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Purification et caractérisation spectroscopique de cytochrome c oxydases.

Abstract : Cytochrome c Oxidase (CcO) membrane protein complexes catalyse the reduction of oxygen that takes place in the respiratory chains of eukaryotes and aerobic bacteria. During this reaction, that requires four protons and four electrons, four additional protons are pumped across the membrane and these participate in the formation of the proton motive force that is required for ATP synthesis. The active site of CcO aa3 contains a high-spin heme, heme a3, and a copper atom, CuB. These cofactors can bind, apart from O2, other diatomic molecules involved in signalling such as NO or CO. My thesis work concerns both CcO-ligand interactions and internal electron transfer (ET), in mitochondrial CcO and in bacterial oxidases aa3 and ba3. These oxidases accommodate four redox centres, heme a3 and CuB as well as heme a (resp. b for ba3) and CuA, involved in ET from the donor, Cytochrome c to the acceptor, the oxygen bound to the active site. The reversible inhibition of CcO by NO is involved in the regulation of respiration. By studying the influence of the NO concentration on the dynamics of NO in the oxidases aa3 from P. denitrificans and ba3 from T. thermophilus, we have demonstrated that multiple ligand interact in the active site. For aa3, geminate recombination of NO, after its photo dissociation from heme a3, occurs in two phases of 200 ps and 20 ns. The amplitude of this phase increases with suprastoichiometric NO concentrations. In contrast, no effect of the NO concentration is observed for Oxidase ba3, where the NO-reductase activity prevents the stable co presence of two NO molecules. Altogether, these results imply that a second NO molecule can be accommodated in/near the active site of CcO aa3 favouring geminate recombination of the first NO molecule, rather than its motion out of the protein. In order to determine the nature of this second NO-binding site, EPR experiments were performed. A spectral change as a function of the NO concentration was only observed for aa3 Oxidase. At low concentrations (CcO: NO <1:1), the signal, very similar to that of ba3, is specific for an NO molecule bound to a histamine coordinated heme. However, at higher concentrations, the spectrum resembles that of a five coordinated nitrosylated heme. As the visible spectrum does not indicate a rupture of the heme-NεHis bond, we propose that the second NO molecule induces a rotation of the heme-bound NO from an orientation parallel to the histamine ring to a more perpendicular one. Such a rotation would disrupt the paramagnetic interaction between the histamine ring and NO. Our interpretation is strengthened by molecular modelling studies of the active site of CcO aa3, with one and two NO molecules bound. These studies indicated that the presence of a second NO molecule, bound to CuB, induces a ~70° rotation of the heme-bound NO. In addition, at high NO concentrations, a signal characteristic of an NO-metal interaction appear that can be attributed to CuB. The assessment of the simultaneous presence of two NO molecules in the active site of CcO aa3, even at low NO concentrations, has implications for understanding the mechanism of CcO inhibition by NO. In order to study the influence of the active site environment modifications on NO dynamics, the amino acid V279 has been substituted. Preliminary studies of strains expressing the mutants indicate modification of their O2 reductase activities. Along a different line, we investigated the reduction kinetics of the active site by heme a. As this reaction occurs too fast to be observed by electron injection into the system, we studied reverse electron transfer. The experiments were performed on mammalian CcO aa3 with heme an oxidised, heme a3 reduced and CO-bound. After the cleavage of the CO-Fe bond by a light pulse, the electrons can equilibrate between the two hemes which have close-lying redox potentials. Spectroscopic ally, we measured that 13 ± 3 % of this transfer takes place in 1.2 ns; this time corresponds to the intrinsic transfer. The fastest rate previously measured for interheme ET in CcO was 3 µs. In the light of our results, this phase can be explained by a modification of the heme a3 redox potential due to CO leaving CuB. The very fast electron exchange determined between the two hemes may help to increase the oxygen trapping efficiency under physiological conditions ([O2] low and weak efflux), by decreasing the duration of the periods where heme a3 is not reduced.
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Eric Pilet. Purification et caractérisation spectroscopique de cytochrome c oxydases.. Sciences du Vivant [q-bio]. Ecole Polytechnique X, 2004. Français. ⟨pastel-00002298⟩

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