 Welcome back to the NPTEL lecture series on bioelectricity. So, let us resume the story of photosynthesis. So, we have already discussed about some of the initial plan of nature or how nature has evolved its complete light to chemical energy transformation system. So, one thing which is very remarkable to observe the most remarkable feat of nature is the way this whole chlorophyll, the assembly of the chlorophyll and the whole chloroplastor structure, this whole evolution itself because since during the last century when the microscopic techniques developed and we are trying to understand the intricate details and the molecular self assembly of this whole process, more and more we are learning about it, more and more we are looking at it with amazement and all that I mean what a wonderful photovoltaic system nature has developed over a period of billions of years of evolution. So, today what we will do we will talk about in this lecture this is the thirty third lecture. So, we will be talking about some of the intricate details at the level of chloroplast membrane and the level of the stromal membrane and how the electron transfer takes place, how the light energy gets converted and how this leads to a proton gradient which eventually leads to the synthesis of the energy stored by that process eventually leads to the synthesis of energy rich molecules. So, let us move step by step. So, coming to the first slide just a bit of a recap if you want to you know kind of get a global feel of the whole phenomena. So, here is a leaf and if you take a cross section of the leaf this is how it looks like a cross section of the leaf and within that there are something called mesofil cells and these mesofil cells are rich in chloroplast. If you follow this slide you will see they are rich in chloroplast and if you could take a cross section of the chloroplast in the microscope this is how will you see the stroma and the green um and the thylakoid membrane and the thylakoid compartments and these different thylakoid membranes are connected by the connecting tubes and this is a double layer structure in which this thylakoid membrane is being housed. So, if you even just by looking at this self assembly you will be able to appreciate that how wonderfully this self assembly has been made out there and it is on those thylakoid membrane the actual molecular assembly or the molecular machinery of photosynthesis is housed which is playing a critical role in the energy transfer process. So, and there is one more picture here in black and white which essentially shows actual micro photograph of the chloroplast. So, if you go in a further little bit of a detail just to get a much more better view of the mesofil cell you can go through this slide where you will see the cross section in a much better way and you see the cell wall the vacuole and the chloroplast and you see the nucleus and you could observe one very interesting feature that um along that small organ which it is believed that is that it has been parasitized this. So, at the level of self assembly the level of kind of you know what I should say the level the dimension at which all these very precise self assembly of these structures have taken place this is exceptionally important and one really needs to appreciate that if some day we really can like even if we try to mimic these kind of structures using nanotechnology tools we really have to learn much better ways how we can manipulate molecules to have such a wonderful self assembly. The guiding principle for self assembly now coming to the light. So, you have to realize that this is structure. So, this is structure what you see here is equipped with three interesting features. So, for synthetic machinery the three important features what is equipped with is the light harvesting component that is what we are going to discuss in the next slide. Then you have power system two followed by power system one and then underneath power system one you have water splitting cluster and from power system one of course, the NADPH sorry NADPH reductives what we are not dealing with in this synthesis of energy molecules. So, this is the overall component of this naturally evolved system. So, the first level is the light harvesting. So, this light harvesting phenomena. So, you always exclusively talk about there you have the chlorophylls which are present there I am just putting CHL for the chlorophyll. So, it is not the chlorophyll which is exclusively doing the task chlorophyll is being supported by several other molecules which helps to funnel the light to chlorophyll something there are series of proteins which are sitting like this when the light energy falls on them they kept on funneling this thing all the way and it finally, reaches the chlorophyll molecule and what you see out here in the next slide is some of those scheme. So, these are like on the surface of the these are acting as antennae molecules that is what you see that which is written like the antennae pigments or antennae molecules which are present there for system and it is light harvesting antenna. So, it is just like on the top of a building have this antenna to you know catch the or a any kind of electronic gadgets like TV or you know radio whatever you have this antenna which is catching the radio signals. So, same way there are the molecule at the molecular level there are molecules which is the ability to capture the sunlight and not only they could capture they are arranged in a three dimensional geometry in such a way they transmit it to the final candidate which is chlorophyll of course, there is some degree of loss, but this what ensures that there is a maximum capture of sunlight as possible and if you look at center like this there are several molecules that support the light trapping of course, you always talk about chlorophyll and chlorophyll A chlorophyll B, but there you have lutein you have zeaxanthine you have beta carotene you have lycopene there are series of antenna pigment complex which help in funneling the electron to their specific site. Now, coming to the specific site what we meant by the specific site. So, the antenna molecule has funneled it here, but is it this chlorophyll immediately ejects an electron and gets activated actually