No / Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C→A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells. / CAEB, Balearic Islands and Yorkshire Cancer Research

Le photosystème II (PSII) est un complexe multi-protéique qui utilise l'énergie solaire pour oxyder l'eau et réduire des quinones. Le site catalytique d'oxydation de l'eau est localisé coté lumen du complexe, alors, que le site de réduction comprenant deux quinones (QA et QB) et un fer non-hémique est localisé sur le coté stromal du complexe membranaire. Dans cette thèse j'ai étudié les deux cotés accepteur et donneur d'électrons du PSII.QA•- et QB•- sont couplés magnétiquement au fer non-hémique donnant de faibles signaux RPE. Le fer non-hémique possède quatre ligands histidines et un ligand (bi)carbonate échangeable. Le formate peut échanger le ligand (bi)carbonate induisant un ralentissement dans le transfert d'électrons. Ici, je décris une modification du signal RPE de QB•- Fe2+ lorsque le formate est substitué au (bi)carbonate. J'ai aussi découvert un second signal RPE dû à la présence du formate à la place du (bi)carbonate lorsque QB est doublement réduit. De plus, j'ai trouvé que les signaux RPE natifs de QA•- Fe2+ et QB•- Fe2+ possèdent une signature intense encore jamais détectée. Tous les signaux RPE rapportés dans cette thèse devraient faciliter le titrage redox de QB par RPE. J'ai aussi observé que QB•- peut oxyder le fer non-hémique à l'obscurité en anaérobie. Cette observation implique qu'au moins dans une fraction des centres, le couple QB•-/QBH2 possède un potentiel redox plus haut que supposé. La quantification du nombre de centres où cette oxydation du fer se produit par le couple QB•-/QBH2 reste à faire. La réduction du PSII par le dithionite génère un signal modifié de QA•-Fe2+, un changement structural du PSII observé par électrophorèse. Cela peut indiquer la réduction d'un pont disulfure à l'intérieur du PSII. Concernant le site d’oxydation de l'eau, j'ai étudié la première étape de l'assemblage du site catalytique (Mn4Ca), en suivant l'oxydation du Mn2+ par RPE en bande X et haut champ. J'ai mis au point des conditions expérimentales permettant le piégeage du premier intermédiaire et j'ai aussi trouvé une incohérence avec des travaux publiés dans la littérature. J'ai aussi trouvé que le dithionite pouvait réduire le site catalytique Mn4Ca, en formant des états sur-réduits qui peuvent correspondre aux intermédiaires de l'assemblage du cluster Mn4Ca. / Photosystem II (PSII) uses light energy to oxidise water and reduce quinone. The water oxidation site is a Mn4Ca cluster located on the luminal side of the membrane protein complex, while the quinone reduction site is made up of two quinones (QA and QB) and a non-heme Fe2+ located on the stromal side of the membrane protein. In this thesis I worked on both oxidation and reduction functions of the enzyme. QA•- and QB•- are magnetically couple to the Fe2+ giving weak and complex EPR signals. The distorted octahedral Fe2+ has four histidines ligands and an exchangeable (bi)carbonate ligand. Formate can displace the exchangeable (bi)carbonate ligand, slowing electron transfer out of the PSII reaction centre. Here I report the formate-modified QB•- Fe2+ EPR signal, and this shows marked spectral changes and has a greatly enhanced intensity. I also discovered a second new EPR signal from formate-treated PSII that is attributed to formate-modified QA•- Fe2+ in the presence of a 2-electron reduced form of QB. In addition, I found that the native QA•- Fe2+ and QB•- Fe2+ EPR signals have a strong feature that had been previously missed because of overlapping signals (mainly the stable tyrosyl radical TyrD•). These previously unreported EPR signals should allow for the redox potential of this cofactor to be directly determined for the first time. I also observed that when QB•-Fe was formed; it was able to oxidise the iron slowly in the dark. This occurred in samples pumped to remove O2. This observation implies that at least in some centres, the QB•-/QBH2 couple has a higher potential then is often assumed and thus that the protein-bound semiquinone is thermodynamically less stable expected. It has yet to be determined if this represents a situation occurring in the majority of centres. Treatment of the system with dithionite generated a modified form of QA•-Fe2+ state and a change in the association of the proteins on gels. This indicates a redox induced modification of the protein, possibly structurally important cysteine bridge in PSII.On the water oxidation side of the enzyme, I studied the first step in the assembly of the Mn4Ca cluster looking at Mn2+ oxidation using kinetic EPR and high field EPR. Conditions were found for stabilising the first oxidised state and some discrepancies with the literature were observed. I also found that dithionite could be used to reduce the Mn4Ca, forming states that are formally equivalent to those that exist during the assembly of the enzyme.

