 Thank you for your nice presentation and it's really a pleasure to be here. Good afternoon to everybody. Good morning from those that are on this side of the day. And thank you for your presentation. It's a challenging situation. I am presenting here something that is much slower than what I've been hearing up to now. So what I will be presenting you is the application of the wonderful presentation that the previous speaker gave us. And it was an interesting talk by Julia who did present the basic points of everything that is fast. Well, we are here in a word that is a slightly slower, but not so slow. But I don't want to dwell in too much of introduction. So the mighty power of photosynthesis is because exactly like any well-being is based on the use of energy resources. Earth is also based on the use of energy resources. The energy is sun. The car is the planet. And of course, engine are plants, the photosynthetic organisms. And photosynthesis has shaped life and the structure of our planet. Since the beginning of the formation of the planet Earth, we know that some microorganisms able to take advantage of solar radiation appeared. And initially Earth was not populated much and was definitely not an oxygenic planet. But then evolution brought in oxygenic species, the so-called oxygenic photosynthetic bacteria that started poisoning the atmosphere, shifting from a reducing atmosphere to an oxygenic one. And actually if you can see this curve here at a certain point, at a certain point, the evolution on Earth of this species, the appearing of new and new photosynthetic species brought us to the Cambrian explosion, where the oxygen level reached roughly 30% of the overall composition of atmosphere. And at that time, the amount of oxygen and the amount, the dimension of animal on Earth, where it was so big that you can see is colopander, which is a small box that's now populating Earth. And they are mostly, at the most five centimeters, were longer than two meters. And dragonfly, you can hold them in a hand and not completely cover them. Well, you know how small dragonfly are today. So, yes, photosynthesis is also responsible, is responsible for shaping the Earth, is also responsible of how we, how to say, how we survive consuming energy, because roughly three to four hundred million years ago, a large number of trees and plants and also animals landed on the floor of, oceans. And then in time, we're covered by sand and silt. And at the end, due to pressure, high temperature, we got oil and natural gases. These two things are directly bound to photosynthesis. And of course, we all know that, and we all know that there is a need, absolutely a need for substituting fossil fuels. And photosynthesis is also involved in this idea of substituting fossil fuels. So, just by quoting Steven Gust, I would say that photosynthesis is arguably the most important biological process on Earth. It's so because the amount of energy that is played in this process, it's humongous. So, theoretical potential of solar power is given by the integral of the flux over the surface of the Earth. And if you just make this nice calculation, you end up with roughly 90 terawatts. Now, this represents the amount, even in an hour, this represents roughly the amount of energy that was used in the entire year of 2001 from all sorts of combined energy. So we have here a powerful energy source and possibly a system to convert it in other forms of energy that would be useful for us. What we need is to understand the mechanism and how to do it. But this will be the second part of the talk. What I want to do now is just introducing you to photosynthesis through its applications by starting with presenting a few molecular aspects of photosynthesis. And these are mostly based on the use of pigments. Pigments are the most important players in photosynthesis. Very important are these pigments based on these two tetrapyros, the chlorine and the bacterial chlorine. The difference by simply one double bond in one of the pyrorines. And these two molecules are the heart of the photosynthetical photochemical machinery that converts solar light into free energy. And this heart is specially formed by the chlorophylls, which are an extension of the chlorins that we saw in the previous transparency. There is some other substituents on the external part of the ring and the Fittil group here on the bottom where bound to the de-cycle of the de-porphyry. And so the Fittil chain is absolutely important because it helps the system to introduce itself inside the proteins. And you will see this in a moment. I just wanted to show you that there is a second here in cycle B. There is a double bond, which can be present or not present. And again, this is the difference between chlorophyll and bacterial chlorophyll. While insisting so much on this molecule, well, because if you look at the absorption of this molecule, you can see that it spans from the entire spectrum, a visible spectrum, starting from the low wavelengths, around 400 nanometers to the high wavelengths. And it spans from roughly 680 nanometers, which is the reddish color, and ends up to 800, 812. Now, this is very, very important because the most important part of photosynthesis is the light goes from 600 to 900 nanometers. And we'll see this little later. On the top of this absorption spectra is the absorption, the flux of photons coming from the sun and eventually being absorbed on earth by photosynthetic corgis. And you can see that most of the absorption of the chlorophylls fall in the red near IR part of the spectrum. There are many different chlorophylls. But together with chlorophylls, we have carotenoids. Now carotenoids fill a hole, which, if we can go back to this transparency, is the hole between 500 and 600 nanometers. And carotenoids are fully colored molecules that are also inserted within the proteins, as we will see in a few minutes. And differently from the chlorophylls, these molecules do not play a central role in the conversion, but do play a role in harvesting light. So we just saw a few pigments, but these pigments that we saw were presented as single pigments in solution, typically organic solution. What happens when they are inserted in biological