Desulfoferrodoxin is a small protein found in sulfatereducing bacteria that contains two independent mononuclear iron centers, one ferric and one ferrous. Expression of desulfoferrodoxin from Desulfoarculus baarsii has been reported to functionally complement a superoxide dismutase deficient Escherichia coli strain. To elucidate by which mechanism desulfoferrodoxin could substitute for superoxide dismutase in E. coli, we have purified the recombinant protein and studied its reactivity toward O 2 . . Desulfoferrodoxin exhibited only a weak superoxide dismutase activity (20 units mg ؊1 ) that could hardly account for its antioxidant properties. UVvisible and electron paramagnetic resonance spectroscopy studies revealed that the ferrous center of desulfoferrodoxin could specifically and efficiently reduce O 2 . , with a rate constant of 6 -7 ؋ 10 8 M ؊1 s ؊1. In addition, we showed that membrane and cytoplasmic E. coli protein extracts, using NADH and NADPH as electron donors, could reduce the O 2 . oxidized form of desulfoferrodoxin.Taken together, these results strongly suggest that desulfoferrodoxin behaves as a superoxide reductase enzyme and thus provide new insights into the biological mechanisms designed for protection from oxidative stresses.Desulfoferrodoxin (Dfx) 1 is a small, nonsulfur iron protein that has been isolated from several strains of anaerobic sulfatereducing bacteria (1, 2). Although no enzymatic activity could be associated to Dfx, the physicochemical properties of its iron centers have been well documented (1-3). Recently, the threedimensional structure of Dfx from Desulfovibrio desulfuricans has been solved at a resolution of 1.9 Å (4). Dfx is a homodimer with a molecular mass of 2 ϫ 14 kDa. The monomer is organized in two protein domains, each with a specific mononuclear iron center named center I or center II. Center I contains a mononuclear ferric iron coordinated by four cysteines in a distorted rubredoxin-type center. Center II has a ferrous iron with square pyramidal coordination to four nitrogens from histidines as equatorial ligands and one sulfur from a cysteine as the axial ligand (4). The midpoint redox potentials have been reported to be 2-4 mV for center I and 90 -240 mV for center II (2, 3). The high redox potential value for center II explains the stability of the ferrous ion in the presence of oxygen.Initially, the structural dfx gene was cloned and sequenced from Desulfovibrio vulgaris Hildenborough and was named rbo (5). rbo was found upstream of the rubredoxin gene, forming an operon. The encoded 14-kDa protein was tentatively named rubredoxin oxidoreductase (Rbo) because it was likely to function in oxidation-reduction with rubredoxin as a redox partner (5). Independently, a protein isolated from D. desulfuricans and D. vulgaris and named Dfx was found to be encoded by the rbo gene (1, 2, 6). However, up to now, Dfx did not show any evidence for a rubredoxin oxidoreductase activity, and its physiological role remains unclear. Consequently, the name of the corresponding ...

Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.