Molybdenum K-edge X-ray absorption and Mo(V) electron paramagentic resonance (EPR) spectroscopies have been used to probe the metal coordination in oxidized and reduced forms of both wild-type and a site-directed mutant of Rhodobacter sphaeroides dimethyl sulfoxide (DMSO) reductase. We confirm our earlier findings (George, G. N.; Hilton, J.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 1113−1117) that the molybdenum site of the oxidized Mo(VI) enzyme possesses one terminal oxygen ligand (MoO) at 1.68 Å, four thiolate ligands at 2.44 Å, and one oxygen at 1.92 Å and that the dithionite-reduced Mo(IV) enzyme possesses a desoxo species with three or four Mo−S at 2.33 Å and two different Mo−O ligands at 2.16 and 1.92 Å. Mo(V) EPR indicates the presence of one exchangeable oxygen ligand, most likely an Mo−OH, in the signal-giving species, probably originating from the MoO of the oxidized enzyme (E m8.5(IV/V) = +37 mV, E m8.5(V/VI) = +83 mV). The addition of dimethyl sulfide, in the reverse of the physiological reaction, reduces the enzyme. In this form, the enzyme contains a desoxo active site with four Mo−S at 2.36 Å and two different Mo−O ligands at 1.94 and 2.14 Å. Recombinant wild-type R. sphaeroides DMSO reductase expressed in Escherichia coli initially has a dioxo structure (two MoO at 1.72 Å and four Mo−S at 2.48 Å) but assumes the wild-type Mo(VI) structure after a cycle of reduction and reoxidation. The site-directed Ser147→Cys mutant possesses a monooxo active site in the oxidized state (MoO at 1.70 Å) with five sulfur ligands (at 2.40 Å), consistent with cysteine 147 coordination to Mo. The dithionite reduced form of the mutant possesses a desoxo site also with five Mo−S ligands (at 2.37 Å) and one Mo−O at 2.12 Å. The variant has substantially different Mo(V) EPR and electrochemistry (E m8.5(IV/V) = −43 mV, E m8.5(V/VI) = +106 mV). The active-site structure and catalytic mechanism of DMSO reductase are discussed in the light of these results.

During the past four years, a substantial amount of structural information has been accumulated on the molybdoenzyme dimethyl sulfoxide (DMSO) reductase from purple bacteria. This enzyme contains a mononuclear Mo coordinated by two molybdopterin guanine dinucleotides as its single cofactor. Crystallographic studies on the enzyme from Rhodobacter sphaeroides and Rhodobacter capsulatus revealed substantial differences in the Mo coordination environment in the oxidized Mo(VI) state, despite a close structural similarity in the overall fold of the protein. The crystal structure of DMSO reductase from R. sphaeroides identified a Mo environment with a mono-oxo ligation and an asymmetric coordination by the two molybdopterins, with three short and one very long Mo−S bond. In contrast, two independent crystallographic studies of the enzyme from R. capsulatus revealed two additional Mo coordination environments:  a pentacoordinated dioxo metal ligation sphere in which one molybdopterin is completely dissociated from the Mo and a heptacoordinated environment with symmetrical metal coordination by both molybdopterins and two oxo ligands. In all three structures the side chain of a serine was a ligand to the Mo. Adding to the controversy, EXAFS studies on the R. sphaeroides enzyme suggested a hexacoordinated active site geometry, whereas the same technique indicated seven ligands for the R. capsulatus enzyme. The 1.3 Å resolution crystal structure of oxidized DMSOR from R. sphaeroides presented here reveals plasticity at the active site. The Mo is discretely disordered and exists in a hexacoordinated and a pentacoordinated ligation sphere. The hexacoordinated model reconciles the existing differences in active site coordination of R. sphaeroides DMSO reductase as studied by crystallographic and EXAFS techniques. In addition, the pentacoordinated structure closely resembles one of the reported R. capsulatus crystal structures. In retrospect, the active site geometry in the previously reported 2.2 Å crystal structure of R. sphaeroides DMSO reductase appears to represent an average of the two conformations described here. Thus, structural flexibility at the active site appears to give rise to the observed differences in the Mo coordination environment.