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The respiratory DMSO reductase from Rhodobacter capsulatus catalyzes the reduction of dimethyl sulfoxide to dimethyl sulfide. Herein, we have utilized this Mo enzyme as an enantioselective catalyst to generate optically pure sulfoxides (methyl p-tolyl sulfoxide, methyl phenyl sulfoxide and phenyl vinyl sulfoxide) from racemic starting materials. A hexaaminecobalt coordination compound in its divalent oxidation state was employed as the mediator of electron transfer between the working electrode and DMSO reductase to continually reactivate the enzyme after turnover. In all cases, chiral HPLC analysis of the reaction mixture revealed that the S-sulfoxide was reduced more rapidly leading to enrichment or isolation of the R isomer.

The bacterial molybdoenzyme dimethyl sulfoxide (DMSO) reductase from Rhodobacter capsulatus catalyzes the reduction of DMSO to dimethyl sulfide in anaerobic respiration. In its native state, DMSO reductase is reduced to its active state by a pentaheme cytochrome (DorC). Alternatively, we show that DMSO reductase catalysis may be driven electrochemically using a series of homologous coordination compounds as mediating synthetic electron donors. All mediators are macrocyclic hexaaminecobalt(II) complexes in their active form, differing principally in their redox potentials over a range of about 250 mV. Thus, each complex presents a different reductive driving force to DMSO reductase and this leads to pronounced differences in the electrocatalytic behavior as measured by cyclic voltammetry. Digital simulation of the experimental voltammetry enables the critical features of the catalytic cycle to be extracted.

Electrochemically driven catalysis of the bacterial enzyme dimethyl sulfoxide (DMSO) reductase (Rhodobacter capsulatus) has been studied using the macrocyclic complex (trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) as a mediator. In the presence of both DMSO and DMSO reductase, the normal transient Co(III/II) voltammetric response of the complex is transformed into an amplified and sigmoidal (steady-state) waveform characteristic of a catalytic EC' mechanism. At low concentrations of DMSO (approximately K (M)) or high mediator concentrations (more than the concentration of DMSO reductase), the steady-state character of the voltammetric response disappears and is replaced by more complicated waveforms that are a convolution of transient and steady-state behavior as different steps within the catalytic cycle become rate limiting. Through digital simulation of cyclic voltammetry performed under conditions where the sweep rate, DMSO concentration, DMSO reductase concentration and mediator concentration were varied systematically, we were able to model all voltammograms with a single set of rate and equilibrium constants which provide new insights into the kinetics of the DMSO reductase catalytic mechanism that have hitherto been inaccessible from steady state or stopped flow kinetic studies.

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo(VI) form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo(VI) form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo(VI) species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion.

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.

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

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum. Dimethyl sulphide dehydrogenase was shown to contain bis(molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdopterin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB, ddhD and ddhC. DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c2 mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

Regulation of the expression of dimethylsulfoxide (DMSO) reductase was investigated in the purple phototrophic bacterium Rhodobacter capsulatus. Under phototrophic, anaerobic conditions with malate as carbon source, DMSO caused an approximately 150-fold induction of DMSO reductase activity. The response regulator DorR was required for DMSO-dependent induction and also appeared to slightly repress DMSO reductase expression in the absence of substrate. Likewise, when pyruvate replaced malate as carbon source there was an induction of DMSO reductase activity in cells grown at low light intensity (16 W m(-2)) and again this induction was dependent on DorR. The level of DMSO reductase activity in aerobically grown cells was elevated when pyruvate replaced malate as carbon source. One possible explanation for this is that acetyl phosphate, produced from pyruvate, may activate expression of DMSO reductase by direct phosphorylation of DorR, leading to low levels of induction of dor gene expression in the absence of DMSO. A mutant lacking the global response regulator of photosynthesis gene expression, RegA, exhibited high levels of DMSO reductase in the absence of DMSO, when grown phototrophically with malate as carbon source. This suggests that phosphorylated RegA acts as a repressor of dor operon expression under these conditions. It has been proposed elsewhere that RegA-dependent expression is negatively regulated by the cytochrome cbb3 oxidase. A cco mutant lacking cytochrome cbb3 exhibited significantly higher levels of phi[dorA::lacZ] activity in the presence of DMSO compared to wild-type cells and this is consistent with the above model. Pyruvate restored DMSO reductase expression in the regA mutant to the same pattern as found in wild-type cells. These data suggest that R. capsulatus contains a regulator of DMSO respiration that is distinct from DorR and RegA, is activated in the presence of pyruvate, and acts as a negative regulator of DMSO reductase expression.

