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 ...

Iron-peroxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges superoxide radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded in crystallo revealed iron-(hydro)peroxo intermediates with the (hydro)peroxo group bound end-on. The dynamic SOR active site promotes the formation of transient hydrogen bond networks, which presumably assist the cleavage of the iron-oxygen bond in order to release the reaction product, hydrogen peroxide.