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J. Biol. Chem., Vol. 278, Issue 41, 39662-39668, October 10, 2003

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Kinetics of the Superoxide Reductase Catalytic Cycle^*,

Joseph P. Emerson, Eric D. Coulter, Robert S. Phillips and Donald M. Kurtz, Jr. 

From the Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602

The steady state kinetics of a Desulfovibrio (D.) vulgaris superoxide reductase (SOR) turnover cycle, in which superoxide is catalytically reduced to hydrogen peroxide at a [Fe(His)₄(Cys)] active site, are reported. A proximal electron donor, rubredoxin, was used to supply reducing equivalents from NADPH via ferredoxin: NADP⁺ oxidoreductase, and xanthine/xanthine oxidase was used to provide a calibrated flux of superoxide. SOR turnover in this system was well coupled, i.e. reduced:NADPH oxidized over a 10-fold range of superoxide flux. The reduction of the ferric SOR active site by reduced rubredoxin was independently measured to have a second-order rate constant of 1 x 10⁶ M^–1 s^–1. Analysis of the kinetics showed that: (i) 1 µM SOR can convert a 10 µM/min superoxide flux to a steady state superoxide concentration of 10^–10 M, during which SOR turns over about once every 6 s, (ii) the diffusion-controlled reaction of reduced SOR with superoxide is the slowest process during turnover, and (iii) neither ligation nor deligation of the active site carboxylate of SOR limits the turnover rate. An intracellular SOR concentration on the order of 10 µM is estimated to be the minimum required for lowering superoxide to sublethal levels in aerobically growing SOD knockout mutants of Escherichia coli. SORs from Desulfovibrio gigas and Treponema pallidum showed similar turnover rates when substituted for the D. vulgaris SOR, whereas superoxide dismutases showed no SOR activity in our assay. These results provide quantitative support for previous suggestions that, in times of oxidative stress, SORs efficiently divert intracellular reducing equivalents to superoxide.

Received for publication, June 18, 2003 , and in revised form, August 1, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table 1S, Figs. S1–S3, and Equations 1S–2.

To whom correspondence should be addressed. Tel.: 706-542-2016; Fax: 706-542-9454; E-mail: kurtz{at}chem.uga.edu.

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