Dissertations / Theses on the topic 'Photosystem II'

Protocols were developed to fractionate each of the five protein components present in the PSII reaction centre complex, isolated from the thylakoid membrane of pea plants. The precise molecular weights of all the purified components were then successfully determined, for the first time by electrospray and fast atom bombardment mass spectrometry. Discrepancies between the molecular weights assigned and those calculated from the respective cDNA sequences were observed for all of the reaction centre component proteins. Application of novel mapping and sequencing strategies on these proteins has assured the elucidation of full primary structures of and subunits of cytochrome b559 and the majority of the structures of the D1 and D2 proteins. In the case of the subunit to cytochrome b559 an N-terminal processing event, involving the removal of the initiating formyl-methionine residue was found. The transformations on the subunit of cytochrome b559 were characterised as a N-terminal modification, entailing the processing of the formyl-methionine residue followed by an acetylation of the amino terminus of the mature protein, and a sequence change at residue twenty six, were a serine has been replaced by a phenylalanine residue. This was presumed to be due to an error in the gene sequence, but is more probably a result of the recently described phenomenon of mRNA editing. Currently, the nucleotide sequence of the pshI gene from pea plants is not available. Using a combination of mass spectrometric techniques and automated gas phase sequencing the primary structure of this component has been obtained in these studies. In contrast to the smaller subunits, data obtained on the D1 and D2 proteins indicated that both these components exist in heterogeneous forms. Mapping studies on these subunits have identified phosphorylation and oxidation, as at least two sources of this microheterogeneity.

Photosystem II (PSII) is the multi-subunit pigment-protein complex which catalyses water oxidation in higher plants, algae and cyanobacteria. Its atomic structure and protein cofactor organisation has not yet been determined. Elucidation of the structure would aid our understanding of PSII function and regulation, and may provide us with the knowledge to design new solar driven catalysts for the production of renewable energy. This thesis details the biochemical and structural studies on PSII complexes differing in subunit composition that have been isolated in order to enhance our current understanding of PSII structure. The largest of several novel PSII complexes isolated was a 725 kDa PSII-LHCII complex from the higher plant, spinach (Spinacea oleracea), consisting of the light harvesting proteins Lhcb1, 2, 4 and 5 bound to a dimeric oxygen evolving PSII core. The Lhcb proteins, 33 kDa and 23 kDa extrinsic proteins were sequentially removed, allowing their positions to be determined at a resolution of 25 Å by producing enhanced images from electron micrographs of negatively stained particles. A three-dimensional reconstruction was also achieved with these images at a resolution of 30 Å. The results suggest that the dimeric 725 kDa particle bound one LHCII trimer per reaction centre, connected to a centrally located PSII core dimer by one copy of Lhcb4 and 5. It is further concluded that CP43 is located to the outer side of the central dimer core and that there is a single copy of the 33 kDa and 23 kDa proteins per reaction centre. The significance of the dimeric organisation is discussed in terms of the in vivo structure. A similar complex of the PSII-LHCII type was also isolated from the green alga, Chlamydomonas reinhardtii and enhanced images obtained to enable comparison with the higher plant data. A smaller spinach CP47-RC dimeric PSII complex (390 kDa), was subjected to single particle image averaging with the view to more closely identifying the position of the reaction centre proteins within the PSII-LHCII complex.

Thesis advisor: William H. Armstrong
The Oxygen-Evolving Complex (OEC) of Photosystem II (PSII) utilizes a Mn4Ca cluster to catalyze the conversion of water to dioxygen within plant chloroplasts. The active site of the water oxidase is found on the lumenal side of the thylakoid membrane. For many years, the nature of this convoluted system, including the unresolved structural arrangement of the OEC manganese-oxo aggregate, stimulated on-going research projects in a diverse set of scientific fields. A tetranuclear oxo-bridged manganese complex associated with calcium [Ca] and chloride [Cl], along with a redox active tyrosine (Tyr), is thought to be the center of this remarkable and unique biological machinery. An illustrious catalytic cycle, known as the Kok cycle, progresses through a series of five intermediate states (Si, i = 0-4) to conduct water oxidation and dioxygen evolution. A tentative structural proposal based on the single crystal X-ray diffraction (XRD) crystallographic measurements introduced a CaMn3 cubane cluster and an appended fourth manganese atom. It was proposed that water binds between the “dangling” Mn atom and the Ca atom, and that is where the O-O bond formation is proposed to occur, followed by O2 release without structural rearrangement of the cubane core. The plausible manganyl (MnV=O) species was also suggested as an intermediate in the S4 state for the O-O bond formation and release O2. We have examined plausible reactive manganyl species as are proposed to exist at the OEC S4 state. The existence of manganyl in synthetic model systems will be presented in Chapter 2. In this study, we utilized stopped-flow UV-vis spectroscopy and mass spectrometry to investigate the formation and the nature of the intermediate in the reaction between mononuclear Schiff base manganese complexes and a reagent that is often used for O atom transfer reactions. Chapter 3 involves establishment of a logical synthetic method to prepare the related complexes, Mn2O2(bpy/dmb)2(ArRCOO)2 [R = 2,6-diphenyl, 2,6-ditolyl]. The dimanganese-oxo center is considered as a basic unit on the path toward the construction of higher nuclearity of Mn aggregates, preferably Mn4 clusters to be used for OEC catalytic cycle mimicry. Controlled ligand exchange synthesis of this type of carboxylate-rich/bridged {Mn2O2} dimers will provide an alternate pathway toward obtaining the Mn aggregates that are not attainable by direct ‘self-assembly’ synthetic methods. In Chapter 4, we will describe a novel mixed-ligand tetranuclear Mn cluster of the adamantane core type, [Mn4(μ-O6)(bpy)4(py)4](ClO4)4. This cluster was synthesized by using a simple reaction and its spectroscopic characterization will be discussed. We will also demonstrate chromatographic behavior of the Mn clusters that we encountered in this work (see Appendix A)
Thesis (PhD) — Boston College, 2008
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy.

