Reaction of Desulfovibrio vulgaris Two-Iron Superoxide Reductase with Superoxide:  Insights from Stopped-Flow Spectrophotometry

• Published in: Biochemistry (Easton) Vol. 46; no. 40; pp. 11342 - 11351
• Main Authors: Huang, Victor W, Emerson, Joseph P, Kurtz, Donald M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 09.10.2007
• Subjects: Desulfovibrio vulgaris - enzymology; Iron - chemistry; Iron - metabolism; Kinetics; Models, Chemical; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Spectrophotometry - methods; Superoxides - chemistry; Superoxides - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi700450u

Stopped-flow mixing of the Desulfovibrio vulgaris two-iron superoxide reductase (2Fe-SOR) containing the ferrous active site with superoxide generates a dead time intermediate whose absorption spectrum is identical to that of a putative ferric−hydroperoxo intermediate previously observed by pulse radiolysis. The dead time intermediate is shown to be a product of reaction with superoxide and to be generated at a much higher proportion of active sites than by pulse radiolysis. This intermediate decays smoothly to the resting ferric active site (∼30 s-1 at 2 °C and pH 7) with no other detectable intermediates. Deuterium isotope effects demonstrate that solvent proton donation occurs in the rate-determining step of dead time intermediate decay and that neither of the conserved pocket residues, Glu47 or Lys48, functions as a rate-determining proton donor between pH 6 and pH 8. Fluoride, formate, azide, and phosphate accelerate decay of the dead time intermediate and for azide or fluoride lead directly to ferric−azido or −fluoro complexes of the active site, which inhibit Glu47 ligation. A solvent deuterium isotope effect is observed for the azide-accelerated decay, and the decay rate constants are proportional to the concentrations and pK a values of HX (X- = F-, HCO2 -, N3 -). These data indicate that the protonated forms of the anions function analogously to solvent as general acids in the rate-determining step. The results support the notion that the ferrous SOR site reacts with superoxide by an inner sphere process, leading directly to the ferric−hydroperoxo intermediate, and demonstrate that the decay of this intermediate is subject to both specific- and general-acid catalysis.