Kinetics of the Superoxide Reductase Catalytic Cycle

• Published in: The Journal of biological chemistry 2003-10, Vol.278 (41), p.39662-39668
• Main Authors: Emerson, Joseph P., Coulter, Eric D., Phillips, Robert S., Kurtz, Donald M.
• Format: Article
• Language: English
• Subjects: Animals; Catalytic Domain; Desulfovibrio gigas; Desulfovibrio vulgaris; Desulfovibrio vulgaris - enzymology; Escherichia coli - enzymology; In Vitro Techniques; Kinetics; NADP - metabolism; Oxidation-Reduction; Oxidoreductases - metabolism; Recombinant Proteins - metabolism; Rubredoxins - metabolism; Sodium Azide - pharmacology; Sodium Cyanide - pharmacology; Superoxide Dismutase - metabolism; superoxide reductase; Superoxides - metabolism; Treponema pallidum; Treponema pallidum - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M306488200

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.∼202·¯ 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 × 106m–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 10–10m, 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.