Ce manuscrit décrit d'une part la synthèse et la fonctionnalisation de dérivés de types azacalix[4]arènes et d'autre part l'élaboration d'une nouvelle voie de synthèse des ligands benzoquinonediimines.La première partie, consacrée aux azacalix[4]arènes, détaille les différents procédés utilisés pour fonctionnaliser des azacalix[4]arènes après leur macrocyclisation. L'introduction de groupes amides a généré des récepteurs d'anions qui présentent une sélectivité non usuelle pour les ions chlorures. Les complexes hotes-invités formés reposent sur des intercations supramoléculaires et constituent l'une des principales thématiques de ce manuscrit.La seconde part du manuscrit porté sur une nouvelle voie de synthèse des ligands benzoquinonediimines qui reposent sur la transamination du 1,2,4,5-tétraaminobenzène en présence d'un excès d'amine primaire. La simplicité de cette procédure permet d'obtenir une grande variété de benzoquinondiimines diversement substiuées. L'impact de cette substiution est illusté via l'étude de nouveaux complexes bimétalliques contenant un ligand benzoquinonediimine pontant. / The first pat of this manuscrit deals with the synthetis and fonctionalization of azacalix[4]arnes derivatives while the second part describes a new syntetic access of benzoquinonediimines ligands.In the first part, differents approaches were used to introduce fonctional groups on the peripheral positions of alrealdy formed azacalix[4]arenes. the introduction of hydrogen donor groups such as amides groups generated azacalix[4]arnes based anions receptor that preferentially bind chloride anions. These host-guest complexes are built on supramolecular interations and are one of the main topic of this manuscrit.In the second part, a new synthetic procedure of benzoquinonediimines is described and studied. The simple and efficient one-pot reaction involves a transamination between 1,2,4,5-tetraaminobenzene and primary amine and allows to prepare a large range of N-substituted benzoquinonediimines derivatives. The impact of the N-substitution is enlightened through the study on bimetallic complexes containing a bridging benzoquinonediimine unit.

La quinone réductase 2 ou QR2 est une enzyme qui, comme son homologue QR1, joue un rôle de détoxification des quinones, molécules fortement réactives, en les réduisant en hydroquinones. Cependant, il a été observé au niveau cellulaire et tissulaire que l'activité de cette flavoprotéine pouvait avoir des effets délétères en déclenchant une surproduction d'espèces réactives de l'oxygène (ROS). D'autre part, on observe une surexpression ou une sous expression de QR2 dans certaines maladies neurodégénératives comme la maladie de Parkinson et la maladie d'Alzheimer. Dans ce contexte, ce travail a porté sur l'étude des espèces oxygénées réactives produites lors du cycle redox quinone / QR2 et leurs variations en fonction de la nature de la quinone, sur protéine purifiée et sur modèles cellulaires comparativement à QR1. Les propriétés d'oxydo-réduction des substrats, co-substrats et inhibiteurs de QR2 étudiées par électrochimie ont permis de les classer en fonction de leur capacité à être réduits. L'activité enzymatique de la protéine, qu'elle soit purifiée ou intracellulaire, a été suivie par différentes méthodologies (résonance paramagnétique électronique, spectroscopie UV-visible et de fluorescence, U(H)PLC-MS, microscopie confocale de fluorescence). La production du radical superoxyde est observée en présence de lignées cellulaires surexprimant ou non QR1 et QR2. Les quinones sont réduites enzymatiquement pour donner des hydroquinones via l'activité des quinones réductases (QR1 et QR2) et des semiquinones via l'activité de réductases à un électron (CytP540 réductase par exemple). La réoxydation de ces produits est responsable d'une production plus ou moins forte de radicaux superoxydes selon la structure initiale de la quinone et l'affinité pour les différentes réductases. La ménadione provoque une production cellulaire de superoxyde plus importante en l'absence de QR1 et QR2. Ces analyses ont également démontré que, comme son homologue QR1, QR2 est capable de réduire les ortho-quinones dont certaines catécholquinones (aminochrome, dopachrome, adrénochrome) reconnues pour leur toxicité neuronale. / Quinone reductase 2 or QR2 is an enzyme that, like its counterpart QR1, plays a role in detoxification of the highly reactives quinones by reducing them into hydroquinones. On one hand, it has been observed at the cellular and tissue level that the activity of this flavoprotein could have deleterious effects by triggering an overproduction of reactive oxygen species (ROS). On the other hand, overexpression or under