double bond in a rational manner you try to draw it usually in the exam it is given ok draw porphyrin or you will be asking a question in a way that you have to draw porphyrin draw the porphyrin what is not important is these are what substituents are there. Of course, these substituents these R group will determine whether a complex is going to be soluble or how soluble they have a role, but for this class you do not have to understand or know what the role they are actually playing. By changing the R group you can change or modulate the properties to some extent ok. Now, this is the porphyrin you should be very familiar with this is nothing, but a ligand this is a kilator it kilates the metal center actually it is a tetradentate ligand usually what we have seen so far in the amino acid backbone is mono dentate ligand usually right means only one coordination sites are there. One let us say phenol phenol oxygen can bind right imidazole imidazole nitrogen can bind, but porphyrin these 1 2 3 4 4 nitrogen can bind and if you put a metal center over there you can see 1 2 3 4 5 6, 6 membered metal that ring will be formed. You have seen the bidentate ligand how they are forming dent I mean how they are biting it bidentate they are biting it this is like tetradentate and this is a very good kilate. It can you know it is almost like those EDTA we were talking EDTA can extract out porphyrin is a strong ligand it can extract out iron let us say or any metal center very easily ok not I mean of course, not neutral form let us say iron 2 plus or iron 3 plus these are these can be extracted or these can be put very easily over here of course, there is a size right there is a cavity. So, anything that does not fit into that cavity will not have strong binding with the porphyrin. So, the size of the iron let us say iron 2 plus versus iron 3 plus will matter whether it can fit in here or not properly ok. High spin and low spin will matter and thereby of course, high spin low spin size will be different right and thereby you can see whether metal can be sitting over there or not ok. Now, this is what I was talking about iron binding you see 6 membered rings 6 membered rings 6 membered ring and 6 membered ring it is holding it very tightly you do not have to memorize this side chain you have to just remember the core structure core structure includes these pinhole ring right. So, these side chain whether this is a double bond or a methyl double bond or methyl methyl or carboxylic acid these are the one you do not have to remember anything ok, but the porphyrin ring itself porphyrin ring means without those substituent that is the simple porphyrin ok you can then substitute. So, you should be very familiar with this this is the imidazole which also binds at the. So, the if porphyrin ring is actually flat like this flat. So, iron is right let us say in the middle of my pump and then imidazole is right over here in the middle. So, below this plane this is a plane below this plane imidazole is bind like that is bound like that ok. So, it is a tetradentate ligand porphyrin is a tetradentate ligand this is the fifth coordination imidazole ok. The sixth coordination is over here which is open or almost let us say something is there very far interacting not too close this is the center where oxygen binds when we are inhaling right oxygen gets in this is the site on top of it on top of the flat porphyrin ring that binds the oxygen and that determines the life. You can now imagine I will come to that if this oxygen binding site this is imidazole this is oxygen binding site if this oxygen binding site is occupied by carbon monoxide or cyanide I will see you again after life ok. So, that is what happens those bindings are going to be stronger right compared to oxygen binding those bindings are very strong. So, oxygen cannot displace them the ligand exchange between oxygen and CO will not happen other or cyanide will not happen other way around will may happen oxygen can go off cyanide can come in that is why it becomes so poisonous. So, deadly carbon monoxide cyan and so on it is also one of your tutorial question ok. Now, I will go into again once again do not get I mean sometime we do get very well there is feedback also right. Do not please give your feedback not at the end of the class at the usually I think at the end of the semester or something you are allowed to evaluate us as a teacher. The way we do also get an opportunity to evaluate your performance by taking exam I think that is kind of our exam it determines a lot of things for us. So, take it seriously for all of all of you or for all of the teachers because it determines of course, you know not too serious it is not life threatening, but it did determines something ok. So, the major one of the major objective or topic of this chapter is myoglobin and hemoglobin how molecular oxygen or oxygen which is in the air how it binds in the blood. In the blood we have porphyrin center the porphyrin center we were discussing over here right. So, this is there in the blood and how oxygen from air binds with this center and what happens during this binding what happens when oxygen gets released ok. So, as you know usually what we have learned or what happens oxygen goes in for our body we are showing just human it goes in in the lungs it gets in and then it gets delivered from all of body parts right. Now, let us say in the muscles it gets stored right it gets stored in muscle. So, how oxygen is going to get transported and then how it is get stored in the muscle that is the one kind of the major things we will be discussing today. So, the storage protein let us say in muscle what we have is myoglobin where that is what we are trying to say iron storage is there some other metal storage is there then oxygen storage is there oxygen transport is there right. So, hemoglobin is the one which transports oxygen myoglobin is a single unit means one porphyrin center is there only one hemoglobin is the one where 4 units are there 4 iron centers are there 4 porphyrins are there ok. Where are they roughly again you do not have