Spectroscopic and kinetic studies of mononuclear molybdenum enzymes of the DMSO reductase family

Spectroscopic and kinetic studies of arsenite oxidase and DMSO reductase, both members of the DMSO reductase family of mononuclear molybdenum enzymes, have been undertaken to provide a better understanding of how the physical and electronic structure of the molybdenum center relates to the oxygen atom transfer chemistry catalyzed by each enzyme.; Protein film voltammetry of arsenite oxidase shows that the molybdenum center displays highly concerted two-electron oxidation reduction activity with a reduction potential of +254 mV vs. SHE and spectrophotometric reductive titrations of arsenite oxidase have revealed the heretofore unresolved reduction potentials of the [3Fe-4S] and [2Fe-2S] Rieske-type iron sulfur centers of the enzyme.; Stopped-flow kinetic studies of the reaction of oxidized R. sphaeroides DMSO reductase with the general reductant sodium dithionite is biphasic, with an intermediate species formed that is determined to be that of the catalytically relevant MoV enzyme form. UV-visible spectral deconvolution of enzyme-monitored turnover reactions using DMSO reductase with the oxidizing substrates (DMSO or TMAO) and the reductant sodium dithionite clearly reveals the mechanistic differences of catalytic oxygen atom abstraction between them where a stable Ered-substrate complex is only observed for reaction with DMSO.; The pH-dependence of UV-visible spectra for DMSO reductase intermediates was also examined and only the spectrum of reduced enzyme is found to be particularly sensitive to pH. The spectrum of the high pH reduced enzyme implies dissociation of the Q pterin in reduced enzyme and further studies suggest that this Q pterin dissociation may stabilize an Eox•DMS intermediate which accumulates to an increasing degree as pH increases and is responsible for the observed pH-dependence of steady-state enzyme activity. Spectroscopic and kinetic analysis of the W116F mutant shows that, in contrast to previous studies, the functional form of the enzyme has a bisdithiolene molybdenum center. Resonance Raman has also been employed to further characterize W116F DMSO reductase forms and shows that both oxidized and reduced species are vibrationally analogous to those of the wild-type enzyme. This provides further evidence of full bis-dithiolene coordination to the molybdenum in the mutant active center.