SHP (Sphaeroides Heme Protein) is a monoheme cytochrome c of unknown function. In general, ligands cannot bind to ferric SHP, but some diatomic molecules, such as O2 or NO, can bind to ferrous SHP. The gene encoding SHP and genes encoding a diheme cytochrome c (DHC) and a b-type cytochrome (Cyt-b) are found in the same chromosome region in different species. In the case of Shewanella oneidensis MR-1, mRNA levels for SHP, DHC, and Cyt-b are up-regulated by nearly 10-fold when grown under anaerobic conditions using nitrate as the electron acceptor. Thus it is possible that the physiological role of SHP may be in nitrate metabolism. However, nitrate is too big to be a candidate substrate for SHP, and some nitrification steps need more than one electron transfer (SHP is a monoheme cytochrome). Therefore, we will focus on the nitrite reductase, nitric oxide reductase and nitric oxide dioxygenase activities of SHP. In this thesis it is shown that SHP can catalyse the reaction between oxygen and nitric oxide to give a nitrate ion as the final product. Thus a possible aerobic function for SHP as a nitric oxide dioxygenase is proposed. Aerobically, SHP is proposed to be a nitric oxide dioxygenase which utilizes the same mechanism as other NO dioxygenases, flavohemoglobin (HMP) and neuroglobin (Ngb). This mechanism is proposed to proceed via an oxy-ferrous complex (SHP2+-O2) which reacts with nitric oxide. A mechanism for the catalytic reaction with ferrous-NO complex is described. SHP2+-NO can be quickly converted back to ferrous SHP by reacting with superoxide liberated by SHP2+-O2 or from another source. In addition it is also found that Shewanella MR-1 wild type reveals a higher NO tolerance than the SHP knockout strain in aerobic conditions. The catalytic mechanism of NO dioxygenase is oxygen-dependent, but the SHP mRNA up-regulation in Shewanella oneidensis MR-1 grown with nitrate under anaerobic conditions indicates that SHP may also perform some anaerobic function and may possibly be involved in nitrate metabolism. This work found that SHP reveals anaerobic nitrite reductase activity. However, the catalytic efficiency of SHP is considerably lower than other nitrite reductases. This infers that although SHP can reduce nitrite in vitro, it is unlikely to function as a nitrite reductase in vivo. Ferrous SHP binds NO with a Kd of less than 1 μM, and does not auto-oxidise. Therefore, under anaerobic conditions SHP2+-NO must be processed by some other mechanism. In addition, biochemical results reveal that the SHP/DHC complex has NO reductase activity under anaerobic conditions. Unfortunately, this function was not proved in vivo. SHP was initially isolated from Rhodobacter sphaeroides and its structure was reported in 2000. Based upon this structure, SHP is clearly a class I cytochrome c with one axial histidine ligand to the heme iron. Unusually, however, it has an asparagine residue as the other axial heme ligand, and as such is unique among cytochromes c. For this reason it may be assumed that the asparagine plays a special role. This study reveals several potential reasons why SHP utilises asparagine as a heme ligand. Firstly, in the ferric form, asparagine 88 binds to the heme iron to prevent small molecules binding. Secondly, in the ferrous form it moves to allow oxygen to bind and form the oxy-ferrous complex, using hydrogen bonding for stability. Thirdly, using asparagine as a heme ligand creates a suitable redox potential for reduction by DHC, thus allowing NO dioxygenation.

