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Florence Bonnot

Superoxide reductase: Mechanism of electron transfer to the active site and the role of lysine 48 in catalysis

Published on 25 November 2009

Thesis presented November 25, 2009

Superoxide reductase (SOR) is a metalloprotein which catalyzes the reduction of the radical superoxide into hydrogen peroxide. Its active site is constituted by a mononuclear iron center pentacoordinated [FeHisN4CysS1]. For its catalytic activity, SOR requires physiological partners as electron donor (cellular reductases). SORs of Class 1, such as that of Desulfoarculus baarsii, possess besides the active site, an additional iron center, rubredoxin [Fe(SCys)4] type, which does not react with the superoxide and with an unknown function. We showed that this rubredoxin center plays a role of electronic relay between reductases and the active site of the SOR. However, we show that this electron transfer between the rubredoxin center and the active site is not intramolecular but intermolecular, between two molecules of SOR. We propose that the presence of this iron center allows the SOR to adapt to various cellular reductases in order to optimize its detoxification activity.
The catalytic mechanism of the SOR had been studied by pulsed radiolysis. We show that the previous studies on the SOR from D. baarsii were perturbed by a photochemical phenomenon, resulting from a particular property of one of the key reaction intermediates of the catalytic cycle. The reaction mechanism has been reinvestigated in the absence of this photochemical effect and allowed us to propose a new reaction mechanism of reduction of the superoxide by the SOR of D. baarsii.
Our studies on the SOR mutant K48I of D. baarsii allowed us to bring to light the essential role of that lysine 48 in the protonation of the reaction intermediate Fe3+-hydroperoxyde. In absence of this lysine, we observed a modification of the reactivity of this reaction intermediate, which does not drive any more to the formation of the product of the reaction H2O2. We showed that the species Fe3+-hydroperoxide formed within this mutant is then capable to carry out specific reactions of oxidation, such the transformation of thioanisole methyl phenyl sulfoxide.
Our data strongly suggest that in absence of this lysine, the intermediate Fe3+-hydroperoxide evolves towards a high degree oxidation species, iron-oxo type, strongly oxidizing, responsible for these oxidations. So, the lysine 48 appears as an essential residue, which allows the SOR to direct the evolution of the intermediary Fe3+-hydroperoxide to the production of H2O2, rather than to the formation of high-valent iron-oxo species, as observed in the cytochrome P450 oxygenases​.

Superoxide, oxidative stress, pulse radiolysis, hydrogen peroxide, anaerobiosis, superoxide reducatase, Desulfoarculus baarsii, iron hydroperoxide

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