In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function.

I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained.

The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light.

Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role.

The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants.

A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.

La photosynthèse est un processus biologique naturel qui convertit l'énergie lumineuse en énergie chimique par l'action de centres réactionnels photosynthétiques. L'énergie convertie est stockée sous forme de produits de haute énergie synthétisés par la branche réductive du processus photosynthétique. Les électrons nécessaires à ces réactions sont fournis par des molécules d'eau lors de leur oxydation par le centre de dégagement de l'oxygène (Oxygen Evolving Complex: OEC) pour le système de photosynthèse II (PSII). La photosynthèse artificielle cherche à reproduire les réactions qui se produisent dans les organismes naturels afin de i) de mieux comprendre les processus chimiques qui se déroulent dans les systèmes naturels, et ii) de parvenir à exploiter l'énergie solaire pour le développement de carburants propres et renouvelables. Chaque étape qui survient dans le processus de photosynthèse naturelle, telle que la capture de lumière, le transfert d'énergie, le transfert d'électron, la séparation de charge, l'activation du catalyseur et la réaction catalytique doit se produire au sein du système artificiel. La photosynthèse artificielle cherche à reproduire les réactions qui se produisent dans les organismes naturels afin de i) de mieux comprendre les processus chimiques qui se déroulent dans les systèmes naturels, et ii) de parvenir à exploiter l'énergie solaire pour le développement de carburants propres et renouvelables. Chaque étape qui survient dans le processus de photosynthèse naturelle, telle que la capture de lumière, le transfert d'énergie, le transfert d'électron, la séparation de charge, l'activation du catalyseur et la réaction catalytique doit se produire au sein du système artificiel. Avec ces concepts en vue, nous avons conçu, synthétisé et caractérisé des molécules qui imitent les réactions réalisées par les antennes et les centres réactionnels présents dans le photosystème II. Ces molécules sont capables de reproduire la séparation de charges induite par la lumière, le transfert d'électrons et l'accumulation d'équivalents oxydo-réducteurs observés pendant la photosynthèse naturelle. Les antennes artificielles se constituent de caroténoïdes et phthalocyanines. Ces molécules présentent des profiles d'absorption large avec des coefficients d'extinction élevés, et sont capables de supporter des transferts d'énergie ultra rapides qui permettent l'état de séparation de charges. En faisant varier la longueur de la chaine conjuguée des caroténoïdes de neuf à onze liaisons doubles, nous avons pu mettre en évidence comment ces molécules peuvent agir aussi bien comme donneurs que comme agents dissipateurs d'énergie, effet caractéristique qui s'apparente au processus de trempe non-photochimique (Non Photochemical Quenching: NPQ) qui se produit dans le cycle de la zéaxanthine. Les mimiques des agents donneurs du photosystème II ont aussi été étudiées. Ces systèmes supramoléculaires contiennent une partie photoactive liée de façon covalente par un intermédiaire à une cavité contenant un ion ou un agrégat d'ions métalliques. La photosensibilisateur utilisé est un complexe du ruthénium [Ru(bipy)3]2+ (bpy = 2,20-bipyridine), homologue du P680, qui absorbe la lumière dans le spectre visible et déclenche le transfert d'électron. Les espèces RuIII résultantes ont un potentiel d'oxydation réversible de 1.3 V vs SCE, comparables à celui de P680 (1.25 V vs NHE) et présentent donc la possibilité d'oxyder à la fois un complexe manganèse ainsi qu'une source d'électron. Concernant les molécules imitant le coté donneur du PSII, nous avons synthétisé des paires ruthénium-phénol, ainsi que des systèmes ruthénium-manganèse bimétalliques. Parmi ces dernières, nous avons étudié celles présentant des cavités de coordination constituées de terpyridines, vu qu'il a déjà été montré que les dimères Mn-di-μ-oxo-Mn de ce type peuvent catalyser l'oxydation de l'eau en oxygène moléculaire. Des salènes et salophènes ont aussi été examinés étant donné que de tels groupes peuvent accomplir l'oxydation à deux électrons de substrats organique. Dans la littérature, ces réactions sont toutes conduites par l'action d'oxydants chimiques externes, tandis que nous avons pour but d'utiliser des espèces oxydantes induites par l'action de la lumière.