systems? Well, the differences are striking. Most of the system have their maximum absorption shifted toward the red. And indeed, you can actually reach in some specific microorganism absorption of the bacterial chlorophyll within the protein that reach 100,000, 1100 nanometers. This offers your coverage of the full spectrum from 400 to 1200 nanometers. It's so important because it covers all the visible and infrared light at the most, the largest amount of the photos that reaches Earth. Well, this is just another view of, say, of viewing things and skipping it. So very quickly, who does photosynthesis on Earth? Well, there are, of course, higher and lower plants. We all know the green plants that are surrounding us. But then there are the algae and the bacteria. I will mostly concentrate myself on the bacteria because it's the simplest. And in 30 hours, we cannot have a complete course on photosynthesis. So bacteria are divided into two classes, the one identified by a blue dot and those by a red dot. The difference, the main difference is that the blue dots are typically oxygenic species. So photosynthetic microorganisms that do photosynthesis, oxygenic. So they are appeared later on the planet. And the red dots identify species that do an oxygenic photosynthesis. So the photosynthesis that appeared on Earth more than 3.7 billion years ago. They are all negative, gram-negative bacteria, although there is one gram positive, but none of them is pathogen. So if you look the diversity of the photosynthetic organism, it's very striking that there is only a single mechanism that is able to convert the solar light into other forms of energy. And that is the photochemical mechanism. But of course, this may come as a surprise, but it's not since they all share the same evolutionary path. And the evolutionary path is the best way of converting solar energy into energy, viable for us and for other inhabitants of planet Earth. So I'm just will focus on one single guy. This will be our host, actually our guest, the photosynthetic bacterium rodubactose ferrarius. It's an important bacterium because it's been a model for many, many years, model of photosynthesis for many, many years. And I just wanted to show how it's organized. Biology is important to see how things work and how things are made up. So you can see here an external membrane, an outer membrane that divides the growing milieu from the internal of the cell. Within immediately below there is this internal membrane. In this internal membrane, there are this invagination of the membrane. These are not untouched. They are attached to the upper part of the cell, so they seem to be floating in the cell, but they are not. And these are called interacetoplasmic membrane. Within this interacetoplasmic membrane, they are located the most, actually the incomplete photosynthetic apparatus of the photosynthetic bacterium. And if you take the bacterium and you squeeze it under high pressure and then release it at atmospheric pressure, the bacterium explodes. And those interacetoplasmic membrane break up and then seal again, forming the circular vesicles that are called chromatophores. And the sealed vesicles are formed by four types of protein, five types of protein at the most. So the antenna proteins here shown in green, the second kind of antenna showed is red rings around the central bacteria reaction center, which is this molecule here, protein here in bluet. The BC1, so the counterpart for the establishment of the delta pH across the membrane. And finally the ATPase. Now the ATPase is the important protein that uses the energy converted by the reaction center to synthesize ATP, adenosine triphosphate, which is the exchange money for energy in biological system. If you have ATP, then you can do almost any reaction you can think in a biological system. Now if you open up the vesicle and make it flat, you can see a representation, a simpler representation with the reaction center sitting here. LAC1, which is here represented as a red and shown here as a circular surrounding the reaction center. And then this small molecules that are called LAC2 where LCH says for light harvesting. So this part harvests light and the reaction center instead converts it from photons to charge separated state. We will see this in very, very soon. Now this is the center of the protein portion. And these two are two different enzymes, which are responsible of the conversion, the absorption of the photons and its conversion in charge separated state. On the left hand side, you, you're seeing one that is the first one that was crystallized. It's from a bacteria called blasterchloris viridis. On the left, on the right hand side, you see the one from Rhodobacter spheroidus, our guest, I'd say. And the way it works, light is absorbed by light coming from directly from the sun or from excitation transfer from the antennas arrive to this protein. The system works by circulating electrons within its internal part and then protons get transferred from the inner part. So from the Periplus out on the cytoplasm and can will be used then to synthesize ATP. I added the transparency because I saw that Julia was using so many Nobel prizes in her talk. And we also have our, our, and that's crystallization of the first photosynthetic protein that was awarded to. That is an offer Huber and Artwood Michael in 1988 for crystallizing the first protein on the left, the one, the most complicated one here, the one on the left. So we know we have an hour Nobel Prize. We have very interesting thing because it was showing that they showed for the first time that you can actually crystallize crystallize membrane protein, integral membrane protein. And that's why they won the Nobel Prize, but their work was extremely nice because we have now a precise description of what is the reaction center. The reaction center took his terrible name because the person who first isolated and individuated as a central enzyme for the conversion was a guy called Roderick Clayton who wanted to leave a mark on the, on history and actually he did because it's RC is exactly the words, the letters that form his name and last name. And this molecule here is composed by has a rough weight of 100 kilodalton