The first non-turnover voltammetric response from a molybdenum enzyme: direct electrochemistry of dimethylsulfoxide reductase from Rhodobacter capsulatus

Abstract

The first direct voltammetric response from a molybdenum enzyme under non-turnover conditions is reported. Cyclic voltammetry of dimethylsulfoxide reductase from Rhodobacter capsulatus reveals a reversible Mo(VI/V) response at +161 mV followed by a reversible Mo(V/IV) response at -102 mV versus NHE at pH 8. The higher potential couple exhibits a pH dependence consistent with protonation upon reduction to the Mo(V) state and we have determined the p K(a) for this semi-reduced species to be 9.0. The lower potential couple is pH independent within the range 5<pH<10. The optical spectrum of the Mo chromophore has been investigated with spectroelectrochemistry. At high potential, in its resting state, the enzyme exhibits a spectrum characteristic of the Mo(VI) form. This changes significantly following bulk electrolysis (-400 mV versus NHE) at an optically transparent, indium-doped tin oxide working electrode, where a single visible electronic maximum at 632 nm is observed, which is comparable with spectra reported previously for the dithionite-reduced enzyme. This two-electron process is chemically reversible by reoxidizing the enzyme at the electrode in the absence of mediators or promoters. The activity of the enzyme has been established by observation of a catalytic current in the presence of DMSO at pH 8, where a sigmoidal (steady state) voltammogram is seen.

Mechanistic Studies of Rhodobacter sphaeroides Me2SO Reductase

“…At pH 6.0 and 5°C, rates were estimated to be 1 s Ϫ1 for the Mo(VI)/ Mo(V) reduction and 0.2 s Ϫ1 for the Mo(V)/Mo(IV) reduction, although the k app determined for the second couple slightly overestimates the effective rate of Mo(V) reduction. These results are consistent with the reported reduction potentials reported by Aguey-Zinsou et al (28) for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples at pH 6 (ϩ260 and Ϫ130 mV versus NHE, respectively), offering a thermodynamic basis for the slower rate observed for Mo(V) reduction. The reaction of TMAO with the now fully reduced Me 2 SO reductase is quite facile with extrapolated, limiting rate constants at high [TMAO] range from 134.5 to 2300 s Ϫ1 (29,30), significantly faster than either reductive step.…”

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…258 This occurred for a bacterial reaction center, 287 photosystems I 288 and II, 289 bovine milk xanthine oxidase, 290 Rhodobacter capsulatus xanthine dehydrogenase 183 and DMSO reductase, [291][292][293] For adsorbed enzymes, a preliminary diagnosis of health can come from noncatalytic electrochemical studies themselves, since the conformity of the peaks to ideal predictions (section 2.1) reports on the homogeneity of reduction potentials, and the values of the measured reduction potentials can be compared to those measured under potentiometric, equilibrium titrations. Ideally, both values should agree within a few millivolts, noting that the accuracy of redox titrations is rarely better than (10 mV.…”

Enzyme Electrochemistry — Biocatalysis on an Electrode

“…In fact this simplicity enable clear resolution, for the first time of non-turnover Mo VI/V and Mo V/IV signals from a molybdenum enzyme active site (in the absence of substrate). [200] Optical spectroelectrochemistry was also employed to observe the enzyme in its fully oxidized and reduced forms. Upon addition of DMSO, a catalytic wave was seen.…”