Reaction of Desulfovibrio vulgaris Two-Iron Superoxide Reductase with Superoxide: Insights from Stopped-Flow Spectrophotometry
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- 作者：Victor W. Huang ; Joseph P. Emerson ; Donald M. Kurtz ; Jr.
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：October 9, 2007
- 年：2007
- 卷：46
- 期：40
- 页码：11342 - 11351
- 全文大小：1188K
- 年卷期：v.46,no.40(October 9, 2007)
- ISSN：1520-4995
文摘
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 absorptionspectrum is identical to that of a putative ferric-hydroperoxo intermediate previously observed by pulseradiolysis. The dead time intermediate is shown to be a product of reaction with superoxide and to begenerated at a much higher proportion of active sites than by pulse radiolysis. This intermediate decayssmoothly 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 ofdead time intermediate decay and that neither of the conserved pocket residues, Glu47 or Lys48, functionsas a rate-determining proton donor between pH 6 and pH 8. Fluoride, formate, azide, and phosphateaccelerate 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 isobserved for the azide-accelerated decay, and the decay rate constants are proportional to the concentrationsand pKa values of HX (X- = F-, HCO2-, N3-). These data indicate that the protonated forms of theanions function analogously to solvent as general acids in the rate-determining step. The results supportthe notion that the ferrous SOR site reacts with superoxide by an inner sphere process, leading directlyto the ferric-hydroperoxo intermediate, and demonstrate that the decay of this intermediate is subject toboth specific- and general-acid catalysis.