Kinetics and Mechanism of Superoxide Reduction by Two-Iron Superoxide Reductase from Desulfovibrio vulgaris

Abstract

Superoxide reductases (SORs) contain a novel square pyramidal ferrous [Fe(NHis)(4)(SCys)] site that rapidly reduces superoxide to hydrogen peroxide. Here we report extensive pulse radiolysis studies on recombinant two-iron SOR (2Fe-SOR) from Desulfovibrio vulgaris. The results support and elaborate on our originally proposed scheme for reaction of the [Fe(NHis)(4)(SCys)] site with superoxide [Coulter, E. D., Emerson, J. E., Kurtz, D. M., Jr., and Cabelli, D. E. (2000) J. Am. Chem. Soc. 122, 11555-11556]. This scheme consists of second-order diffusion-controlled formation of an intermediate absorbing at approximately 600 nm, formulated as a ferric-(hydro)peroxo species, and its decay to the carboxylate-ligated ferric [Fe(NHis)(4)(SCys)] site with loss of hydrogen peroxide. The second-order rate constant for formation of the 600-nm intermediate is essentially pH-independent (pH 5-9.5), shows no D(2)O solvent isotope effect at pH 7.7, and decreases with increasing ionic strength. These data indicate that formation of the intermediate does not involve a rate-determining protonation, and are consistent with interaction of the incoming superoxide anion with a positive charge at or near the ferrous [Fe(NHis)(4)(SCys)] site. The rate constant for decay of the 600-nm intermediate follows the pH-dependent rate law: k(2)(obs) = k(2)'[H(+)] + k(2)' ' and shows a significant D(2)O solvent isotope effect at pH 7.7. The values of k(2)' and k(2)' ' indicate that the 600-nm intermediate decays via diffusion-controlled protonation at acidic pHs and a first-order process involving either water or a water-exchangeable proton on the protein at basic pHs. The formation and decay rate constants for an E47A variant of 2Fe-SOR are not significantly perturbed from their wild-type values, indicating that the conserved glutamate carboxylate does not directly displace the (hydro)peroxo ligand of the intermediate at basic pHs. The kinetics of a K48A variant are consistent with participation of the lysyl side chain in directing the superoxide toward the active site and in directing the protonation pathway of the ferric-(hydro)peroxo intermediate toward release of hydrogen peroxide.

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…Indeed, Tp, usually classified as a microaerophile, is able to receive electrons from the different rubredoxin-like proteins, with high k app values varying from 156 to 213 min À1 , with the notable exception of Dv rubredoxin. As already described, superoxide reacts with the active site of the three SORs studied with the same rate of 10 9 M À1 s À1 [10,19,[28][29][30][31], and therefore our results could suggest that Tp needs a faster electron transfer system to be able to deal with the relatively high concentrations of oxygen encountered during the dissemination of the spirochete into the tissues, lowering the superoxide concentration to nonlethal levels. Inversely, aerotolerant organisms such as Dv and Dg are supposed to be exposed more briefly to oxygen and at much lower concentrations, as reflected in the lower k app values observed.…”

“…Under our experimental conditions, we can assume there to be a steady-state concentration of superoxide, saturating concentration of rubredoxin, and less than saturating concentrations of SOR, so the rate-limiting step of the process must be the reaction of SOR with rubredoxin, knowing that superoxide has been shown to react with the active site of each of the three SORs studied with the virtually diffusion controlled rate of 10 9 M À1 s À1 [10,19,[28][29][30][31].…”

“…Therefore, the presence of an electron donor is necessary to regenerate the ferrous active form and complete the catalytic cycle of the enzyme [10,19,[28][29][30][31]. Recently, several groups, including ours, have clearly established rubredoxin as the proximal electron donor to SORs in the case of Tp, Dv, P. furiosus, and A. fulgidus [4,[32][33][34][35][36].…”

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site

“…Center II has an open coordination site, which represents the site where superoxide binds and is reduced by the ferrous iron to H 2 O 2 (eq. 2) [16][17][18][19][20][21] Two main classes of SORs have been described, associated with the presence of an additional N-terminal domain, which chelates an additional mononuclear iron center. When present, this iron center, named center I, is chelated by four cysteine residues in a distorted tetrahedral similar to arrangement of rubredoxin, in a fold very similar to that found for the small electron transfer protein desulforedoxin [22].…”

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.…”