does not work like that it is very interesting to note at this level also there are some very interesting processes which taking place. So, these what I am circling are all chlorophyll molecule now what happens is this energy is transmitted to this, this is transmitted to this, this is transmitted to this, this is transmitted to this. So, there is a kind of hopping motion if you see the slide you will see there is a hopping motion of the ejected electron because of the action of a photon at the reaction center. So, it is never remember one molecule of light say one molecule of or one molecule or one photon say for example, leading to one electron it is never like that it is multiple photon leading to finally, one activated chlorophyll molecule. You might wonder why nature has designed a system which is from a simple perspective will sounds very once again will sounds very inefficient possibly one of the possible thing which I can upfront tell if there is a continuous ejection of electron from this chlorophyll molecule because as soon as the chlorophyll molecule ejects an electron it behaves like a free radical. So, if there are too many at a place then the funneling or it may cause the self damage. So, possibly this is all speculation possibly in order to avoid such self damage nature has designed it in such a way that not all chlorophylls are reaction centers there are a specific reaction centers where the electron is ejected out and that electron is accepted by several series of carriers. The one which is say for example, if this one the one which I am shedding is a reaction center in an around this there are series of other proteins which are there, but again what we do not know say for example, in a pool of chlorophyll is the reaction center unique does it remain unique throughout the life of that particular cell or does it change you know we do not have an answer to that, but we indeed know if there are 10,000 or 20,000 or 1 million chlorophyll molecule present here it is not each one of them are reaction centers there is specific moieties which are reaction center and nobody knows really what decides which one will become a reaction center this is something still mind boggling thing. So, the take home message at this stage is we indeed have reaction centers if you see this slide there are so many in the dark green sphere you see lot of chlorophyll molecules, but the reaction center is in this picture it is shown only once I mean likewise you can you know imagine there are there is only one specific there are multiple such reaction centers. So, this is the first take home message that we indeed have reaction centers which are which has unique properties and they have the ability to you know initiate the whole cascade of reactions which eventually leads to the formation of the sugar molecules and everything. Now, just I put a picture on the slide a side by side. So, light is there carotenoids chlorophyll B chlorophyll A and the light reaches the reaction center at the reaction center. Now, what is happening at the molecule level as I have already told you there is a motion of this in the antenna complex. So, at the reaction center the reaction is getting initiated from here. So, you can now get a better view of exactly what is happening and exactly where it is happening. So, you have to realize that this structure what you see on the slide now which is a single chloroplast. So, on these chloroplast at every side you have these accessory molecules in the form of lupine carotenoids likewise which acts in as a the antennae they are funneling the electron to the chloroplast pool within the chloroplast within sorry within the chlorophyll pool sorry within the chlorophyll pool within the chlorophyll there is a hopping taking place and then what you see out here is something like this there is a specific reaction center where the reaction gets initiated. So, where the reaction is getting initiated what does that mean. So, now you have talked about light has reached. So, there are specific reaction centers. So, at the reaction center what is happening this is very interesting to understand. So, here there is a chlorophyll molecule and it has I am just putting as r c there are two reaction centers in this whole process one is called photosystem 2 which is also in short called ps 2 and there is another one which is called ps 1 and they are spatially separated from each other. So, if you see a pool like this of chlorophyll this is a pool of chlorophyll. So, there is specific ps 1 and there is specific ps 2 and they are spatially different and they have a different name one is called p 6 80 and other one is called p 700 we will get into all those things 6 87 and like this do not get worried about the numbers what is important here is you understand there are two specific reaction centers which are distinct from each other and they have different kind of surrounding complexes around it. So, now if this is a reaction center say for example, I call this as reaction center 1 or 2 what is over you want to call that. So, what is the exact chemical event what is taking place here. So, this chlorophyll molecule gives out an electron and become positive. So, now while it is devoid of this chlorophyll molecule is one second while this chlorophyll molecule is devoid of an electron this essentially got oxidized. Now, this one has already donated an electron in order to bring it back to its ground state this has to be supplied with an electron. Once it gets an electron it will come back to its ground state as long as you understand this basic phenomena accepting an electron or getting oxidized and getting reduced the whole force system 1 or 2 is all about who is. So, remember this donating electrons and who is accepting electrons as a matter of fact for whole story about evolution is about who is donating electron and who is accepting electron and which is third question here is which is the most abundant source of electron the section where I will be dealing with iron disulfide molecule and ancient molecule I will highlight that fact that very early in the evolution if you are aware about iron sulfur theory of evolution you will see very early in the evolution nature was using hydrogen sulfide H 2 S as the major source of