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Mise en évidence, par spectroscopies de Résonance Paramagnétique Electronique et d'absorption électronique UV-visible, de la formation de radicaux Tryptophanyles et Tyrosyles par transfert d'électron intramoléculaire vers l'hème dans les catalases monofonctionnelles et peroxydases bifonctionnelles

Abstract : Catalases, peroxidases and catalase-peroxidases (KatGs) regulate the cellular concentration of hydrogen peroxide. We investigated the reactive intermediates of these enzymes by combining multifrequency (9-285 GHz) Electron Paramagnetic Resonance spectroscopy, UVvisible electronic absorption, deuterium labelling and site-directed mutagenesis. We identified tyrosyl and tryptophanyl radicals as alternative reactive intermediates to the [Fe(IV)=O Por•+]. The comparative study of eight catalases allowed us to demonstrate that temperature, pH and oxidant concentration favour the formation of the [Fe(IV)=O Tyr•] species in beef liver, human erythrocytes, B. abortus and M. lysodeikticus catalases. These findings would explain the different specific activities with H2O2 of these enzymes. If considering that the spontaneous electron transfer between tyrosine and the porphyrin would be in competition with the H2O2 disproportionation. KatGs are bifunctional enzymes, which disproportionate the H2O2 as efficiently as monofunctional catalases, due to specific structural features. Our EPR study showed that the reaction mechanism of KatGs is more complex than that of peroxidases and catalases. One tyrosyl and two tryptophanyle radicals were detected in B. pseudomallei KatG. Substrates tests performed with ABTS and isoniazid (front-line pro-drug used in the treatment of tuberculosis) revealed that these protein-based radicals could play the role of cofactors. Comparison of the protein-based intermediates formed by seven KatGs, showed that the tryptophan radical site is unique but not conserved. The localisation and the nature of the protein-based radicals may influence the reactivity of KatGs towards substrates.
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Submitted on : Wednesday, September 1, 2010 - 7:00:21 AM
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Julie Colin. Mise en évidence, par spectroscopies de Résonance Paramagnétique Electronique et d'absorption électronique UV-visible, de la formation de radicaux Tryptophanyles et Tyrosyles par transfert d'électron intramoléculaire vers l'hème dans les catalases monofonctionnelles et peroxydases bifonctionnelles. Sciences pharmaceutiques. Université Pierre et Marie Curie - Paris VI, 2008. Français. ⟨tel-00326339⟩

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