to even attempt to memorize any of this structure please do not ok. This is a hem center hem center this is another hem center this is another hem center this is another hem center. Hem means porphyrin ring plus iron that is called hem that porphyrin ring flat plus iron that is the hem. So, we have a hem center in here we have 4 hem centers are in there how they are connected they are connected by this protein backbone ok. It could be let us say I mean hem center is here another hem center is here in between hem center is there another hem center is there in between there is those protein backbone amino acid side chain lots of amino acid side chain putting together of course, is peptide backbone those protein backbone peptide put together is forming protein those protein backbones are together. It is not like all of them has to be covalently linked covalent bond we know some of them are covalent bond some of them are just linked by hydrogen bonding. Let us say this is a protein this is another protein they are linked by hydrogen bonding right. So, hem center hem center they are linked by let us say simply said amino acid amino acid residue which is having let us say your covalent bonding ok. So, all of them are connected if not directly none of them are directly connected like iron center bound with nitrogen that is a direct coordination ok, but there is indirect coordination such as hydrogen bonding and other loose forces which becomes important. So, they are connected they can communicate with each other they can talk it is just like we can communicate they can communicate with each other that is essential ok. Communicate means what happens to this center when something happens to this center this center comes to know. If something happens to this center this center comes to know and it relays that is that relay of information will be learning in form of cooperativity. They cooperate with each other ok that is the cooperativity that thing is missing from myoglobin because they have nothing to no one to communicate they are isolated kind of system one porphyrin center it is not like they are unhappy just they do not want to communicate ok. Now, of course, if you want to compare hemoglobin and myoglobin first thing I said is this is the myoglobin one hemoglobin one having 4 iron center. So, 4 unit versus 1 unit of course, their molecular weight is going to be you know related by 4 times. So, myoglobin sorry hemoglobin is approximately 4 times compared to myoglobin ok. Of course, both of them are having porphyrin both of them are red in color in the oxy form. What is oxy form? Oxy form simply means that oxygen is bound deoxy form means that oxygen is not bound period ok oxy hemoglobin deoxy hemoglobin deoxy hemoglobin oxy myoglobin deoxy myoglobin these terms will be used deoxy means no oxygen is there ok. What is there to remember? I do not see anything to remember it is this what I am saying you remember this nothing to remember this is a porphyrin center iron is there porphyrin you have to remember yes porphyrin you have to remember that is the only thing I am asking you to remember rest of the things you can just read or you know understand as a story I think that is good enough story we do remember I guess we can tell a tale right. Now, so the color as you know the blood color is red it is simple right it is going to be red. Now, deoxy form when if you do not have any oxygen in it it is going to be red purple if you do not want to remember do not remember ok. Now, I did not mean in that way of course, iron is in reduced form you are going to add oxygen. So, iron will be in reduced form what will not be discussing today is the redox chemistry what happens to the iron is it going to iron 3 plus yes it goes, but that information we will not be discussing for the class. What we will discuss is initially what happens initially redox chemistry does not happen redox chemistry happen right after it we do not need to know this. We will discuss that oxygen comes and binds with iron center that is it that is the syllabus right after that binding oxygen actually gets reduced iron gets oxidized that is not part of the syllabus do not even write in the exam ok. I will be discussing whatever part in the syllabus ok this is the same thing again 1, 2, 3, 4 iron center nothing else ok 1, 2, 3, 4 iron center we are discussing myoglobin no hemoglobin see you understand by now ok. Now, see something like if you put a lot of oxygen in a lot of oxygen if or other way around if lot of oxygen is not there you will be suffocating right you feel breathless that means oxygen is not there breathless situation is less oxygen right that situation and then if you compare to breathless situation if you want to pressurize oxygen too much oxygen is there that oxygen pressure you can vary and then you can measure how oxygen is uptake uptake means how it is taking how porphyrin center in hemoglobin and myoglobin is taking up oxygen with respect to oxygen pressure. You pressurize oxygen say gas cylinder let us say you can pressurize gas cylinder you can have not so pressurize gas cylinder with respect to partial pressure of oxygen as you increasing the pressure of oxygen and you see how oxygen pickup is happening at the myoglobin and hemoglobin. What you do this is the curve for hemoglobin this is the curve for myoglobin and this is the curve for hemoglobin at pH 7.6 hemoglobin pH 6.8 is red curve lot of things are red this one sigmoidal curve is at pH 6.8 hemoglobin pH 6.8 hemoglobin 7.6 is the blue curve for hemoglobin and then myoglobin that is the curve. Now, so what we are seeing as you increase the partial pressure of oxygen uptake goes up percentage saturation means you can keep on bind up iron center you have more oxygen pressure it will go up sigmoidal curve you are saying it will go up and after certain point it will saturate it will not take up any more oxygen that means that is the maximum that is that is almost like the equilibrium is reached after that no more oxygen pickup will happen. So, you pressurize you know you take pure oxygen we have 16 percent oxygen