The organisation of P511 components of a higher plant (lettuce) has been investigated using chloroplasts and isolated 0 2—evolving PSII preparations. Both energy transfer and molecular aspects of P511 have been investigated. The initial reactions of energy transfer that occur within the first few nanoseconds of excitation were measured by time resolved fluorescence using the technique of single photon counting. Fluorescence lifetimes from lettuce chloroplasts and P511 particles prepared from these chloroplasts were measured along with PSI preparations from Chlorogloea fritschii. PSI from lettuce were too unstable for our purposes. It was hoped to obtain an insight into the light—harvesting chlorophyll—reaction centre organisation and energy transfer mechanisms by this technique. The chlorophyll protein complexes of the whole chloroplast system is very complex and so in order to simplify the situation isolated PSI and PSII preparations were used. It was shown that in chloroplasts there are at least three fluorescing components and that they originate mainly from the PSII complex. These components are characterised by their lifetimes into fast, middle and long fluorescing components of approximately 100, 500 and 1500 psec respectively. The fast and long fluorescing components seem to be closely coupled to the reaction centre as the proportions of these two components are dependent on the redox state of the reaction centre. The contribution by the fast component increases whilst that of the long component decreases in the presence of an electron acceptor. The reverse occurs when the reaction centres are closed by DCMIJ. The middle component seems to be a component loosely coupled to the reaction centre and could be mobile PSIIPSII light harvesting chlorophylls. The differing lifetimes of these components could reflect the relative sizes of the chlorophyll pools. Our P811 data was analysed employing a number of currently proposed models, in particular the bipartite model used by Schatz and Holzvarth (1986). However, none of the models applied to our data could adequately explain them. The lifetime data from PSI was dominated by a 20 psec component which was not seen when the chloroplast measurements were taken. Thus a lot more work is needed to be dane before energy transfer reacti one within the chlorophyll complexes can be fully understood. On the molecular aspects of P811 organisation a calcium—binding protein (CaRP) has heen isolated from this complex. This CaBP shows similarities to a well characterised regulator CaRP called calmodulin. Thus the isolated protein could be involved in regulation of some aspect of the photosynthetic apparatus. Evidence has also been gathered implicating Ca2+ as being involved in the OEC possibly in a structural role to organise the components into an efficient 02—evolving configuration. It would also seem that as the isolated P811 preparation ages it undergoes a series of conforniational changes which alters the system. The response to the addition of Pb2 ' in the presence of Ca 2t. This could have significant implications for experimental protocols when using isolated P811 particles.

Nous avons synthétisé et caractérisé des molécules qui imitent les réactions réalisées par les antennes et les centres réactionnels présents dans le photosystème II. Ces molécules sont capables de reproduire la séparation de charges induite par la lumière, le transfert d’électrons et l’accumulation d’équivalents oxydo-réducteurs observés pendant la photosynthèse naturelle. Les antennes artificielles se constituent de caroténoïdes et phthalocyanines. Ces molécules présentent des profils d’absorption large avec des coefficients d’extinction élevés, et sont capables de supporter des transferts d’énergie ultra rapides qui permettent l’état de séparation de charges. En faisant varier la longueur de la chaine conjuguée des caroténoïdes, nous avons pu mettre en évidence comment ces molécules peuvent agir aussi bien comme donneurs que comme agents dissipateurs d’énergie, effet qui s’apparente au processus de Non Photochemical Quenching. Les mimes des agents donneurs du photosystème II ont aussi été étudiés. Ces systèmes supramoléculaires contiennent une partie photoactive liée de façon covalente à une cavité contenant un ion ou un agrégat d’ions métalliques. Le photosensibilisateur utilisé est un complexe du ruthénium [Ru(bipy)3]2+, homologue du P680, qui absorbe la lumière dans le spectre visible et déclenche le transfert d’électron. Les espèces RuIII résultantes ont un potentiel d’oxydation réversible de 1. 3V/SCE, comparables à celui de P680 et présentent donc la possibilité d’oxyder à la fois un complexe manganèse. Concernant les molécules imitant le coté donneur du PSII, nous avons synthétisé des paires ruthénium-phénol, ainsi que des systèmes ruthénium-manganèse
We have designed, synthesized and characterized molecules that mimic the reactions performed by antennas and reaction centers present in Photosystem II. These molecules are able to undergo light-induced charge separation, electron transfer, and accumulation of oxidizing/reducing equivalents that mimic the processes occurring in natural systems. The artificial anntenas are composed of carotenoid and phthalocyanin groups. These molecules show large absoption profiles with high extinction coefficients, and are capable of ultra-fast energy transfer processes which lead up to charge separation states. Varying the conjugation length of the carotenoid molecules form 9 double bonds to 11 double bonds, we can show how these molecules may act as energy donors as well as energy dissipators, a process akin to the Non Photochemical Quenching (NPQ) processes which happen during the zeaxanthin cycle. The donor side mimics of Photosystem II have also been studied. These supramolecular systems contain a photoactive component covalently linked through a spacer to a cavity where a metal ion or cluster is located. The photosensitizer used is a [Ru(bpy)3]2+ (bpy = 2,2’-bipyridine) analogue, a counterpart to P680, which absorbs light in the visible region and triggers an electron transfer process. The resulting RuIII species has a reversible oxidation potential of 1. 3 V vs. SCE, similar to that of P680, and is, in theory, capable of oxidizing a Manganese cluster and an electron source. Among the molecules mimicking the donor side of PSII we have synthesized ruthenium-phenol pairs, as well as bimetallic Ruthenium-Manganese systems

This Thesis aims to increase the understanding of the way in which herbicides inhibit plant photosynthesis by interference at the site of photosystem II. Two structurally dissimilar series of compounds, provided by Schering Agrochemicals (Saffron Walden, U.K.), one rigid, one flexible, both series showing varying herbicidal activity, were submitted to X-ray crystallography, NMR spectroscopy and molecular modelling studies. As a result of these investigations, an explanation is given for the observed trend in activity of both series of compounds. A template for the herbicide binding site is also presented. The most active molecules in the two series of' compounds were compared using molecular modelling. A model is presented which suggests that both molecules may interact with the same binding site at the photosystem II reaction centre.