electron as earth cool down and that is the time when we were living in an anaerobic environment as earth cool down the next source of electron which is also currently existing is water. And the by product of these water is nothing but oxygen and this is how we have evolved from an anaerobic world or without oxygen world to an aerobic world which is rich in oxygen and I will also highlight that is still there are zones on the floor of earth where this kind of situation exist specially this kind of situation you will observe in the deep down inside the ocean in the hydrothermal vents. And we will talk more about this while we will be talking about iron disulfide as energy molecule in one of our I think probably that we are 38 lecture where we will be talking about Fe S 2. So, having said this there is something called this kind of complexes are called OEC complex oxygen evolving complex just let me oxygen and I will put all of them in perspective sorry it is not system it is complex. So, now the question is who is donating electron here chlorophyll is donating electron there must be an acceptor who are the acceptors will talk about them. Now who is donating this chlorophyll because you have to realize in this pool you cannot keep on you know damaging yourself because if you damage this is good enough a free radical which could damage rest of the system. So, it has to be immediately brought back. So, who is donating electron to this so that it comes back to its ground state we will talk about that. So, this is where this whole scheme of things have developed and always remember in this whole process there are only three things you have to understand very like clearly who is donating electron who is accepting electron and who is the most abundant source of electron. And we are going to answer these three fundamental question through the fort synthesis and this is this whole process and if you see this this is what mankind or the whole humankind is trying to emulate which will be our next lecture essentially where we will be talking about how we are trying to emulate this electron transport phenomena because it is the electron which is the key to all the power what we use across the world apart from the fuels and everything now. So, this is the overall situation of the fort system how far we have gone. So, there is an electron donor there is an electron acceptor lost electron is to be placed by one from the will come to that water breakdown and that is what is you see water breakdown is about this situation what I highlighted which is the donor of electron excitation energy is transferred between molecules. So, with this overall geometry let us see the overall map where these different reaction centers are there. So, there are two basic reaction center if you look at it one is called fort system two on your left hand side you could see on the slide the another called fort system one fort system two is a complex assembly. So, as I was trying to tell you each one of these are complex assemblies of several proteins and will come in depth about those what are those complex assemblies of protein. So, now what is happening there is a light you see on the left hand side keep on following me from the left hand side there is something written in red circle one that is where the photon is falling on. So, photon is cascading if you go through the slide very carefully you will see photon is photon has ejected an electron that electron is cascading through the chlorophyll molecule eventually it reach to something called p 6 8 t if you remember what I was trying to tell you p 6 8 t which is the reaction center at fort system two at p 6 8 t an electron is being ejected. So, now what is happening is p 6 8 t is a chlorophyll molecule unique chlorophyll molecule at the reaction center. So, this has donated an electron and electron has been donated and now p 6 8 t is a free radical which has excess positive charges essentially this is an oxidation reaction which is taken place. So, now p 6 t a 6 t is the first donor of electron now see the slide you will see p 6 8 t then you see there are three different complexes where you see the number four is being shown and that arrow is showing how the electron is getting transferred. So, now follow what I was trying to tell you who is donating electron who is accepting electron. So, what you see now all those three complexes with the yellow arrow on the slide are who are the acceptors of electron and then this electron eventually reaches. So, in the meantime there is another simultaneously another event has taken place in another reaction center which is called photo system one and photo system one parallely which I was trying to tell you that p 700. So, which is p s 1 which is p 700 simultaneously another chlorophyll molecule out here because of the light got oxidized. So, there are two electrons which have been given out by the two different photo systems at p 6 8 t there is an electron which has been ejected simultaneously at p 700 there is another electron which has been ejected. Now you have two free radical dangling out there one is p 6 8 t asterisks and the other one is p 700 asterisks. Now what happens in nutshell before then I will show you all the whole process from p s 2 which is p 6 8 t the electron eventually brings this p 700 free radical back to it is reduce the state and this p 6 8 t asterisks which is in a excess positive state is supplied with an electron from water which brings it back to its ground state. Now coming back to the slide if you look at it. So, here now follow me at p 6 6 p at p 6 8 t which is short system to because of the light there is an electron which is ejected that electron traverse through three different complexes which is called electron transport chain provides the energy for the synthesis of biocheme osmosis of 8 p will come to that how that is being done and then this electron brings back the p 70 which simultaneously at number 5 if you look at it which simultaneously got it into the excited state by you know getting oxidized and the electron which is ejected by 4 system 1 is then used by another enzyme called NADPH reductase to make energy rich molecule called NADPH and if you see that there are two dots. So, basically it has got two electrons