expression of QR2 has been observed in some neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In this context, this work focused on the study of reactive oxygen species produced during the quinone / QR2 redox cycle and their variations depending on the nature of the quinone, on both purified protein and cell models, in comparison to QR1. The redox properties of the substrates, co-substrates and inhibitors ok QR2 studied by electrochemistry allowed to classify them according to their capacity to be reduced. The enzymatic activity of the protein, either purified or intracellular, was followed by various methodologies (electron paramagnetic resonance, UV-visible and fluorescence spectroscopy, U(H)PLC-MS, confocal fluorescence microscopy). Production of superoxide radical is observed in the presence of cell lines overexpressing or not QR1 and QR2. Quinones are reduced enzymatically to form hydroquinones via the activity of quinone reductase (QR1 and QR2) and semiquinone via the activity of one electron reductases (e.g. CytP540 reductase). Reoxidation of these products is responsible for a greater or lesser production of the superoxide radical, according to the initial structure of the quinone and the affinity for different reductases. Menadione causes a higher production of cellular superoxide in the absence of QR1 and QR2. These analyzes have also shown that, like its counterpart QR1, QR2 is capable of reducing ortho-quinones including catecholquinones (aminochrome, dopachrome, adrenochrome) known for their neuronal toxicity.

This thesis concerns the use of various potent oxidants in organic synthesis. The main focus is directed at selectively introducing trifluoromethyl groups into compounds containing double or triple bonds. All reactions proceed under mild conditions and can in most cases be performed on the bench-top. We have developed three different procedures for transformations of activated alkenes and alkynes as well as quinones. In paper I the selective introduction of a trifluoromethyl group together with an oxygen functionality to double and triple bonds is demonstrated. Paper II is focused on the related chemoselective cyanotrifluoromethylation in which a cyano group is added instead of the oxygen functionality. Paper III describes a new procedure for C–H trifluoromethylation of quinones. Our studies on the mechanistic aspects of the above reactions are described in Paper IV. In these studies we investigated the ligand and substituent effects in Cu-catalyzed reactions. Paper V is focused on a conceptually new palladium-catalyzed allylic C–H acyloxylation of olefins under oxidative conditions. The procedure uses an inexpensive, safe and environmentally benign oxidant, sodium perborate, which is activated with acetic anhydride. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.</p>

This research has investigated proton-coupled electron transfer (PCET) of quinone/hydroquinone and other simple organic PCET species for the purpose of furthering the knowledge of the thermodynamic and kinetic effects due to reduction and oxidation of such systems. Each of these systems were studied involving the addition of various acid/base chemistries to influence the thermodynamics and kinetics upon electron transfer. It is the expectation that the advancement of the knowledge of acid/base catalysis in electrochemistry gleaned from these studies might be applied in fuel cell research, chemical synthesis, the study of enzymes within biological systems or to simply advance the knowledge of acid/base catalysis in electrochemistry. Furthermore, it was the intention of this work to evaluate a system that involved concerted-proton electron transfer (CPET), because this is the process by which enzymes are believed to catalyze PCET reactions. However, none of the investigated systems were found to transfer an electron and proton by concerted means. Another goal of this work was to investigate a system where hydrogen bond formation could be controlled or studied via electrochemical methods, in order to understand the kinetic and thermodynamic effects complexation has on PCET systems. This goal was met, which allowed for the establishment of in situ studies of hydrogen bonding via 1H-NMR methods, a prospect that is virtually unknown in the study of PCET systems in electrochemistry, yet widely used in fields such as supramolecular chemistry. Initial studies involved the addition of Brønsted bases (amines and carboxylates) to hydroquinones (QH2’s). The addition of the conjugate acids to quinone solutions were used to assist in the determination of the oxidation processes involved between the Brønsted bases and QH2’s. Later work involved the study of systems that were initially believed to be less intricate in their oxidation/reduction than the quinone/hydroquinone system. The addition of amines (pyridine, triethylamine and diisopropylethylamine) to QH2’s in acetonitrile involved a thermodynamic shift of the voltammetric peaks of QH2 to more negative oxidation potentials. This effect equates to the oxidation of QH2 being thermodynamically more facile in the presence of amines. Conjugate acids were also added to quinone, which resulted in a shift of the reduction peaks to more positive potentials. To assist in the determination of the oxidation process, the six pKa’s of the quinone nine-membered square scheme were determined. 