Photosystem II is a unique enzyme which catalyzes light induced water oxidation. This process is driven by highly oxidizing ensemble of four Chl molecules, PD1, PD2, ChlD1 and ChlD2 called, P680. Excitation of one of the Chls in P680 leads to the primary charge separation, P680+Pheo-. Pheo- transfers electrons sequentially to the primary quinone acceptor QA and the secondary quinone acceptor QB. P680+ in turn extracts electrons from Mn4CaO5 cluster, a site for the water oxidation. There are two redox active tyrosines, TyrZ and TyrD, found in PSII. They are symmetrically located on the D1 and D2 central proteins. Only TyrZ acts as intermediate electron carrier between P680 and Mn4CaO5 cluster, while TyrD does not participate in the linear electron flow and stays oxidized under light conditions. Both tyrosines are involved in PCET. The reduced TyrD undergoes biphasic oxidation with the fast (msec-sec time range) and the slow (tens of seconds time range) kinetic phases. We assign these phases to two populations of PSII centers with proximal or distal water positions. We also suggest that the TyrD oxidation and stability is regulated by the new small lumenal protein subunit, PsbTn. The possible involvement of PsbTn protein in the proton translocation mechanism from TyrD is suggested. To assess the possible localization of primary cation in P680 the formation of the triplet state of P680 and the oxidation of TyrZ and TyrD were followed under visible and far-red light. We proposed that far-red light induces the cation formation on ChlD1. Transmembrane interaction between QB and TyrZ has been studied. The different oxidation yield of TyrZ, measured as a S1 split EPR signal was correlated to the conformational change of protein induced by the QB presence at the QB-site. The change is transferred via H-bonds to the corresponding His-residues via helix D of the D1 protein.

Photosystem II is a membrane-bound complex found in plants, eukaryotic algae and cyanobacteria which converts photo-excitation energy into chemical energy in the form of both oxidising and reducing power, catalysing the oxidation of water and reduction of quinone. The structure of this enzyme is tuned to balancing thermodynamic and quantum efficiency while minimising photodamage. This thesis tests a number of hypotheses regarding structural and functional aspects of this enzyme, addressing (1) the importance of structural differences between normal- and high-light-induced protein isoforms, (2) the binding mode of the inhibitor DCMU, (3) electron transfer from the primary quinone acceptor QA to the terminal quinone acceptor QB and (4) the structural origins of functional differences between redox-active tyrosines YZ and YD, using the thermophilic cyanobacterium Thermosynechococcus elongatus as a model organism. A crystal structure of PSII containing the PsbA3, the high-light isoform of the D1 protein which binds cofactors involved in the active electron transfer chain, is presented, demonstrating structural similarities with PsbA1. Crystallographic evidence is also presented which supports the binding of DCMU to D2-Ser264 and D2-Phe265 in this isoform, similar to predictions from PsbA1. Kinetic studies show that the half-times of electron transfer from QA- to QB and QA- to QB- are around 400 μs and 1 ms respectively, and the implications of these rates for structural studies are discussed. Finally, electron paramagnetic resonance experiments provide evidence that a hydrogen-bonding network linking YD and CP47-Glu364 is not a major determinant of functional differences between YZ and YD.

With a surge in future demand for hydrogen as a renewable fuel, the specific aim of this study was to develop a novel strategy in photosynthetic hydrogen production from green algae, which is one of the cleanest processes among existing hydrogen-production methodologies currently being explored. The novel strategy designed was a spectral-selective PSI-activation/PSII-deactivation protocol that would work to maintain a steady flow of electrons in the electron transport system in the light-dependent part of photosynthesis for delivery of electrons to hydrogenase for photo-hydrogen production. The strategy would work to activate PSI to assist in driving the electron flow, while partially deactivating PSII to a degree that it would still supply electrons, but would limit its photosynthetic oxygen production below the respiratory oxygen consumption so that an anoxic condition would be maintained as required by hydrogenase. This study successfully showed that the implementation of the spectral-selective PSIactivation/ PSII-deactivation strategy resulted in actual and relatively sustained photohydrogen production in Chlamydomonas reinhardtii cells, which had been dark-adapted for three hours immediately prior to exposure to a PSI-spectral selective radiation, which had a spectral peak at 692 nm, covering a narrow waveband of 681-701 nm, and was applied at 15 W m⁻². The optimal condition for the PSI-spectral-selective radiation (692 nm) corresponded with low cell density of 20 mg chlorophyll L⁻¹ ("chl" henceforth) with cells grown at 25⁰C. At this condition, the PSI-spectral-selective radiation induced the maximal initial hydrogen production rate of 0.055 mL H² mg⁻¹ chl h⁻¹ which statistically the same as that achieved under white light of 0.044 mL H² mg⁻¹ chl h⁻¹, a maximal total hydrogen production of 0.108 mL H² mg⁻¹ chl which significantly exceeded that under white light of 0.066 mL H² mg⁻¹ chl, and a maximal gross radiant energy conversion efficiency for hydrogen production of 0.515 μL H² mg⁻¹ chl L⁻¹ that statistically matched that under white light of 0.395 μL H² mg⁻¹ chl L⁻¹. The study also successfully demonstrated the reversibility feature of the novel strategy, allowing for the cells to alternately engage in photo-hydrogen production and to recover by simply switching on or off the PSI-spectral-selective radiation.