formed by roughly 1000 amino acids, then it has three sub units, which are here color that three three three different colors. And nine factors that are shown here on the light on the right. For which are bacteria chlorophyll is we saw already color with our chlorophyll is two of them are to bacteria pfio fighting and pfio fighting are just chlorophyll deprived of the center magnesium, then to quinones. Quinones are very important as an electron acceptor we already heard it today and also yesterday in the nice talks and one iron iron not him iron iron that sits here and mostly plays a role in the structure of the system. So, this is how the, the cofatos are organized in the in the reaction center. This is the three sub units. What happens is that along one of the branches that the electron is shuttle from the dimer here, which are two material chlorophyll overlapping one to each other to the quinone. And this overlapping here is actually a pardon this electron transfer here is a hopping between the dimer the bacteria chlorophyll a the bacteria pfio fighting a and the quinone a and finally to the quinone b. Actually the system works like a semiconductor so light is absorbed and promotes electrons from the balance bond to the conduction bond, and then the electrons will reach eventually do catalytic side one you reach in the other. E poor where electron get transferred and moving all the energetic of the system the energetic of the system is so nice, because the engineered system is so well organized that before any charge the combination takes place. Forward electron transfer reaction happens so and the forward electron transfer reaction is at least two three times faster than the back reaction or the fluorescent so from the star which is the dimer excited state chlorophyll electrons get transferred to the bacteria chlorophyll a in 10 to the minus 12 second arrives with the bacteria pfio fighting a and the recombination will be 10 to the minus eight but in 10 to the minus 10 seconds reaches the other. Electron scepter and then again faster than the recombination reaches reaches the last electron scepter so what you actually have done you have moved by roughly. Almost four nanometers the electrons from the position of the dimer to the final position of the quinone and this allows you to move around the protein to move around the. Move around the electron from one side to the other side of the membrane this is amazingly done by the system and is sort of a. A paradise for anyone who won't wishes to do photo chemistry and let study later in class. The optical spectrum of the proton is very important for us and you have several peaks some of them are associated to the dimer 865 nanometers then to bacteria of chlorophylls or bacteria pfio fighting, then you have the second. Q bands of bacteria chlorophyll and bacteria pfio fighting, then the B bands of the bacteria chlorophyll and bacteria pfio fighting a plus the final absorption band that includes quinones and aromatic amino acids, you can use it and have the difference between the. dark and the light spectrum and you can see that they speak, which is at under 65 is complete and at under 65 nanometers is completely bleached when it's illuminated that's because all the electrons get promoted to the. First excited state and you can actually use this ban here to check the. kinetic cover reaction these are very the one I'm talking here is very slow electron transfer reaction that go from milliseconds to. Seconds actually, but they are very, very important because they allow and similar chemistry to take place after the photo chemical acts that generates charge separated state so we're talking about chemical. Time domain that is. Order of magnitude slower than what Julia was showing and but also order of magnitude more efficient in transferring them the charge separated state to chemical reactions. And here is one of the examples that they wanted to give you can see here are two different two different charge recombination experiment, you can see here the red line here as an experimental that was obtained by. shooting a flesh of light here to the system is at 865 nanometers, then the system is fully bleached, and you can follow the charge recombination of the system after the light is being removed in this red trace here is actually. The electric reaction center to which the QB side was removed, so you only had electrons reaching here so the charger combination is faster because has to go from here to here. And then take either another reaction center with the QB or reconstitute the QB functionality. The electron has to travel all the way from the D plus to the QB minus and amazingly enough, there is no electron direct charger combination for QB to D plus but it repopulates. The QA side and then recombines from here so that's you actually see a very slow decay that has an average time between one and 1.2 seconds and of course in one to 1.2 seconds you cannot you can do much much nicer thing than in in chemistry than in. picoseconds and that is the purpose. Of the following presentation but before going to the following presentation just want to indicate and this probably my last but one transparency, the effect of the chemical environment if you look at this graph here, the black line is represented by. Is exactly what we saw in the previous one, so is this one represented here, and then you can actually solubilize this protein in different kind of systems so detergent. Liposomes or liposomes made of negatively charged, negatively charged lipids and you can see that you can slow down the reaction the recombination reaction up to ending up to a recombination reaction that takes about three, four seconds. This is a humongous amount of time for such a system to be used for chemical reaction of course there is trade off the slower the system, the lower the energy story in that system, but you can play with trade off there is always a way to play if you have a trade off. Let me just show you I hope I'm in time, but let me show you just show my very last, my very last transparency, my very last slide sorry, it's half Italian and half English I don't know why, but that's the application of photosynthesis in the living words of