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 (DMSO R) with dimethyl sulfoxide (DMSO) and with dimethyl sulfide (DMS) as substr ates are described. Maximum activity was observed at pH 6.5 and below and a t 8.3, respectively. Rapid-scan stopped-flow spectrophotometry has been use d 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. Mel. Biol. 275, 613-623], and its subsequent reoxi dation by DMSO. Both these two-electron reactions were faster than enzyme t urnover under steady-state conditions, indicating that one-electron reactio ns 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 t han in DMSO reduction. Reduction of DMSOR by DMS was incomplete below simil ar to 1 mM DMS but complete at higher concentrations, implying that the enz yme'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 incomp lete, regardless of the DMSO concentration. Evidence for the existence of a spectroscopically indistinguishable reduced state, which could not be reox idized by DMSO, was obtained. Brief reaction (less than similar to 15 min) of DMS with DMSOR was fully reversible on removal of the DMS. However, in t he presence of excess DMS, a further slow reaction occurred aerobically, bu t not anaerobically, to yield a stable enzyme form having a lambda(max) 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 r eoxidation with DMSO. We suggest that in this enzyme form two of the dithio lene ligands of the molybdenum have dissociated and formed a disulfide. The implications of this new species are discussed in relation both to conflic ting published information for DMSOR from X-ray crystallography and to prev ious spectroscopic data for its reduced forms.