Reactions of dimethylsulfoxide reductase from Rhodobacter capsulatus with dimethyl sulfide and with dimethyl sulfoxide: complexities revealed by conventional and stopped-flow spectrophotometry.
Improved assays for the molybdenum enzyme dimethylsulfoxide reductase (DMSOR) with dimethyl sulfoxide (DMSO) and with dimethyl sulfide (DMS) as substrates are described. Maximum activity was observed at pH 6.5 and below and at 8.3, respectively. Rapid-scan stopped-flow spectrophotometry has been used to investigate the reduction of the enzyme by DMS to a species previously characterized by its UV-visible spectrum [McAlpine, A. S., McEwan, A. G., and Bailey, S. (1998) J. Mol. Biol. 275, 613-623], and its subsequent reoxidation by DMSO. Both these two-electron reactions were faster than enzyme turnover under steady-state conditions, indicating that one-electron reactions with artificial dyes were rate-limiting. Second-order rate constants for the two-electron reduction and reoxidation reactions at pH 5.5 were (1.9 +/- 0.1) x 10(5) and (4.3 +/- 0.3) x 10(2) M-1 s-1, respectively, while at pH 8.0, the catalytic step was rate-limiting (62 s-1). Kinetically, for the two-electron reactions, the enzyme is more effective in DMS oxidation than in DMSO reduction. Reduction of DMSOR by DMS was incomplete below approximately 1 mM DMS but complete at higher concentrations, implying that the enzyme's redox potential is slightly higher than that of the DMS-DMSO couple. In contrast, reoxidation of the DMS-reduced state by DMSO was always incomplete, regardless of the DMSO concentration. Evidence for the existence of a spectroscopically indistinguishable reduced state, which could not be reoxidized by DMSO, was obtained. Brief reaction (less than approximately 15 min) of DMS with DMSOR was fully reversible on removal of the DMS. However, in the presence of excess DMS, a further slow reaction occurred aerobically, but not anaerobically, to yield a stable enzyme form having a lambdamax at 660 mn. This state (DMSORmod) retained full activity in steady-state assays with DMSO, but was inactive toward DMS. It could however be reconverted to the original resting state by reduction with methyl viologen radical and reoxidation with DMSO. We suggest that in this enzyme form two of the dithiolene ligands of the molybdenum have dissociated and formed a disulfide. The implications of this new species are discussed in relation both to conflicting published information for DMSOR from X-ray crystallography and to previous spectroscopic data for its reduced forms.
Adams
B
B
School of Biological Sciences, University of Sussex, Brighton, U.K.
Smith
A T
AT
Bailey
S
S
McEwan
A G
AG
Bray
R C
RC
D001594:Benzyl Viologen / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004121:Dimethyl Sulfoxide / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods; D013440:Sulfides / Q000737:chemistry*
D007506:Iron-Sulfur Proteins; D013440:Sulfides; D001594:Benzyl Viologen; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase; C004784:dimethyl sulfide; D010100:Oxygen; D004121:Dimethyl Sulfoxide