today, we can have it in biosensors. Generation from bio biofuels of third or fourth generation production of bio hydrogen bio electricity biosensor cellular in the cellular farm and bio catalysis it's important and growing fields and artificial photosynthesis. You already heard Julia showing some ideas on artificial photosynthesis we will apply this in the next talk, and you can do this kind of things either using the whole organism of portion of the organisms, and to do this you have to be a visionary but as young Sheldon Cooper says there is a fine line between being wrong and being visionary and unfortunately you have to have to be a visionary to see it. I hope I can see it otherwise. It's a bad thing for me. So thank you for your attention and I'm open to question and sorry if I was too long. No, Massimo you were perfectly on time. So we have now a little time for questions so I don't see any question in the chat but you can switch on your microphone and ask Massimo directly if you want just write something in the chat. Too much biology. Too much biology today. I can break the ice possibly asking, you know, having gathered the Julia talk just before your talk. I realize that you have this in this reaction center you have these two branches, very similar to what Julia has in her beautiful molecule. So I'm wondering first, there is a reason for having two branches and not a single branch why nature chooses this complex architecture and then who the one possibly think about an experiment like Julia did putting some vibrational energy or some energy, disturbing one of the two branches. Okay, the answer for the first question is why nature. Made up this thing in this way it's probably was a trial and error way in evolving. And at the end they found out to have a B plan. So, in a photosynthetic organism, something can always go bad and one of the two branches may not work. So you have a backup one that can work so it's evolutionary and struggling for life. Yes, yes, redundancy it's very important biological system there isn't no history of theoretical biology on redundancy. The other one is that, of course you can do this, and there is a fantastic group, there are several groups to do this. The most advanced one is the group by Van Grondel in at the university. What he does is exactly what you represented here I are in in in reaction center and in biology we have the lack of having the possible of making a mutation within a protein so it was not only able to produce the chlorophyll and bacteria chlorophyll within the protein, but also to maintain this molecule and change small amino acid that would move energy around. And he has a so many understanding of this system that it will take like three days only to present his work. Van Grondel is an institution in the bacteria reaction center and fast for the physics and for the chemistry. There is an end rise by Ali. Yes. So, so thanks for your talk. I had a more general broad curiosity about what's understood. You know, you showed these very nice pictures of the photosynthetic system. And, of course, this is a very static picture of, you know, the system and I imagine that there's a lot of conformational heterogeneity dynamics. What's, you know, what's the extent what role does conformational fluctuations play in the various processes of photosynthesis and is this is this under completely understood what are the open questions there. Thank you. This is a very nice question. Saying that something is completely understood is overstating. But in terms of what goes on, what goes on in the very first femtosecond, the fluctuation. That's all within the dimer. So the two bacteria clover facing each other. And what happens is that that if you saw that system, it looks almost symmetric, very symmetric, but that's not exactly true. There is a break in the symmetry particularly around the dimer and the breaking symmetry is exactly in the composition of amino acid that sits with two one chlorophyll on the other one chlorophyll. And that's how nature chooses to use a branches because the energy of the B branches is unfavorable compared to the energy of the B branches because the excited states has the letter and sitting most of the time on the a branch a material chlorophyll of the dimer. So actually that's absolutely true. And this was shown by contemporary experiment by experiment done with the EPR and or and fast fast for the physics and photochemical experiments. And the other thing is that you also have a very nice description of the conformational changes because you can do what is called the time resolved x-ray crystallography. By the advent of free electron lasers as breakthrough from for biology, you throw so much energy that the molecule breaks up, but this happens within a few, almost 10th of picoseconds, but then you can take x-ray images in time between zero and 10 picoseconds and reconstitute the movement of all amino acid before the system explodes. And actually that's that's something that they're doing very nicely in Arizona in Temple University. And there is this group that is building a fellow electron laser x-ray facility. And if it wasn't for pandemic, I would be there since last year, because I got a position there, but they never was never allowed to reach them because of COVID-19 so COVID-19 is a horrible thing. Also for this. Thank you. Thank you very much. Sure. Fascinating. Fascinating. Thank you. Other questions from the audience, please write on the chat or raise your hand. I will try to follow. May I ask a question? Sure. Yeah, that was an excellent talk, Professor Trotter. Thank you. And the direction that you know, in the synthetic or semi-synthetic approach to mimic photosynthesis, one of the challenges that exists. And you know, what constituents of chromophoric systems are required in order to nearly reach there or like have some similarity to what photosynthetic reaction center would do? Well, most of the things were explained extremely carefully and detailed by Julia. Julia showed you that if you take the simple system, then just the model system, the first model system that appeared, the trade-off for having a simple system is that the charger combination has a time that is so fast that you cannot do