Oxygenic photosynthesis in plants, algae and cyanobacteria converts sunlight into chemical energy. In this process electrons are transferred from water molecules to CO2 leading to the assembly of carbohydrates, the building blocks of life. A cluster of four manganese ions and one calcium ion, linked together by five oxygen bridges, constitutes the catalyst for water oxidation in photosystem II (Mn4CaO5 cluster). This cluster stores up to four oxidizing equivalents (S0,..,S4 states), which are then used in a concerted reaction to convert two substrate water molecules into molecular oxygen. The reaction mechanism of this four-electron four-proton reaction is not settled yet and several hypotheses have been put forward. The work presented in this thesis aims at clarifying several aspects of the water oxidation reaction by analyzing the mode of substrate water binding to the Mn4CaO5 cluster. Time-resolved membrane-inlet mass spectrometric detection of flash-induced O2 production after fast H218O labelling was employed to study the exchange rates between substrate waters bound to the Mn4CaO5 cluster and the surrounding bulk water. By employing this approach to dimeric photosystem II core complexes of the red alga Cyanidoschyzon merolae it was demonstrated that both substrate water molecules are already bound in the S2 state of the Mn4CaO5 cluster. This was confirmed with samples from the thermophilic cyanobacterium Thermosynechococcus elongatus. Addition of the water analogue ammonia, that is shown to bind to the Mn4CaO5 cluster by replacing the crystallographic water W1, did not significantly affect the exchange rates of the two substrate waters. Thus, these experiments exclude that W1 is a substrate water molecule. The mechanism of O-O bond formation was studied by characterizing the substrate exchange in the S3YZ● state. For this the half-life time of this transient state into S0 was extended from 1.1 ms to 45 ms by replacing the native cofactors Ca2+ and Cl- by Sr2+ and I-. The data show that both substrate waters exchange significantly slower in the S3YZ● state than in the S3 state. A detailed discussion of this finding lead to the conclusions that (i) the calcium ion in the Mn4CaO5 cluster is not a substrate binding site and (ii) O-O bond formation occurs via the direct coupling between two Mn-bound water-derived oxygens, which were assigned to be the terminal water/hydroxy ligand W2 and the central oxo-bridging O5. The driving force for the O2 producing S4→S0 transition was studied by comparing the effects of N2 and O2 pressures of about 20 bar on the flash-induced O2 production of photosystem II samples containing either the native cofactors Ca2+ and Cl- or the surrogates Sr2+ and Br-. While for the Ca/Cl-PSII samples no product inhibition was observed, a kinetic limitation of O2 production was found for the Sr/Br-PSII samples under O2 pressure. This was tentatively assigned to a significant slowdown of the O2 release in the Sr/Br-PSII samples. In addition, the equilibrium between the S0 state and the early intermediates of the S4 state family was studied under 18O2 atmosphere in photosystem II centers devoid of tyrosine YD. Water-exchange in the transiently formed early S4 states would have led to 16,18O2 release, but none was observed during a three day incubation time. Both experiments thus indicate that the S4→S0 transition has a large driving force. Thus, photosynthesis is not limited by the O2 partial pressure in the atmosphere.

Con la elucidación de la estructura cristalina del Fotosistema II (PSII) se ha dado un paso importante en la búsqueda de nuevas alternativas de energía ambientalmente amigables. El intento de imitar la reacción que caracteriza a la fotosíntesis (para poder generar combustibles poco contaminantes), podría representar una nueva oportunidad en la reducción de nuestra dependencia de los combustibles fósiles.
With the elucidation of the crystal structure of photosystemII (PSII), an important step in the search for new environmentally friendly energy alternatives has been taken. The attempt to imitate the characteristic reaction of photosynthesis (in order to manufacture ecologically friendly fuels) could represent a new opportunity to reduce our dependence on fossil fuels.

