Kinetics and mechanism of superoxide reduction by two-iron superoxide reductase from Desulfovibrio vulgaris.

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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.