anything. So if I may say, what is the drawback at the moment to artificial photosynthesis is complexity. The system we are using, although very complex, they are too simple compared to the natural systems. I only showed you a general image of the complexity of this biological system, but you have to imagine that between each step of the electron transfer, there are at least 10 to 15 amino acid involved. So, and that's why the lambda, the reorganization energy that comes in place is so relevant in determining the rate transfer or charge combination or forward electron transfer. So, in one word, it's the lack of complexity, the real drawback at the moment for artificial photosynthesis. Thank you. Once again, I very much enjoyed your talk. Thank you. Thank you. Thank you very happy. I just want to comment on this complexity thing is very, very interesting. So, basically, we have to start to study systems where there are several chromophores because you need this cascading. And most probably also, as also Julia pointed out, you need also vibrational coupling or conformational coupling, call it as you want some relaxation of your system as to avoid going back. Very, very interesting consideration. I was in general. I have more questions, comments. If not, I think we can go straight to the second part of the presentation. And let's see what Massimo has in store for us. In the comment of what Fatima wrote, she said that just the thanks slide strange that even it contains many languages, but it contains Arabic words. Fatima, please send me the Arabic word. I will insert it. I apologize for being so I'm polite to you. If you do send it, the Arabic word that will immediately insert. So please do so. Okay, yeah, let's go to the second part. Okay, I'm sharing the other one. And just anticipate to Fatima will have the same problem in the thank you transparency, also the second part. Coming up, you should see it now. Don't you? Yes, yes. Oh, thank you Fatima. She sent me the word. Okay. Again, applications. I was asked to give a general part general topic and then application of the in my research. Of course, we have to go back to the center for the chemical apparatus which is built as we were saying by a protein scaffolding which the the pigments are inserted that I showed you are only the head of the pigments. I cut away the idrophobic chains because they will confuse the scheme, but each of those pigment has or quinone has a long chain, at least 15 to 50 carbon atoms because these molecules have to be very stably bound to the reaction center but not covalently bound. Now, given that I will just go forward and mention this think of complexity. So solar driven chemistry is the game to play here. Well, as I was saying, one of the very first example of artificial photosynthetic molecules is this triad. And you can see you have a carotenoid on the end, a central porphyrin and a quinone. This is a very bright way of thinking. You can just make up a light absorbing part that distributes excitation to the carotenoid, which gives an electron to the quinone. And of course, if you wait long enough, the electron of the quinone will recombine with a charge left on the carotenoids and the system will go back to the ground state. Well, the point is that the lifetime of the cells and although very high in yield in generating that charge separated state is 60 seconds, 60 per nanoseconds, which is, as I was saying, too short for any ancillary chemistry. And so people understood that there was needed for something else. So they added in a second porphyrin and a second quinone. You can see that the pentad elongates the pathway between the electron donor and the electron receptor. And so, effectively, the charge of combination got much, much longer. The yield was comparable. But still 200 microseconds is too short. So to make this, I was asked this question before. So what is the difference between this natural, the natural system and this system here is complexity and you need complexity exactly as you need complexity in a soccer game. You know, artificial photosynthesis, the system just been played by the two goalkeeper who kicked the ball hoping to make goal and the other to the adversary to the opponent while when and that would be very boring as a game. But then if you had the other players, the complexity of the game increases and we like it more and more, at least I like it more and more. That's exactly the difference between artificial photosynthesis matters for the synthesis at the moment. So artificial photosynthesis adding is adding players but naturally biological photosynthesis has already the players in their plays and they're playing very nice. We were saying that it's so well designed that the forward electron transfer is always faster than the charger combination reaction, and you can actually have a conversion of 100%. So each absorbent photon from the reaction centers is converted in a charge separated state. And that is what we are looking in also natural system that's does not happen. Contemporarily you have high high efficiency conversion and a very slow recombination rate. So these two are typical of photosynthetic natural system. Biological system. And as I mentioned is because of evolution between each electron transfer step we have so many amino acids that have an interplay in the energy distance between the pigments and they may have also an interplay. For example here these are 15 angstrom. There is no way that the electron would rally from here to here just by that simply. There are at least four histidine amino acids here that land their own orbitals to allow the movement of the electrons from one quinoa to another. So complexity is the goal and is what we do and and is what we need. So what we decided to do is to take advantage of the chemical complexity that comes directly from nature and eventually assemble it with some inorganic patterns to to generate the so called hybrid organic biological assembly. And if you look at the energy conversion efficiency. We already mentioned that grows going from artificial to natural system because a structural complexity grows from artificial to natural system. Of course chemical robustness instead decreases. If you increase complexity