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

• Published in: Biochemistry (Easton) Vol. 46; no. 40; pp. 11342 - 11351
• Main Authors: Huang, V W, Emerson, J P, Kurtz, DM Jr
• Format: Journal Article
• Language: English
• Published: 09.10.2007
• Subjects: Desulfovibrio vulgaris
• ISSN: 0006-2960
• 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 ( similar to 30 s super(-1) at 2 degree 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 sub(a) values of HX (X super(-) = F super(-), HCO super(-) sub(2), N super(-) sub(3)). 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.