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.

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.

Superoxide reductase (SOR) is a small metalloenzyme that catalyzes reduction of O(2)(*)(-) to H(2)O(2) and thus provides an antioxidant mechanism against superoxide radicals. Its active site contains an unusual mononuclear ferrous center, which is very efficient during electron transfer to O(2)(*)(-) [Lombard, M., Fontecave, M., Touati, D., and Nivière, V. (2000) J. Biol. Chem. 275, 115-121]. The reaction of the enzyme from Desulfoarculus baarsii with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bimolecular reaction of superoxide reductase with superoxide, with a rate constant of (1.1 +/- 0.3) x 10(9) M(-1) s(-1). A first intermediate is formed which is converted to a second one at a much slower rate constant of 500 +/- 50 s(-1). Decay of the second intermediate occurs with a rate constant of 25 +/- 5 s(-1). These intermediates are suggested to be iron-superoxide and iron-peroxide species. Furthermore, the role of glutamate 47 and lysine 48, which are the closest charged residues to the vacant sixth iron coordination site, has been investigated by site-directed mutagenesis. Mutation of glutamate 47 into alanine has no effect on the rates of the reaction. On the contrary, mutation of lysine 48 into an isoleucine led to a 20-30-fold decrease of the rate constant of the bimolecular reaction, suggesting that lysine 48 plays an important role during guiding and binding of superoxide to the iron center II. In addition, we report that expression of the lysine 48 sor mutant gene hardly restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions.

The active site of superoxide reductase SOR consists of an Fe2+ center in an unusual [His4 Cys1] square-pyramidal geometry. It specifically reduces superoxide to produce H2O2. Here, we have reacted the SOR from Desulfoarculus baarsii directly with H2O2. We have found that its active site can transiently stabilize an Fe3+-peroxo species that we have spectroscopically characterized by resonance Raman. The mutation of the strictly conserved Glu47 into alanine results in a stabilization of this Fe3+-peroxo species, when compared to the wild-type form. These data support the hypothesis that the reaction of SOR proceeds through such a Fe3+-peroxo intermediate. This also suggests that Glu47 might serve to help H2O2 release during the reaction with superoxide.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPH or NADH. Overexpression in E. coli now allows the preparation of large amounts of pure protein. Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain. Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein. On the other hand, the presence of the 2 -OH of the ribityl chain stimulates the NADPH-dependent reaction significantly. Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin. Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E. coli growing cells. As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.Flavins are well known as key prosthetic groups of a large number of redox enzymes named flavoproteins. More recently, protein-free flavins, riboflavin, FMN, or FAD, were also suggested to play, as electron transfer mediators, important biological functions, for example during ferric iron reduction (1-3), activation of ribonucleotide reductase (4, 5), bioluminescence (6, 7), and oxygen activation (8) (Scheme 1).The reduction of free flavins by reduced pyridine nucleotides NADPH or NADH is not an efficient reaction. The kinetics is slow unless very high nonphysiological concentrations of both reactants are present in the reaction mixture (8). As a consequence, living organisms have evolved enzymes that catalyze the reduction of riboflavin, FMN, and FAD by NADPH and NADH and are called NAD(P)H:flavin oxidoreductases or flavin reductases. It is now well established that such enzymes are present in all microorganisms, including the luminous marine bacteria, and also in mammals (1). A recent study has shown that flavin reductase activities are present in erythrocytes and in various human tissues (liver, heart, kidney, and lung) (9).In most cases, a single living organism contains multiple flavin reductases different in enzymatic nature and molecular mass. The luminous bacteria, Vibrio harveyi, contains at least three types of FMN reductases (10 -14). In Escherichia coli at least two flavin reductases have been isolated. One, named Fre, is a 26.2-kDa enzyme using both NADH and NADPH as electron donors (4), whereas the other is the sulfite reductase, a 780-kDa enzyme using NADPH exclusively (15). Still very little is known on the structure and the mechanisms of flavin reductases. No three-dimensional structure of such an enzyme is available yet, and only recently...

Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-). SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 A resolution, respectively. The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge. The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays. The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site.

The redox-induced structural changes at the active site of the superoxide reductase (SOR) from Desulfoarculus baarsii and Treponema pallidum have been monitored by means of FTIR difference spectroscopy coupled to electrochemistry. With this technique, the structure and interactions formed by individual amino acids at a redox site can be detected. The infrared data on wild-type, Glu47Ala, and Lys48Ile mutants of the SOR from D. baarsii provide experimental support for the conclusion that the two different coordination motifs observed in the three-dimensional structure of the SOR from Pyrococcus furiosus [Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. (2000) Biochemistry 39, 2499-2508] correspond to the two redox forms of the SOR iron center. We extend this result to the center II iron of SOR of the desulfoferrodoxin type. Similar structural changes are also observed upon iron oxidation in the SOR of T. pallidum. In D. baarsii, the IR modes of the Glu47 side chain support that it provides a monodentate ligand to the oxidized iron, while it does not interact with Fe(2+). Structural changes at the level of peptide bond(s) observed upon iron oxidation in wild-type are suppressed in the Glu47Ala mutant. We propose that the presence of the Glu side chain plays an important role for the structural reorganization accompanying iron oxidation. We identified the infrared modes of the Lys48 side chain and found that a change in its environment occurs upon iron oxidation. The lack of other structural changes upon the Lys48Ile mutation shows that the catalytic role of Lys, as evidenced by pulse radiolysis experiments [Lombard, M., Houée-Levin, C., Touati, D., Fontecave, M., and Nivière, V. (2001) Biochemistry 40, 5032-5040], is purely electrostatic, guiding superoxide toward the reduced iron.