Photosynthesis is indisputably the primary biological process to introduce chemical energy and biomass into ecosystems by oxidizing water and reducing carbon dioxide into organic compounds. Photosystem II (PSII) is a unique protein complex, present in thylakoid membranes of all oxygenic photosynthetic organisms, able to catalyze the water-splitting reaction using sunlight as driving force, thus being responsible for the generation of all the molecular oxygen accumulated in the atmosphere for over three billion years. Although its catalytic core has been extremely conserved throughout evolution, from cyanobacteria to higher plants, the necessity of different photosynthetic organisms to cope with ever-changing environmental light conditions led to the emergence of a great variability among its peripheral antenna systems, differentiating in extrinsic phycobilisomes in cyanobacteria and intrinsic light harvesting complexes (LHCII) in green algae and higher plants. LHCII are integral membrane proteins that occur as heterotrimers of Lhcb1-2-3 subunits and monomeric Lhcb4-5-6 polypeptides and associate peripherally with the PSII core in variable numbers, thus forming large supramolecular assemblies called PSII‐LHCII supercomplexes. The minimal functional unit, found in all light conditions, consists of a dimeric PSII core (C2) with two strongly bound LHCII trimers (S2), made of Lhcb1 and Lhcb2, connected by two monomeric Lhcb4 and Lhcb5 subunits, and is called C2S2. In limiting light conditions, the C2S2 can further associate with one or two moderately bound LHCII trimers (M2) which consist of Lhcb1, Lhcb2 and Lhcb3 proteins connected by the monomeric Lhcb6, a peculiar subunit found only in higher plants, originating supercomplexes of type C2S2M1-2. A further supramolecular organization is due to the lateral association of PSII-LHCII supercomplexes within the thylakoid membrane plane, forming PSII-LHCII megacomplexes, or even higher ordered arrays. The LHCII fulfill a dual role by either quenching the excess light energy, often occurring in natural environments, or optimizing its harvesting in ecosystems where there is competition and mutual shading. The rearrangement of the PSII’s modular antenna system through its dynamic interaction with the PSII core, therefore, appears to be a key process in light harvesting regulation. Moreover, plant’s PSII and LHCII are spatially and functionally segregated into piled discs of thylakoid membranes (grana), where they occupy 80% of the surface. Their structural arrangement into PSII-LHCII supercomplexes interacting dynamically with each other appears to be critical in determining the overall membrane architecture and ultimately the efficiency of photosynthesis. Although the overall structure of the basic C2S2 supercomplex in plants has been recently resolved at nearly atomic resolution, there is still a lack of knowledge regarding its structural rearrangement in different light conditions as well as its specific interaction within the membrane plane and between adjacent membranes. During this thesis’ work we have been able to isolate pure PSII-LHCII super- and megacomplexes from pea plants grown in moderate light by mild solubilization of stacked thylakoid membranes. In order to assess their overall functional architecture, the full biochemical characterization of isolated PSII-LHCII supercomplexes, comprehensive of accurate proteomic analyses, was coupled with structural studies. Their structural characterization, performed by transmission electron microscopy (TEM) in cryogenic conditions (cryo-EM) and subsequent single particle analysis, led to a novel 3D structure at about 14 Å resolution of the supercomplexes of type C2S2M. The obtained electron density map revealed that under normal light conditions most of the supercomplexes within the grana are of type C2S2M and occur as paired supercomplexes, whose interactions are mediated by physical connections across the stromal gap of adjacent membranes. The specific overlapping of LHCII trimers facing each other in paired supercomplexes, as already observed in other studies, suggests that this conformation might be representative of their native state within the membranes. The physical connections observed across the stromal gap might be attributable to the mutual interaction between the long N-terminal loops of the monomeric Lhcb4 subunits. These subunits occupy a pivotal position in the 3D map of the paired supercomplexes and are clearly bridged across the stromal gap by electron densities attributable to these loops. In addition, despite the its structural flexibility, the remarkable sequence conservation of this region, even in distant phylogenetic photosynthetic organisms, may suggest its major involvement in structural dynamics. The specific interaction observed in paired supercomplexes seems to be mediated by cations present within the chloroplast in relatively low concentrations as their depletion from buffers used for isolation leads to the dissociation of the paired supercomplexes into single ones. Moreover, this evidence was also strongly supported by the decrease in the PSII excitonic connectivity measured in-vivo. The paired behavior has also been observed in higher oligomerization forms of isolated PSII-LHCII supercomplexes in which two paired supercomplexes laterally interact with each other in the membrane plane, thus forming paired megacomplexes. This novel structure has been obtained by EM and 2D reconstruction of negatively stained particles and, despite its low resolution, reveals how PSII-LHCII supercomplexes may laterally and stromally interact with each other in different ways. The observation of the potential overlapping of LHCII trimers in megacomplexes facing each other, as well as the occurrence of different geometries of interaction between supercomplexes within the membrane plane and between megacomplexes in adjacent membranes, provide intriguing insights on how PSII and LHCII might interact in a very stable manner within the thylakoid membrane and between different discs in the grana. In order to study the PSII-LHCII supercomplex remodeling in the context of ever-changing light environmental conditions, PSII-LHCII supercomplexes have been isolated from pea plants grown at different light intensities: low (LL), moderate (CL) and high light (HL). The accurate profiling and quantitation of the LHCII subunits in the isolated supercomplexes and in the native