1H-NMR spectra and diffusion measurements also assisted in determining that none of the added species hydrogen bond with the hydroquinones or quinone. The observed oxidation process of the amines with the QH2’s was determined to be a CEEC process. While the observed reduction process, due to the addition of the conjugate acids to quinone were found to proceed via an ECEC process without the influence of a hydrogen bond interaction between the conjugate acid and quinone. Addition of carboxylates (trifluoroacetate, benzoate and acetate) to QH2’s in acetonitrile resulted in a similar thermodynamic shift to that found with addition of the amines. However, depending on the concentration of the added acetate and the QH2 being oxidized, either two or one oxidation peak(s) was found. Two acetate concentrations were studied, 10.0 mM and 30.0 mM acetate. From 1H-NMR spectra and diffusion measurements, addition of acetates to QH2 solutions causes the phenolic proton peak to shift from 6.35 ppm to as great as ~11 ppm, while the measured diffusion coefficient decreases by as much as 40 %, relative to the QH2 alone in deuterated acetonitrile (ACN-d3). From the phenolic proton peak shift caused by the titration of each of the acetates, either a 1:1 or 1:2 binding equation could be applied and the association constants could be determined. The oxidation process involved in the voltammetry of the QH2’s with the acetates at both 10.0 and 30.0 mM was determined via voltammetric simulations. The oxidation process at 10.0 mM acetate concentrations involves a mixed process involving both oxidation of QH2 complexes and proton transfer from an intermediate radical species. However, at 30.0 mM acetate concentrations, the oxidation of QH2-acetate complexes was observed to involve an ECEC process. While on the reverse scan, or reduction, the process was determined to be an CECE process. Furthermore, the observed voltammetry was compared to that of the QH2’s with amines. From this comparison it was determined that the presence of hydrogen bonds imparts a thermodynamic influence on the oxidation of QH2, where oxidation via a hydrogen bond mechanism is slightly easier. In order to understand the proton transfer process observed at 10.0 mM concentrations of acetate with 1,4-QH2 and also the transition from a hydrogen bond dominated oxidation to a proton transfer dominated oxidation, conjugate acids were added directly to QH2 and acetate solutions. Two different acetate/conjugate acid ratios were focused on for this study, one at 10.0 mM/25.0 mM and another at 30.0 mM/50.0 mM. The results of voltammetric and 1H-NMR studies were that addition of the conjugate acids effects a transition from a hydrogen bond oxidation to a proton transfer oxidation. The predominant oxidation species and proton acceptor under these conditions is the uncomplexed QH2 and the homoconjugate of the particular acetate being studied, respectively. Furthermore, voltammetry of QH2 in these solutions resembles that measured with the QH2’s and added amines, as determined by scan rate analysis. In an attempt to understand a less intricate redox-active system under aqueous conditions, two viologen-like molecules were studied. These molecules, which involve a six-membered fence scheme reduction, were studied under buffered and unbuffered conditions. One of these molecules, N-methyl-4,4’-bipyridyl chloride (NMBC+), was observed to be reduced reversibly, while the other, 1-(4-pyridyl)pyridinium chloride (PPC+), involved irreversible reduction. The study of these molecules was accompanied by the study of a hypothetical four-membered square scheme redox system studied via digital simulations. In unbuffered solutions each species, both experimental and hypothetical, were observed to be reduced at either less negative (low pH) or more negative (high pH), depending on the formal potentials, pKa’s of the particular species and solution pH. The presence of buffer components causes the voltammetric peaks to thermodynamically shift from a less negative