La photosynthèse permet de convertir l’énergie solaire en énergie chimique. Ce processus met en jeu un grand nombre de protéines et complexes protéiques. Le premier complexe de la chaîne photosynthétique est le photosystème II où a lieu l’oxydation de l’eau. Le PSII est composé des protéines D1 et D2. Chez la cyanobactérie thermophile Thermosynechococcus elongatus, il y a trois gènes qui codent trois protéines D1 différentes. La première partie de la thèse décrit le développement d’outils protéomiques basés sur les gels d’électrophorèse 2D pour étudier le protéome des trois différents variants, qui expriment chacun une seule protéine pour D1. Peu de différences ont été trouvées. Toutefois, un seul des variants exprime Tll0287. La deuxième partie de la thèse décrit la caractérisation de Tll0287 avec différents techniques : spectroscopies d’absorption UV-visible ou de résonance Raman et spectro-électrochimie. Tll0287 a été identifié comme un cytochrome de type c, mais il présente beaucoup de caractéristiques inattendues. Les spectres d’absorption UV-visible et de résonance Raman de Tll0287 réduit montrent une dépendance vis-à-vis du pH. Deux formes d’hèmes sont présents dans chacun des états oxydé et réduit. Un changement du ligand cystéine a été observé quand l’hème est réduit. Les titrages redox présentent de multiples potentiels à pH 10 et pH 5. Tll0287 peut fixer une molécule de CO à pH 7,6. Ces caractéristiques suggèrent que Tll0287 pourrait être une protéine senseur. De plus, la structure cristallographique montre que Tll0287 n’a pas un repliement classique d’un cytochrome de type c mais celui d’une protéine senseur. Les mutants de délétion du gène tll0287 ont été construits et aideront à comprendre la fonction de ce nouveau cytochrome. La troisième partie décrit l’étude de PsbV2 : un autre cytochrome de type c. Afin d’obtenir en quantité suffisante la protéine pour permettre sa caractérisation, elle a été surexprimée dans un système homologue en utilisant le promoteur de l’enzyme de la rubisco. Le potentiel redox de PsbV2 a été déterminé, comme étant très bas, inférieur à -460 mV (vs SHE, pH 5). Le spectre d’absorption UV-visible de la forme réduite a été caractérisé. La structure cristallographique de PsbV2 a été résolue et a révélé une cystéine comme ligand axial et un repliement proche de celui de cytochromes connus de T.elongatus. Bien que Tll0287 et PsbV2 présentent une cystéine comme ligand axial, leurs structures et leurs propriétés physico-chimiques suggèrent que leurs fonctions sont bien différentes. Une contribution majeure de cette thèse est la caractérisation d’un nouveau senseur à hème de type c chez les cyanobactéries et le développement d’outils nécessaires pour son étude. / Photosynthesis converts solar energy into chemical energy. This process involves a large number of proteins and protein complexes. The first protein complex in the photosynthetic chain is Photosystem II within the oxidation of water takes place. PSII is composed of the D1 and D2 proteins. In the thermophile cyanobacterium Thermosynechococcus elongatus, three genes encoded three different D1 proteins. The first part of this thesis describes the development of proteomics tools based on 2D gel-electrophoresis to study the proteome of three different variants, each expressing a single different D1 protein. Very few differences were found. However, only one expressed the protein Tll0287. The second part of the thesis describes the characterization of Tll0287. It was characterized using different techniques: electronic absorption and Raman resonance spectroscopies and spectro-electrochemistry. Tll0287 has been previously identified as a c-type cytochrome, but it presents some unexpected characteristics. The UV-visible absorption and Raman resonance spectra of reduced Tll0287 show a pH dependence. The reduced and oxidized states each had two different forms of the heme. A switch of ligands from a cysteine to histidine was observed in the reduced state. Redox titration showed multiple midpoints at pH 10 and 5. Tll0287 was shown to fix a CO molecule at pH 7.6. These physical properties suggested that Tll0287 could be a sensor. The crystallographic studies reveal that Tll0287 does not have a classical c-type cytochrome fold and is similar to other known sensor proteins, strengthening the hypothesis that it is a sensor. Deletion mutants were constructed that will help to better understand the function of this new cytochrome. The third part describes a study of the PsbV2, another c-type cytochrome. In order to obtain sufficient quantities to carry out characterization of this protein, it was overexpressed in a homologous system using the promotor of the rubisco enzyme. The redox midpoint potential of PsbV2 was found to be very low, below -460 mV (vs SHE, pH 5). The UV-visible absorption spectrum of the reduced form was determined. The crystallographic structure of PsbV2 was solved and reveals an axial cysteine ligand. Although both Tll0287 and PsbV2 share this feature, their different structures and physico-chemical properties suggest that their functions are unlikely to be similar. A major contribution of this thesis is the characterization of a new c-type cytochrome sensor in cyanobacteria and the development of proteomic tools required to study it.