Superoxide Reductase from Desulfoarculus baarsii:  Identification of Protonation Steps in the Enzymatic Mechanism

Abstract

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.

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.…”

“…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].…”

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

“…It displays a high redox potential of about + 300 mV (vs. NHE) at neutral pH and remains mainly in a reduced form in the presence of air [15][16][17][18]. 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.…”

“…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.…”