Reaction of the Desulfoferrodoxin from Desulfoarculus baarsii with Superoxide Anion

Abstract

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 ...

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

“…These results show that, in presence of a steady-state concentration of the physiological substrate of SOR, neelaredoxin was able to catalyze the reoxidation of dithionite-reduced rubredoxin, in good agreement with similar experiments performed with P. furiosus SOR [7]. When bovine SOD was added to the system, the rate of rubredoxin reoxidation remained unchanged, because SOD is unable to receive electrons from any component of the reaction system at rates able to outcompete with the catalytic reduction of superoxide (10 9 M À1 s À1 ) [6,16,17,18]. However, when SOD was added in place of SOR, reoxidation of rubredoxin was totally inhibited, which confirmed the superoxidemediated character of the observed electron transfer reaction.…”

“…However, analysis of the full genome indicates that Tp lacks the classic antioxidant defense enzymes such as superoxide dismutase (SOD), catalase or peroxidases [3]. It was only recently reported that Tp presents an alternative way to survive upon transient exposure to oxygen and a reactive oxygen species (ROS) in the environment, by expressing the gene for a superoxide reductase (TP0823) [4,5,6]. Superoxide reductases (SORs), a new class of non-heme iron enzymes initially found in sulfate-reducing bacteria, play a fundamental role in the defense of microorganisms against oxidative stress, by catalyzing the monovalent reduction of the superoxide anion, rather than the dismutation, according to Eq.…”

“…Superoxide reductases (SORs), a new class of non-heme iron enzymes initially found in sulfate-reducing bacteria, play a fundamental role in the defense of microorganisms against oxidative stress, by catalyzing the monovalent reduction of the superoxide anion, rather than the dismutation, according to Eq. 1 [4,5,6,7,8,9,10,11,12]:…”

“…Only the reduced colorless ferrous form of the active site iron is able to react with superoxide, with a virtually diffusion controlled rate of 10 9 M À1 s À1 , leading to the formation of the blue ferric state of the enzyme, which suggests the existence of an electron donor to regenerate the ferrous active form and complete the catalytic cycle of the enzyme [6,16,17,18]. The in vivo electron donor to SOR has generally not been established but previous work has strongly suggested that a rubredoxin (Rd) could be the proximal electron donor to superoxide reductases during periods of oxidative stress [7,8,19,20,21].…”

Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

“…10 The reaction mechanism of SOR, involving transfer of an electron and two protons to superoxide to form hydrogen peroxide, has been studied in detail using kinetic and spectroscopic techniques. 12,13,14,15,16,17,18 Using kinetic data, Nivière et al and Kurtz et al have proposed the presence of at least one Fe III intermediate (henceforth referred to as intermediate I, characterized by a CT band at 600 nm) in the reaction. The formation of the intermediate was diffusion controlled and had no pH dependence or deuterium isotope effect and thus was proposed to be an Fe III -μ 2 -O 2 2− species.…”