out there to make the NADPH molecule. Now what we will do in the meantime if you look underneath p 6 8 t there is water which is getting split and this is the seat for water splitting cluster. Now what we will do I will give you the exact if it is not the exact what is ever we know about the 4 system 2. So, here the how the 4 system 2 looks like p 6 8 t is the reaction center there is a tyrosine residue very close to it and underneath you see there is something written OEC 18 24 OEC 33 this is oxygen evolving complex this oxygen evolving complex is nothing but externally beautiful manganese cluster embedded in a protein surrounding and with calcium close by and this is the cluster which does the magic of nature by splitting water into hydrogen oxygen and electron the most perennial source of electron. So, as I was telling you in the hydrothermal world it was H 2 S and in the modern world it is water. Now look at this these are the OEC is a stand for your oxygen evolving complex which is sitting just underneath it and just follow that there is a stroma and there is a lumen which is telling that is an asymmetric membrane. Then you see few PHO that is few phytin that is another complex protein out there then you see plastic quinone which are Q A and Q B and then what you see cytochrome B 559 I will come to that in the meantime if this this complex not to appreciate it you have to go to this. So, here now all the names are there now I was telling you that how this ATP is being synthesized now follow me this is. So, you have this stromal side and you have the lumen side lumen is the inside the thylakoid membrane which is kind of enclosed side. Now your photosystem P 6 80 is sitting on your left where you see the water is getting split up on the left hand side see the left hand side complex on the slide carefully. So, in the light falls here. So, that it is an ejecting an electron that electron through the cascade of all those different proteins what you see few phytin to plastic quinone likewise. So, you see the plastic quinone P Q it goes to a complex called cytochrome B F 6 complex what you see C Y T there is cytochrome and the electron is travelling all along this membrane. So, then it moves to a PC complex called plastocyanin complex from the plastocyanin complex this electron moves to photosystem 1 which is written out there P S 1 follow me very carefully at P S 1 what is happening P S 1 the P 700 the chlorophyll molecule which got oxidized is brought back to its ground state and the electron which was ejected out there is further used in the initially the electron which before of the oxidation of the P 6 80. So, you have to realize that there is an electron which is donated by P 6 80 this electron is now being utilized in order to reduce the N A D P H. So, it is the N A D P H reductase there you see that enzyme which is sitting there on the right hand corner N A D P reductase the N A D P H reductase basically convert N A D P plus proton to make N A D P H. Whereas, on the other hand what you see is very interesting inside this thylakoid lumen while the water is glating is split up it is evolving oxygen. So, this is what I was trying to tell you that evolution of oxygen is nothing but a by product of water splitting process. So, this is that oxygen is sitting apart from oxygen what it is being generating is a series of protons out there if you see the reaction carefully on the slide you will see H 2 O plus half O 2 plus 2 H. So, now your thylakoid lumen is rich in a proton rich very rich in proton. So, essentially if this is your luminal part of it. So, this has very very high concentration of the protons which are present there. So, now there is a proton gradient across you see a proton gradient across the thylakoid membrane and it is this proton gradient which eventually leads to the synthesis of ATP by ATP synthetase which is present on the thylakoid membrane. So, you see that ATP synthetase on the right hand bottom corner just underneath NADPH reductase. So, this is how this whole transport electron transport and leading to you know the production of energy rich molecule and likewise when and so forth. So, what you can appreciate in this whole process is water here is playing a critical role as the currently as the abundant source of free electrons, but in order to do so nature has to have a very amazing catalytic machinery by which you can you know split water or you can rip water and get the electrons out of it and nature has done it the most elegant way you one can ever think of. So, coming back so these are the standard redox potential of change I will come to that manganese cluster and the how that OEC complex is there. So, just before we get into that. So, see how these electron transport is it is going against the gradient mind it you look at it standard redox potentials are going towards negative and again falling back and then again it is going up. One might wonder that how why this process is like this there is no absolute answer to this question if you look at the slide carefully that as it, but one thing you can appreciate from the slide that possibly in this whole process the system is buying time by doing it in a slightly convoluted way and thereby ensuring these complexes are not getting damaged because of the free radical existence because they are exposed to nature. So, they are purposefully you know increasing the time of doing this reaction with a slight delay and you know kind of changing the redox potential up down likewise. So, possibly again if this is the most correct explanation I would not claim that, but this is something which you know comes in my mind that possibly nature has done something like this in order to you know minimize the damage or you know in order for the whole machinery to function in a much better way, but at the cost of efficiency. So, the efficiency of the system goes down, but maybe within the theoretical I mean within the practical realm or within the practical constraints of working in such a small area of maybe 1 or 2 micron much less than that maybe this is the best nature has evolved till late. So, from here I will just put the port system 1 in place what is happening to that electron from P 700. P 700 is going through again series of routines and