Reaction of Desulfovibrio vulgaris two-iron superoxide reductase with superoxide: insights from stopped-flow spectrophotometry.
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 ( approximately 30 s-1 at 2 degrees 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 pKa 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.
Huang
Victor W
VW
Department of Chemistry, University of Texas at San Antonio, San Antonio, Texas 78249, USA.
Emerson
Joseph P
JP
Kurtz
Donald M
DM
D016969:Desulfovibrio vulgaris / Q000201:enzymology*; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods*; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism*
D013481:Superoxides; D007501:Iron; D010088:Oxidoreductases; C121849:superoxide reductase