but that's a trade off the famous trade off we were talking about so for example PS2 which is this for the chemical core that sits in the plants is so efficient in converting that at a certain point is too efficient. And it can't react with oxygen destroying the proteins itself. So in nature this protein is has its own healing mechanism which one part of the protein destroyed by oxygen is discarded and a new piece of protein is up taken synthesize up taken and inserted in these enzymatic system to allow to repeat the photosynthetic to repeat the photosynthetic process and this takes about half an hour you just take and extract this protein after half an hour it just breaks apart you will not work anymore. So there are best practice for RC exploitation this hybrid system, and I will just show you a few example because I have not so much time. And of course if you extract the protein and you want to be sure that whatever you do is useful, you have a fully vectorial you need a fully vectorial arrangement that's because upon the generation of charge separated state. Every protein is possible to be represented as a dipole. And if you do reconstitution for example in on an electrode to take advantage of the charge separated state of the dipole. Then you want to have configuration of the dipole which is this one all oriented in the same direction, so that they all work cooperatively toward one direction. Instead in the lower case you have 50 50 distribution and each dipole will kill the effect of the dipole of the previous one by listening the whatever effect we are doing so. This was the idea that brought us to do what we will show you and together with the fact that you can actually isolate the reaction center reconstitute in lipo some whatever of vesicle of phospholipids and eventually if you look at this photo cycle here you can allow adding external electron donors and electron acceptors here. Queen so external reductant and internal we known what you can do you can shy light and obtain the first charges separated state. Now, the system is organized in such way that if you have an external electron donors before the combination takes place actually before any forward reaction from the q a minus to the q minus takes place. The diamond radical cation can be read reduced and you end up with a radical anion in the molecule that moves and can be doubly. You can do you can repeat the explain with another flesh, obtain again the radical the anion q a minus to be minus and radical cations, the cations get to gain reduced by the external donors. So you end up with a molecular quinoa and if you remember the position is this is the on the be on the be branch. This is a doubly reduced we know that that takes protons from the external water generates a quinoa that leaves the protein and the substituted from external quinoa. So this you can then repeat the photo cycle. And if you look at the actual result of the photo cycle is that protons are up taken from one part and confined and moved to the other part of the photo cycles. So if you actually have a family of oriented reaction centers like we see here. You can do this in a closet vesicle. Ideally, you could have a change in the pH in the internal side of the closet vesicle and an increase of the pH, but lowering in the of the pH within the the cytoplasmic you are of this. Closet vesicle and an increase in the external. Well, actually, that's exactly what we decided that was very, very interesting to do. And what we did is reconstitute the reaction center within those giant vesicle now giant vesicle are lipo sums that can be obtained quite easily. But their size is gigantic. Sadly, they can go from one to 100 microns. So if you imagine one bacteria would measure roughly one microns you can get large amounts of bacteria or whatever it would in the center and you know this giant lipo sums. So what we did is to bind for four to the reaction center reconstitute the vesicle with the reaction center. And you can see the floor force is mostly is almost exclusively bound to the belayer of the giant vesicle. Then you can upload, you can fill the giant vesicle with the pH indicator. It's at the pyramid in this case, and you can see the fluorescence of the pyramid. And if you combine the two images you have a giant vesicle surrounded by reaction centers and with internal site showing the Presenting the fluorescence of the pyramid, the pH sensitive dye. This is the actual view of the giant vesicle in microscope and this is a fluorescence microscope and you can see there is a lot of variety in the dimension but we set up to choose the dimension of roughly 20 microns. So this is a fast representation of the giant vesicle in which roughly 90% of the reaction center is reconstituted with the dimer, which is this flat part here exposed to the external and the quinone exposed to the internal. There are some that are opposite but they are cancelled out. And if you just look at the photochemistry that I showed you, you can have the electron traveling from the quinone from the dimer to the final quinone. You can re-reduce the reaction center dimer using cytochrome and again cycle the cytochrome from 3 to plus, from 3 plus to 2 plus using ferrocyanide. So you have a full cycle that can be driven by illumination and electrons will be picked up by the from the internal solution from the inside of the vesicle. You can actually show that this is actually taking place by measuring the charge recombination of this charges separated state here and you can see that in absence of cytochrome you have a full recombination rate while the recombination rate dies out if you add cytochrome. That's because this is re-reduced and there is no way that electron can go back to D plus because we have no more D plus. So we showed the orientation, we show that the orientation is corrected for us and then in time we illuminated in 15 minutes we illuminated the different vesicles. These are three different giant vesicles and you can see that the pH inside changes because the pyranine become more and more brilliant and the fluorescence of the pyranine increases because the pH internal pH increases. And you can do this also by showing the change of the pH in time by plotting