pure oxygen you can take or pressurized oxygen you can take. So, therefore, what we have measured here is oxygen pressure is going up how saturation level is going how our hemoglobin and myoglobin is up taking. Now, in lungs what happens in lungs we have this is the partial pressure we have this is the partial pressure. Now, it is almost the it is very, very high oxygen amount we can take it up at the at the at the low pressure if you see at the low pressure you see the myoglobin is having higher affinity that is in the muscles where low pressure of oxygen is there oxygen is going to transfer transfer from hemoglobin to myoglobin in muscles because firstly because at a low pressure of or you know myoglobin has a higher affinity or higher affinity for oxygen compared to hemoglobin. And you know myoglobin also has pH something like 6.8 at a lower pH. So, at a lower pH this is the pH curve with respect to pH hemoglobin oxygen uptake is shown in here at higher pH oxygen uptake is shown in there since in in muscles pH is pH is low then what happens from hemoglobin. So, hemoglobin is over here that much oxygen it can pick up myoglobin right over there is having high affinity for oxygen. Therefore, hemoglobin to myoglobin transfer will happen that is how the transfer mainly happens both at a lower pressure hemoglobin has higher binding and the pH for pH 6.8 this is pH 6.8 curve you see hemoglobin binding is over here myoglobin binding is over there. And therefore, hemoglobin to myoglobin oxygen transfer occurs it is very beautifully written in this here we you can read it is a Stiver Atkins book which is your textbook you can read in there in detail. Now let us go on. So, what is what is essential for oxygen binding irrespective of myoglobin and hemoglobin what is essential? The thing that is most essential is we have to prevent reduction of oxygen. Oxygen can get reduced by 2 electron that is oxygen to peroxo formation can happen iron center can give 1 electron another electron the second electron can also come from either that iron center or from another iron center. Since you are seeing that 2 iron centers are really far apart from each other although they communicate, but the electron transfer will not be that very fast. So, therefore, what happens is oxygen to peroxo formation does not happen that would have been a irreversible thing. So, what all happens in our body is a reversible oxygen binding oxygen does not get reduced completely to peroxide hydrogen peroxide let us say that that reduce reduction does not happen fully. Oxygen to peroxide level reduction is prevented by default by design because the 2 electrons are required oxygen to peroxo that 2 electron reduction cannot be possible because this that reducing equivalent means that you need something to give electron right that is how you will reduce. Reducing equivalent is not available readily there is nothing to reduce very readily right over there that is one aspect. Another aspect is you of course, you have to prevent the oxygen from going out it has to bind those binding even though it is not that very strong what happens over here is let us say it binds over here for example, oxygen is over here this oxygen by will have after binding oxygen will have hydrogen bonding some sort of. So, let us say this is my porphyrin this is my iron center oxygen binds this lone pair right and this is the axial position top the below is again your histidine or histidine unit right this is your porphyrin. Now, this oxygen you will see there is a hydrogen bonding from another histidine. So, this hydrogen bonding also gives the stability to the structure overall it is a bent oxygen geometry and it prevents it prevents the reduction oxygen to peroxide this is peroxide reduction that is what happens in our body when you try to synthesize porphyrin which I have done let us say actually I have done if you take porphyrin and iron synthetically prepared. So, that is why synthetic blood is very difficult to prepare why because the moment you have porphyrin it reacts with oxygen if you have porphyrin synthetically made it reacts with oxygen overall what you get is here iron 3 see what happens here is porphyrin is too far oxygen can come over here it is it cannot bridge between two porphyrin, but in synthetic system two porphyrin can come very close because there is no restriction there is your globular protein that is again another tutorial question which is preventing the dimerization and also providing the stability for the monomeric structure in synthetic system what is happening is or what can happen invariably 99.99 percent cases it happens that porphyrin P means porphyrin oxygen reacts and overall you do not have to remember all these again this one iron 3 iron 3 each iron is giving one electron therefore, iron 2 plus going to iron 3 plus oxygen is getting reduced by 2 unit oxygen 2 peroxo is forming this reaction is killing the porphyrin or the oxygen binding this is not too much of a reversible reaction. So, in our body what happens is it is a reversible oxygen binding oxygen can bind and pop up it can go it can be delivered to myoglobin hemoglobin takes up transport and deliver in synthetic system it is very very rare to deliver how you can deliver that can be done in synthetic system it requires if you put some steric bulk steric bulk means let us say you have you put an umbrella you put an umbrella on top of porphyrin of course, that will take a lot of synthetic effort to put an umbrella on porphyrin if you put an umbrella another umbrella will not be able to come very close and affect. So, another umbrella even if it comes it will come over here, but iron site is here iron site is here. So, they too cannot communicate. So, these are called steric effect you put bulk and thereby you prevent, but that is very very expensive I mean it is a lot of synthetic effort, but by default nature has it in our body we do have that by default we do have what we do have these protein backbone protein backbone prevent these two porphyrin to come close and thereby we do not have any problem to deal with right.