thylakoids, achieved by using a mass-spectrometry based proteomic approach, was coupled with the evaluation in-vivo of their functional antenna size (ASII). At increasing light intensities, the structural remodeling of the modular PSII’s antenna system led to the reduction of the amount of LHCII M-trimers in the isolated complexes, attested by the decreased level of Lhcb3 and Lhcb6. This specific remodeling does not occur at the same rate in the entire thylakoid membrane. The whole LHCII pool is downregulated only in plants grown in HL, suggesting the occurrence of different acclimation strategies. The remarkable decrease of the ASII observed in HL acclimated plants, when compared to LL plants, can be attributed to the significant increase of the Lhcb4 specific isoform Lhcb4.3, occurring both in isolated supercomplexes and in thylakoid membranes. Unlike isoforms Lhcb4.1-2, the Lhcb4.3 isoform, whose transcription is enhanced upon HL exposure, interestingly has a truncated C-terminus that is located at the binding interface with Lhcb6 within the supercomplex structure. The incorporation of Lhcb4.3 in the PSII-LHCII supercomplex might play a major role in decreasing its functional antenna size by reducing its affinity to bind additional M-trimers, thus regulating its light harvesting efficiency even at moderate light intensities. Conversely, the exposure to HL induces the decrease of the PSII antenna cross-section in isolated supercomplexes and the partial depletion of the whole antenna system of PSII in the thylakoid membranes, thus constitutively preventing damages to the reaction center when light continuously exceeds its energy-processing capacity. These results aim at broadening the current knowledge on how the light harvesting antenna system associated with the PSII core is finely regulated upon plants’ long term acclimation to different light intensities. The flexibility of the PSII’s modular antenna system, accompanied by its finely tuned structural interaction with the core complex, pivotal for the 3D organization of plant thylakoid membranes, certainly played a key role in determining its remarkable evolutionary outcome. Taken together, these results may provide new research directions while certainly broadening the knowledge on how PSII-LHCII assemblies and their supramolecular interaction contribute to maintain the complex architecture of thylakoid membranes and the overall efficiency of photosynthesis in ever changing environmental conditions.
La fotosintesi è indubbiamente il processo biologico principale che introduce energia chimica e biomassa negli ecosistemi ossidando l’acqua e riducendo l'anidride carbonica in composti organici. Il fotosistema II (PSII) è un complesso proteico presente nelle membrane tilacoidali di tutti gli organismi fotosintetici, l’unico in grado di catalizzare la reazione di lisi dell'acqua utilizzando la luce solare come forza motrice e di conseguenza responsabile della generazione di tutto l'ossigeno molecolare presente nell'atmosfera da più di tre miliardi di anni. Nonostante il centro catalitico del PSII sia rimasto fondamentalmente inalterato nel corso dell'evoluzione dai cianobatteri alle piante superiori, la necessità di far fronte alla continua variazione delle condizioni di luce ambientali ha portato all’evoluzione di sistemi di antenne periferiche altamente differenziate, distinte in ficobilisomi estrinseci nei cianobatteri e complessi di membrana intrinseci (LHCII) in alghe verdi e piante superiori. Gli LHCII sono complessi proteici di membrana presenti come etero-trimeri composti dalle subunità Lhcb1-2-3 e subunità monomeriche Lhcb4-5-6 associate perifericamente con il centro catalitico del PSII in numero variabile, formando così associazioni supramolecolari chiamate supercomplessi PSII-LHCII. L'unità funzionale minima, presente in ogni condizione di luce, detta C2S2, è costituita da un PSII centro di reazione dimerico (C2) legato strettamente a due complessi antenna trimerici (S2), composti da Lhcb1 e Lhcb2, mediante due subunità monomeriche Lhcb4 e Lhcb5. In condizioni di luce limitante il C2S2 può ulteriormente associare uno o due complessi antenna trimerici legati moderatamente (M2), costituiti dalle subunità Lhcb1, Lhcb2 e Lhcb3, mediante una peculiare subunità monomerica che si trova solo nelle piante superiori, Lhcb6, generando supercomplessi di tipo C2S2M1-2. I supercomplessi PSII-LHCII possono ulteriormente interagire lateralmente all'interno del piano della membrana tilacoidale formando megacomplessi PSII-LHCII o più estesi arrangiamenti ordinati semicristallini. I complessi antenna LHCII svolgono un duplice ruolo, la dissipazione efficiente dell'energia luminosa, spesso in eccesso negli ambienti naturali, e l’ottimizzazione della sua raccolta negli ambienti in cui vi è concorrenza tra organismi e ombreggiatura reciproca. Il riassetto del sistema di antenne modulari del PSII attraverso la sua interazione dinamica con il centro catalitico sembra quindi essere un processo chiave nella regolazione della raccolta della luce. Inoltre, i PSII e LHCII nelle piante sono spazialmente e funzionalmente segregati in dischi impilati di membrane tilacoidi (grana), dove occupano l'80% della superficie. La loro disposizione strutturale in supercomplessi PSII-LHCII che interagiscono dinamicamente tra loro sembra essere determinante per l'architettura complessiva della membrana tilacoidale e quindi per l'efficienza della fotosintesi. Sebbene la struttura del supercomplesso base C2S2 delle piante sia stata recentemente risolta ad una risoluzione quasi atomica, c'è ancora una lacuna conoscitiva riguardo al ri-arrangiamento strutturale dei PSII-LHCII che avviene in diverse condizioni di luce e alla loro interazione reciproca nel piano della membrana e tra membrane adiacenti dei grana. Durante il lavoro svolto in questa tesi, siamo stati in grado di purificare super- e megacomplessi PSII-LHCII isolati da piante di pisello coltivate in luce moderata mediante la completa solubilizzazione delle membrane tilacoidali. La caratterizzazione biochimica dei supercomplessi PSII-LHCII isolati, complementata da accurate analisi proteomiche, è stata accoppiata con studi strutturali al fine di comprendere la loro architettura funzionale. La caratterizzazione strutturale, eseguita mediante microscopia elettronica a trasmissione (TEM) in condizioni criogeniche (cryo-EM) e successiva analisi d’immagine sulle singole particelle, ha portato ad