the intensity of the fluorescence calibrated toward the internal vesicle pH and you can see there is a linear behavior between the amount of light that actually reaches the giant vesicles and the delta pH. So this is important because we were able to obtain a fully oriented system that can be used to re-photochemistry in a closed vesicle. You can imagine this is a very powerful system. The other thing that we showed that was that the other important thing is that we want to increase cross section. As I mentioned, the reaction center is surrounded by light harvesting complexes. These light harvesting complexes are proteins that are generally very tend to degrade under harsh conditions where the reaction center is much more stable. So we like to take them away during isolation and end up only with the reaction center. But that cuts the cross section of the system, so less light is absorbed and the system is less efficient in long accumulation. So what we did is as a principle, we just took this nice molecule that was taken from the lab for organic chemistry. It is built to be a very, very efficient fluorophore. It has two triple bonds that are conjugated to two benzines and there is a central core here that is very fluorescent. This central core here you can play around to have also bound some exigroup that will allow to stay within the membrane since we are thinking to bind it to the reaction center. And then here is the system that we use to conjugate to the license of the reaction center by by conjugation to the via Succinimidil ester. What we do is just we take the spectra of the reaction center in black here, the spectra of the molecule, the one we saw before. This is the fluorescence of the molecule. So, if you look at the peak of the absorption and generate this band of the eye, then you see that there is no absorb correspondence absorption of the reaction center. And so you actually extended the ability to absorb light. But would this be efficient in generating charge separated state? Well, we thought yes, because the emission peak corresponds to one of the absorption peak of the reaction center. So when we do bioconjugation, we actually see that shining light where only the fluorescent molecule absorbs, then you have an increase of five times in the charge separation state production, compared to the native reaction centers at one that lacks the artificial antenna. And similarly, we can drive the photo cycle that I showed in the first in the first transparency in the first slide. And you can see that there is a three to four times increase in the rate of the photo cycle, because the artificial antenna is driving faster and faster the electron, the energy transfer from the 2D reaction center. Well, you can also have some structural information, and we try to have the crystal structure, but since the system is quite disordered, we could only see one binding point to the leasing M1110, sorry, which is close enough to the, but anyway, this shows that the binding molecule is close enough to the pigments to ensure the forced electron energy transfer from the fluorophore to the pigments, explaining why we have such a good converse, such a good increase in the charge separated state. We also then we decided to team up with this organic chemistry group, and you can see here all the molecules that they did for us, and many of them they're used and improved in different way the charge, the yield of the charge separation state. So we actually are also able to, we were also able to increase the yield of conversion in different part of the spectrum. The other application I wanted to show you is that this is probably the most, well very interesting for me, I don't want to say the most interesting. As I was saying earlier in the first part of the talk, we have this closed vesicles, and this is the entire photosynthetic apparatus and you see here the ATP synthase. The ATP synthase, as I mentioned, is the molecule that produces ATP, which is the exchange coin, the exchange currency for energy in biological system. If you have a way to producing ATP, you can drive most of the biological reaction. What we tried to do was to use again giant vesicles, you'll see here this large giant vesicle, isolate the chromatophores, which are naturally organized to have all the photosynthetic apparatus already well ordered in its belayer, and having the ATP synthase protruding towards the outside of the vesicle. You can see here how it works, light, then combining the entire photosynthetic cycle, and finally the synthesis of ATP in yellow here, as molecule that produces energy. If you reconstitute the vesicle called chromatophore within the giant vesicle, then you have a photosynthetic way to produce ATP within a giant vesicle. And if you can have a T7 RNA polymerase, and a template RNA, you can actually synthesize mRNA, just shining light on the system. And you can actually look at the formation of mRNA by looking the fluorescence of the amino-acrydena complex, showing that the entire system can be considered as a minimal cell for biosynthesis of mRNA or proteins or whatever you can think of. Well, this is a proof of concept, so you can think of something that's not sure that we can do, but I have to say so to be cool. Anyway, this is an experimental part, this is the way we isolate the chromatophores, they're all picked at the dimension of 100 nanometers. And if you look here at the microscope, you can see these are time images, you can see here the curvature of the giant vesicle. Here is the chromatophores, some are open, some are broken, but mostly are open, so broken, broken, closed, sealed, sealed. Now, if you take these yellow parts and zoom it out, you can see that in the top part, you have a bubble here that you can look more carefully and reconstitute and recognize as the RTP synthase. So, we know you have sealed the chromatophores showing the ATP and you can use this to synthesize ATP, showing the ATPase and you can use this to synthesize ATP. So, if you do some tricks, you can also, the same trick of the cytochrome I showed before, you can show that roughly 70% of the chromatophores are sealed and functional, so in this fashion here. And if you shine light on the system and you have