potential (low pH buffer) to a more negative potential (high pH buffer). Both of these observations have been previously noted in the literature, however, there has been no mention, to our knowledge, of kinetic effects. In unbuffered solutions the reduction peaks were found to separate near the pKa,1. While in buffered solutions, there was a noted peak separation throughout the pH region defined by pKa’s 1 and 2 (pKa,1 and pKa,2) of the species under study. The cause for this kinetic influence was the transition from a CE reduction at low pH to an EC reduction process at high pH in both buffered and unbuffered systems. This effect was further amplified via the study of the hypothetical species by decreasing the rate of proton transfer. In an effort to further this work, some preliminary work involving the attachment of acid/base species at the electrode surface and electromediated oxidation of phenol-acetate complexes has also been studied. The attachment of acid/base species at the surface is believed to assist in the observation of heterogeneous acid/base catalysis, similar to that observed in homogeneous acid/base additions to quinone/hydroquinone systems. Furthermore, our efforts to visualize a concerted mechanism are advanced in our future experiments involving electromediated oxidation of phenol-acetate complexes by inorganic species. It may be possible to interrogate the various intermediates more efficiently via homogeneous electron-proton transfer rather than heterogeneous electron transfer/homogeneous proton transfer.

A thesis submitted in fulfilment of the requirements for the degree Magister Technologiae (Chemistry) in the Faculty of Applied Sciences, Department of Chemistry, Cape Peninsula University of Technology, 2008 / Green et al. attempted to synthesize linear naphthopyranquinones from a naphthyl dioxolane using a TiCl4 as a catalyst. They managed to synthesise an angular naphthopyran as well as a linear naphthopyran in low yield. They showed that reducing the steric strain at position 1 of the naphthyl dioxolane afforded a low percentage yield of the linear naphthopyran plus an angular one. This thesis describes the synthesis of linear naphthopyrans with an improved percentage yield using TiCl4 as a catalyst. This was achieved by placing a OMe group of less steric hinderance at position 1 and a Br atom at position 4 of a naphthyl dioxolane. The OMe group at position 1 was to allow isomerisation to occur at position 2, and the Br atom was to inhibit isomerisation at position 4, thereby inhibiting the formation of the angular naphthopyran.

A exposição de camundongos neonatos ao poluente 1,2-naftoquinona (1,2-NQ) induziu maior suscetibilidade dos machos jovens (mas não fêmeas) à asma. O objetivo deste estudo foi investigar o impacto da 1,2-NQ no pulmão de camundongos jovens machos e fêmeas e averiguar as associações entre os receptores de potencial transitório TRPA1 e a 1,2-NQ. Camundongos machos e fêmeas C57Bl/6 (2-5 g) foram expostos a 1,2-NQ (100 nM). Maior atividade da catalase e expressão (RNAm) do Nrf2 foi observado no pulmão das fêmeas 24 h após a exposição da 1,2-NQ. O estímulo alérgico na puberdade aumentou a atividade da glutationa peroxidase, redutase e S-transferase nas fêmeas que, diferentemente dos machos, não exibiram exacerbação da asma, mas mostraram maior nitração e carbonilação proteica, expressão da eNOS e TRPA1. O antagonismo dos TRPA1 reduziu a eosinofilia pulmonar nos machos e inibiu a [Ca2+]i em cultura de neurônios frente a 1,2-NQ. A ausência susceptibilidade em fêmeas se deve a maior defesa antioxidante e a maturidade pulmonar destas. / The mice exposure to 1,2-naphthoquinone (1,2-NQ), during postnatal period induced increased susceptibility of males (but not females) to asthma. The aim of this study was to investigate the intensity of lung damage to impact of 1,2-NQ on young mice of both sexes, and to investigate the associations between TRPA1 and 1,2-NQ. Male and female C57Bl/6 mice (2-5g) were exposed to 1,2-NQ (100 nM). After 24 h postnatal exposure to 1,2-NQ, only female lungs showed increased catalase activity and Nrf2 mRNA expression. The allergic stimuli at puberty led to increased glutathione peroxidase, reductase and S-transferase activities only in female lung, which, unlike male, did not exhibit exacerbation of asma, but showed increased pulmonary nitration and protein carbonylation, and increased mRNA expression of eNOS and TRPA1. The TRPA1 antagonist reduced eosinophilia in male lung and inhibited the increased [Ca2+]i in dorsal root ganglion neurons culture to 1,2-NQ. The lack of susceptibility in female might be linked to increased antioxidant defenses and the pulmonary maturity.