it is transferred to f x q x and you could see all those proteins and eventually goes to the NADPH reductase. So, if you see this this is how the whole assembly of this photo system 2 cytochrome b 6 f complex and p s 1 is arranged on the membrane of on the thylakoid membrane separating this trauma and the thylakoid lumen. Now, the overall layout of the system is something like this. So, you see the membrane where you have the photo system 2 you have the photo system 1 you have the electron transport complex. Now, from here I will move on to something called the manganese cluster. So, the critical part of the game where the water is getting split. So, this is the inspiration to make photo electrochemical cells in the modern day, but how this exactly takes place there are several theories, but I will try to simplify it in you know which is understandable way and of course, I will give you the references there are series of references you can go through and you can appreciate it much better. What possibly happens is that manganese remain in multiple oxidation states it remains from 2 to 6 it is multiple oxidation states where it is staying. So, in the in this complex nature has managed and most of the time you will see the manganese in the highest oxidation state, because we are in oxygenated environment. So, you will see the manganese in the state of 6. So, nature has managed to hold manganese these manganese molecules out there at lower oxidation state like 2, 3, 4, 5 likewise and the manganese in that oxidation state could only change to one state change it could you know become 3 by you know throwing an electron it can come back by grabbing an electron it has only that much privilege. So, each one of them if they are at sitting at say manganese 1, manganese say state 2, say state 3, manganese sitting at state 4, manganese state at state 5 and each one of them say for example, can shift 1 degree on both sides you know they can either get reduced or oxidized that is all they cannot fully become 6 or likewise that the one which is standing at 2 will become 3 or likewise and in that whole process they could you know make this movement. So, whenever they grab a water molecule they shift there something like this happens once again something like this happens a water molecule gets trapped and they could you know really split the water molecule by oxidizing it and still the mechanism is yet to be worked out in depth like a universal mechanism which we all can really appreciate, but I will recommend you go through the book of Stryer biochemistry of Stryer you will get much more better feel about it and there is a role of calcium in this whole process. So, since this is out of the periphery of this course I am not really getting in depth with the manganese cluster how it is splitting, but overall this is something like it remains in multiple state and thereby it kind of you know, but one of the challenge of mimicking such cluster is that how you could really hold manganese at a lower oxidation state I mean it is very tough because we are living in an oxygen rich environment. So, this is problem these are the different states or different manganese cluster. So, it is it has been observed that this graph is very important this called how the coke cycle there is something called a coke cycle which evolved it says after every. So, this is the oxidation evolving is that for every flash there will be one oxygen it does not work like that it is actually after every four flash there is one oxygen. So, you see the flash number and you see the oxygen oxygen maximizes after every fourth flash. So, that is basically wanted to say there are four stages in which the water is getting split into you know oxygen and the protons and donating the electron that is why it is shown in the four cycle and if you follow this graph you will see at four at eight at twelve at sixteen at twenty likewise at every fourth interval there is a maximum every maximum emission of oxygen. So, this is the overall how the manganese cluster looks like. So, one of the challenge what I am now moving on to the I will be moving on to the next topic on this should be the next lecture on which will be the thirty fourth lecture where we will be talking about how people are trying to you know utilize this whole idea of generating hydrogen. So, this is a source where you can really generate hydrogen and which could be used as a fuel. So, manganese cluster remain you can say remain dream thing for those who are interested in energy to emulate this kind of structure where by you can split water you will have ready source of electrons ready source of hydrogen and you really can store the energy according to your convenience. So, you want to have the hydrogen energy you get of the electrons you could you know flow them on a circuit likewise several efforts have been made and we will be talk we will be talking a little bit in that context about this in dye sensitized solar cells and likewise. So, I will close in here just to summarize what all we have you know covered today we have talked about let us again go through all the slides real quick. So, we talked about the anatomy of the photosynthetic machinery what are we talked about the stroma and the thylakoid and the granum and we talked the meso the real geometry of the mesofil cells we talked about the light harvesting module where several different molecules are involved in zeaxanthine beta carotene and lycopene and then we talked about the reaction center how the reaction centers when we talked about why possibly nature has made reaction centers and this is side by side view of the whole thing. And then we talked about the two different photo systems which are involved in it photo system 2 and photo system 1 and the electron cascade from photo system 2 to photo system 1 the NADPH reductase adjacent to photo system 1 and the water splitting cluster which is in the form of in the photo system 2. So, which is the oxygen evolving clusters. So, this is the overall what we have talked and what we needed to know. So, the inspiration what are being drawn is out here could we emulate this chlorophyll structure as a photovoltaic material. So, that will be our next lecture where we talked about all these things thank you.