Control of the Evolution of Iron Peroxide Intermediate in Superoxide Reductase from Desulfoarculus baarsii. Involvement of Lysine 48 in Protonation

Florence Bonnot (1) , Thibaut Molle (1) , Stéphane Ménage (1) , Yohann Moreau (1) , Simon Duval (1) , Vincent Favaudon (2) , Chantal Houée-Levin (3) , Vincent Nivière (1)

1 LCBM - UMR 5249 - Laboratoire de Chimie et Biologie des Métaux
2 Génotoxicologie, signalisation et radiothérapie expérimentale
3 LCPO - Laboratoire de Chimie Physique D'Orsay

Superoxide reductase is a nonheme iron metalloenzyme that detoxifies superoxide anion radicals O(2)(•-) in some microorganisms. Its catalytic mechanism was previously proposed to involve a single ferric iron (hydro)peroxo intermediate, which is protonated to form the reaction product H(2)O(2). Here, we show by pulse radiolysis that the mutation of the well-conserved lysine 48 into isoleucine in the SOR from Desulfoarculus baarsii dramatically affects its reaction with O(2)(•-). Although the first reaction intermediate and its decay are not affected by the mutation, H(2)O(2) is no longer the reaction product. In addition, in contrast to the wild-type SOR, the lysine mutant catalyzes a two-electron oxidation of an olefin into epoxide in the presence of H(2)O(2), suggesting the formation of iron-oxo intermediate species in this mutant. In agreement with the recent X-ray structures of the peroxide intermediates trapped in a SOR crystal, these data support the involvement of lysine 48 in the specific protonation of the proximal oxygen of the peroxide intermediate to generate H(2)O(2), thus avoiding formation of iron-oxo species, as is observed in cytochrome P450. In addition, we proposed that the first reaction intermediate observed by pulse radiolysis is a ferrous-iron superoxo species, in agreement with TD-DFT calculations of the absorption spectrum of this intermediate. A new reaction scheme for the catalytical mechanism of SOR with O(2)(•-) is presented in which ferrous iron-superoxo and ferric hydroperoxide species are reaction intermediates, and the lysine 48 plays a key role in the control of the evolution of iron peroxide intermediate to form H(2)O(2).

Domaines: Biochimie [q-bio.BM]; Chimie

Florence Bonnot, Thibaut Molle, Stéphane Ménage, Yohann Moreau, Simon Duval, et al.. Control of the Evolution of Iron Peroxide Intermediate in Superoxide Reductase from Desulfoarculus baarsii. Involvement of Lysine 48 in Protonation. Journal of the American Chemical Society, 2012, 134 (11), pp.5120-30. ⟨10.1021/ja209297n⟩. ⟨hal-01075283⟩