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

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• 作者：Joseph P. Emerson ; Eric D. Coulter ; Diane E. Cabelli ; Robert S. Phillips ; and Donald M. Kurtz ; Jr.
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：April 2, 2002
• 年：2002
• 卷：41
• 期：13
• 页码：4348 - 4357
• 全文大小：201K
• 年卷期：v.41,no.13(April 2, 2002)
• ISSN：1520-4995

文摘

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 studieson recombinant two-iron SOR (2Fe-SOR) from Desulfovibrio vulgaris. The results support and elaborateon 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 ~600nm, 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 ofthe 600-nm intermediate is essentially pH-independent (pH 5-9.5), shows no D₂O solvent isotope effectat pH 7.7, and decreases with increasing ionic strength. These data indicate that formation of the intermediatedoes not involve a rate-determining protonation, and are consistent with interaction of the incomingsuperoxide anion with a positive charge at or near the ferrous [Fe(NHis)₄(SCys)] site. The rate constantfor decay of the 600-nm intermediate follows the pH-dependent rate law: k₂(obs) = k₂'[H⁺] + k₂' ' andshows a significant D₂O solvent isotope effect at pH 7.7. The values of k₂' and k₂' ' indicate that the600-nm intermediate decays via diffusion-controlled protonation at acidic pHs and a first-order processinvolving either water or a water-exchangeable proton on the protein at basic pHs. The formation anddecay rate constants for an E47A variant of 2Fe-SOR are not significantly perturbed from their wild-typevalues, indicating that the conserved glutamate carboxylate does not directly displace the (hydro)peroxoligand of the intermediate at basic pHs. The kinetics of a K48A variant are consistent with participationof the lysyl side chain in directing the superoxide toward the active site and in directing the protonationpathway of the ferric-(hydro)peroxo intermediate toward release of hydrogen peroxide.