your amino-acridine channel open, you can see that shining light for longer and longer time increases the fluorescence of the amino-acridine, which binds to the, which is presented within the sealed vesicle and binds to the mRNA as soon as it's formed. So, in time, what you're seeing is that fluorescence increases and if you do this in a calibrated way, you can measure the amount of RNA that comes directly from the fluorescence of the amino-acridine orange, the amount of ATP that increases and then decreases. Increases as is formed by the chromatophore, chromatophores and then decreases because it's consumed during the reaction of the synthesis of the mRNA and of course the decrease in the ADP, which is the substrate for the ATP synthesis. Now, I hope I didn't go too long again, but here it is, my terrible transparencies again apologize to everybody whose language is not here and thank you for your attention. Thank you Massimo, you stayed on time perfectly, we have time for two questions depending. This one raised the end from Eric Ottunson, please switch on your microphone and ask a question. No, perhaps I was wrong, it was just. Yeah, it was just an applaud. Okay. Okay, thank you. Any other questions, comments? Or any other applause. I don't see any questions for now. I have a, well, perhaps not nice question, nasty question. Let's put this way. I appreciate your idea that borrowing from nature allows you to go towards complexity much easier than going in the chemical lab. The question is, the nasty question is how expensive it is extracting this structure from nature. And if you compare with the cost of chemical reactions and things like that, how difficult it is, how expensive it is. Thank you for the question. That's not a nasty question. In the sense that we ask ourselves, it is worth doing or it is not. At the moment we are playing with the proof of concepts and it's always worth it. The fact that we were published by PMS was really worth it. Sorry about that. Okay. But if you actually think to this proof of concept, what we are trying to do is making this thing simpler and simpler. So we want to use this technique, for example, of ATP synthase in the next step by using the whole bacterium. Now, this is expensive in terms, mostly in manpower because of, because all the isolation, the control, the system that rebuilding is really time expensive and you have to be very careful. So you need fantastic students and postdocs. The thing is that if you can do this with an entire bug, that would be perfect, I imagine. The problem is that entire bugs wants to stay alive. And so the first thing that happens when you have a bacteria within a giant vesicle and that bacteria start to eat the bilayer of that giant vesicle because sees it as food. So that's what we are trying to, we might have the same results in a system formed by a giant vesicle and a bacteria, but we have to try to find a way to avoid bacteria eating up the giant vesicle. That's, and that's, that's the last question. So, yes, when we'll be able to do it, hopefully before retirement. Okay, thank you. I have a question. Must you are good or whatever. Hi, Massimo, I thank for the talk. I have a question actually curiosity. I remember somehow from other sources that the overall efficiency of photosynthesis is not that great, but today I was happy to hear from you that actually in the separation in the process of separate creation of, of charge separated state is very efficient. So it's like 100%. But so where is then the bottleneck. If you might say something about this if there is one or maybe I'm wrong. No, no, you remember very well so it efficiency as a way to measure the capability of our system to convert one thing into the other. So the trick to having the highest figure is to take the right conversion reaction. So if you look at the charge separation that's 100%. If you look at proton transfer, it dropped to 60% because there are a lot of other processes that gets into the way. If you look at the synthesis of carbohydrate, then you drop below 1%. So you're right photosynthesis in its absolute so for example the conversion to from from light to come to carbohydrate is below 1% and that's definitely a non efficient way of of working. So everything depends on when you start to intercept your electrons to make a good use of them. The entire story is that you have to do it before they get converted. So you should intercept your electrons once they're producing from the photosynthetic apparatus and before they go in any further metabolism and for example, throw them out to an electrode. But of course in doing so you don't have to kill the bacteria or the plant or the of the algae, because otherwise it will last a very short time. So what this, I don't know if this makes sense to you but what you're trying to do the community of bio hybrid photosynthesis is to play with the photosynthetic apparatus within the organism in such a way to extract half of the electron from the metabolism and live in the other half for the metabolism. In this way, our idea is to eventually be able to convert sunlight in energy with efficiency of 50%, which is of course not as good as the initial one, but still very good in terms of a system that has a very should be ideally very sustainable for the energy conversion. Hope I did answer your question. Yeah, yes, yes, thank you. I was just thinking that nature, I mean, it's interesting to think that nature did very well on one part, which is the first one and then was not probably, I mean, there must be some reason why the second part is definitely harder to achieve, but I guess this discussion. Yeah, there's a huge field in the theoretical biology that asks why nature is not so good as in photosynthesis, but we could go through it, but I'm sure that there is time for more stuff to come in your lecture, so I will avoid to waste your time. Thank you very much. Thank you. Thank you. There are other questions. It seems not that I guess Massimo will be happy to answer any question. Of course. And so we can thank Massimo again. And close this session, this part of the session, we have a small break for 15 minutes, so we reconvene at 45. No, at 12, sorry, at 12. We did contribute. So thank you.