una nuova struttura tridimensionale (3D) a circa 14 Å di risoluzione del supercomplesso di tipo C2S2M. La mappa di densità elettronica ottenuta ha rivelato che, in condizioni di luce di crescita di intensità moderata, la maggior parte dei supercomplessi è di tipo C2S2M. Essi sono disposti in maniera accoppiata, interagendo mediante collegamenti fisici attraverso l’intervallo stromatico, verosimilmente di membrane adiacenti. La sovrapposizione specifica degli LHCII trimerici, uno di fronte all'altro in supercomplessi accoppiati, come già osservato in altri studi, suggerisce che questa conformazione potrebbe essere rappresentativa del loro stato nativo all'interno delle membrane. I collegamenti fisici osservati nell’intervallo stromatico potrebbero essere attribuibili all'interazione reciproca tra le lunghe porzioni N-terminali di subunità monomeriche Lhcb4 adiacenti. Queste subunità occupano una posizione chiave nella mappa 3D dei supercomplessi accoppiati e le densità elettroniche che attraversano l’intervallo stromatico connettendo i due supercomplessi sono chiaramente attribuibili alle loro porzioni flessibili N-terminali. La sequenza amminoacidica di questa regione, nonostante la sua flessibilità, è sorprendentemente conservata anche in organismi fotosintetici filogeneticamente distanti, il che suggerisce un suo coinvolgimento in dinamiche strutturali fisiologicamente rilevanti per l’apparato fotosintetico. L'interazione specifica osservata nei supercomplessi appaiati sembra essere mediata dai cationi presenti all'interno del cloroplasto in concentrazioni fisiologiche. La loro rimozione dai tamponi utilizzati per l'isolamento, infatti, ne provoca la dissociazione in singoli supercomplessi. Questa evidenza è inoltre sostenuta dalla stima della connettività funzionale misurata in-vivo tramite tecniche di induzione di fluorescenza. Nei supercomplessi appaiati infatti si è evidenziato un potenziale trasferimento di energia maggiore se confrontato con i supercomplessi singolarizzati mediante semplice diluizione dei cationi presenti. L’ appaiamento sul lato stromatico mediato da cationi è stato osservato anche in forme isolate di PSII-LHCII con forme di oligomerizzazione superiore ai supercomplessi, in cui due supercomplessi accoppiati interagiscono lateralmente tra loro nel piano di membrana, formando così megacomplessi appaiati. Questa nuova struttura è stata ottenuta con TEM e ricostruzione bidimensionale a partire da particelle colorate negativamente. Nonostante la bassa risoluzione ottenuta, questa struttura rivela come i supercomplessi PSII-LHCII possono interagire reciprocamente in modi diversi, sia lateralmente che attraverso l’intervallo stromatico. L'osservazione della potenziale sovrapposizione degli LHCII trimerici in megacomplessi accoppiati, così come la presenza di diverse geometrie di interazione tra supercomplessi all'interno del piano di membrana e tra megacomplessi nelle membrane adiacenti, forniscono informazioni interessanti su come PSII e LHCII potrebbero interagire in modo stabile e specifico all'interno della membrana tilacoidale e tra i vari dischi dei grana. Al fine di studiare il rimodellamento dei supercomplessi PSII-LHCII nel contesto di un continuo cambiamento delle condizioni ambientali di luce, sono stati isolati supercomplessi PSII-LHCII da piante di pisello cresciute a diverse intensità di luce: bassa (LL), moderata (CL) e alta (HL). La valutazione in-vivo delle dimensioni dell'antenna funzionale del PSII (ASII) è stata accoppiata con l’identificazione e la quantificazione, mediante analisi proteomiche, delle diverse subunità di LHCII presenti sia nei supercomplessi isolati che nei tilacoidi nativi. All’aumentare dell’intensità di luce di crescita, si evince il rimodellamento strutturale dell’antenna modulare del PSII dovuto alla riduzione della quantità di LHCII trimerici di tipo “M” nei complessi isolati, attestata da una ridotta presenza di Lhcb3 e Lhcb6. Questo rimodellamento specifico non avviene però con le stesse modalità in tutta la membrana tilacoidale. Infatti, la quantità totale di LHCII nei tilacoidi viene significativamente ridotta solo in piante cresciute in HL, suggerendo la presenza di diverse strategie di acclimatazione in grado di ridurre l’antenna funzionale nei tilacoidi. La notevole diminuzione dell’ASII osservata sia nei supercomplessi isolati che nelle membrane tilacoidi di piante cresciute in HL, rispetto alle piante LL, può essere attribuita al significativo incremento di Lhcb4.3, una isoforma di Lhcb4. A differenza delle isoforme Lhcb4.1-2, l'isoforma Lhcb4.3, la cui trascrizione è nota aumentare in seguito all'esposizione ad HL, presenta l’estremità C-terminale troncata. Questa porzione della proteina nella struttura del supercomplesso C2S2M si trova a livello dell’interfaccia di legame con Lhcb6, la subunità monomerica che funge da connettore specifico per l’LHCII trimerico di tipo “M”. L'incorporazione di Lhcb4.3 nel supercomplesso PSII-LHCII sembrerebbe svolgere quindi un ruolo importante nel ridurre le dimensioni dell'antenna funzionale, riducendo l’affinità di legame di antenne aggiuntive (tipo “M”) per ridurre l’efficienza di raccolta della luce già ad intensità moderate. L'esposizione ad HL invece, oltre ad indurre la diminuzione dell'antenna del PSII in supercomplessi isolati, determina anche la riduzione parziale di tutte le antenne del PSII presenti nelle membrane tilacoidi, impedendo quindi danni al centro di reazione quando la luce incidente supera costantemente la sua capacità di utilizzarla efficientemente. Questi risultati contribuiscono ad aumentare le conoscenze su come il sistema di antenne associate al PSII è attivamente regolata a lungo termine modulando l’espressione genica in piante acclimatate a diverse intensità di luce. La flessibilità del sistema modulare di antenne del PSII e la sua interazione strutturale con il centro catalitico, oltre ad essere fondamentale per l’architettura tridimensionale delle membrane tilacoidi delle piante, ha certamente giocato un ruolo chiave nel determinare la loro notevole diversificazione nel corso dell’evoluzione. Nel complesso questi risultati potrebbero fornire nuovi spunti per ampliare la conoscenza di come le associazioni di PSII e LHCII e la loro reciproca interazione contribuiscono a mantenere la complessa architettura delle membrane tilacoidi e quindi l'efficienza complessiva della fotosintesi in condizioni ambientali in continuo mutamento.