The formation of green trihydroxy benzacridine (TBA) derivatives when chlorogenic acid (CGA) quinones and amino acids react can be visually unappealing in some applications where CGA containing ingredients are used. Cysteine was studied as an amino acid anti-greening strategy, because cysteine-CGA conjugates are colorless. Buffered CGA: lysine: cysteine solutions at pH 8.0 and 9.0 were prepared and incubated for a maximum of 48 h at ambient temperature. Color intensity was periodically monitored using a UV-Vis spectrophotometer. Quantification and identification of conjugate formation were conducted by HPLC and LC-MS, while Antioxidant capacity was assessed by Trolox Equivalent Antioxidant Capacity and Folin-Ciocalteu reagent reducing capacity assays. More intense greening was detected at higher pH. Lysyl amine- CGA conjugates were identified as the predominant precursor of green TBA. Concentration-dependent cysteine inhibition of CGA-lysine greening was primarily by redox diphenol regeneration when pH was below cysteinyl thiol pKa 8.3 while primarily by forming cysteinyl-CGA conjugates when pH was above 8.3. Visible greening was fully inhibited with a cysteine: lysine 1:1 molar ratio in pH 9 CGA: Lys: Cys solutions, indicating that cysteinyl thiol was a stronger nucleophile than ε-lysyl amine to react with CGA o-quinones. Mono- and di-cysteine-CGA conjugates contributed to antioxidant capacity. Cysteine concentration, pH and incubation time all significantly affected color intensities and antioxidant capacity (p

Le photosystème II (PSII) est un complexe multi-protéique qui utilise l'énergie solaire pour oxyder l'eau et réduire des quinones. Le site catalytique d'oxydation de l'eau est localisé coté lumen du complexe, alors, que le site de réduction comprenant deux quinones (QA et QB) et un fer non-hémique est localisé sur le coté stromal du complexe membranaire. Dans cette thèse j'ai étudié les deux cotés accepteur et donneur d'électrons du PSII.QA*- et QB*- sont couplés magnétiquement au fer non-hémique donnant de faibles signaux RPE. Le fer non-hémique possède quatre ligands histidines et un ligand (bi)carbonate échangeable. Le formate peut échanger le ligand (bi)carbonate induisant un ralentissement dans le transfert d'électrons. Ici, je décris une modification du signal RPE de QB*- Fe2+ lorsque le formate est substitué au (bi)carbonate. J'ai aussi découvert un second signal RPE dû à la présence du formate à la place du (bi)carbonate lorsque QB est doublement réduit. De plus, j'ai trouvé que les signaux RPE natifs de QA*- Fe2+ et QB*- Fe2+ possèdent une signature intense encore jamais détectée. Tous les signaux RPE rapportés dans cette thèse devraient faciliter le titrage redox de QB par RPE. J'ai aussi observé que QB*- peut oxyder le fer non-hémique à l'obscurité en anaérobie. Cette observation implique qu'au moins dans une fraction des centres, le couple QB*-/QBH2 possède un potentiel redox plus haut que supposé. La quantification du nombre de centres où cette oxydation du fer se produit par le couple QB*-/QBH2 reste à faire. La réduction du PSII par le dithionite génère un signal modifié de QA*-Fe2+, un changement structural du PSII observé par électrophorèse. Cela peut indiquer la réduction d'un pont disulfure à l'intérieur du PSII. Concernant le site d'oxydation de l'eau, j'ai étudié la première étape de l'assemblage du site catalytique (Mn4Ca), en suivant l'oxydation du Mn2+ par RPE en bande X et haut champ. J'ai mis au point des conditions expérimentales permettant le piégeage du premier intermédiaire et j'ai aussi trouvé une incohérence avec des travaux publiés dans la littérature. J'ai aussi trouvé que le dithionite pouvait réduire le site catalytique Mn4Ca, en formant des états sur-réduits qui peuvent correspondre aux intermédiaires de l'assemblage du cluster Mn4Ca.