Expression of the dimethylsulfoxide respiratory (dor) operon of Rhodobacter is regulated by oxygen, light intensity and availability of substrate. Since dimethylsulfoxide reductase contains a pterin molybdenum cofactor, the role of molybdate in the regulation of dor operon expression was investigated. In this report we show that the molybdate-responsive transcriptional regulator, MopB, and molybdate are essential for maximal dimethylsulfoxide reductase activity and expression of a dorA::lacZ transcriptional fusion in Rhodobacter capsulatus. In contrast, mop genes are not required for the expression of the periplasmic nitrate reductase or xanthine dehydrogenase in R. capsulatus under conditions of molybdenum sufficiency. This is the first report demonstrating a clear functional difference between the ModE homologues MopB and MopA in this bacterium. The results suggest that MopA is primarily involved in the regulation of nitrogen fixation gene expression in response to molybdate while MopB has a role in nitrogen fixation and dimethylsulfoxide respiration.

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.

The DMSO reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor (Moco) and is located in the periplasm. DNA sequence analysis identified four genes involved in the biosynthesis of the Moco (moaA, moaD, moeB and moaC) immediately downstream of the dor (DMSO respiratory) gene cluster. Rhodobacter capsulatus MoaA was expressed in Escherichia coli as a His6-tagged protein. Although, the expressed protein formed inclusion bodies, EPR spectroscopy showed that MoaA contains a [3Fe-4S] cluster. A moaA mutant was constructed and its phenotype indicates that the Moco biosynthetic gene cluster downstream of the dor operon is specific for the biogenesis of DMSO reductase. Two forms of DMSO reductase were purified by immunoaffinity chromatography from the moaA mutant. A mature form of DMSO reductase was located in the periplasm and a precursor form was found in the cytoplasm.

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit Mr = 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (alpha-band), 522 nm (beta-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n = 1) with midpoint potentials of -34, -128, -184, -185, and -276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.

The enantioselective reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli, Proteus mirabilis and Proteus vulgaris was investigated. Purified dimethyl sulfoxide reductase from Rhodobacter capsulatus catalysed the selective removal of (S)-methyl p-tolyl sulfoxide from a racemic mixture of methyl p-tolyl sulfoxide and resulted in an 88% recovery of enantiomerically pure (R)-methyl p-tolyl sulfoxide. Rhodobacter capsulatus was shown to be able to grow photoheterotrophically in the presence of certain chiral sulfoxides under conditions where a sulfoxide is needed as an electron sink. Whole cells of Rhodobacter capsulatus were shown to catalyse the enantioselective reduction of methyl p-tolyl sulfoxide, ethyl 2-pyridyl sulfoxide, methylthiomethyl methyl sulfoxide and methoxymethyl phenyl sulfoxide. Similarly, whole cells of Escherichia coli, Proteus mirabilis and Proteus vulgaris reduced these sulfoxides but with opposite enantioselectivity.

The crystal structure of the molybdenum enzyme dimethylsulphoxide reductase (DMSOR) has been determined at 1.9 A resolution with substrate bound at the active site. DMSOR is an oxotransferase which catalyses the reduction of dimethylsulphoxide (DMSO) to dimethylsulphide (DMS) in a two stage reaction which is linked to oxygen atom transfer and electron transfer. In the first step, DMSO binds to reduced (Mo(IV)) enzyme, the enzyme is oxidised to Mo(VI) with an extra oxygen ligand and DMS is released. Regeneration of reduced enzyme is achieved by transfer of two electrons, successively from a specific cytochrome, and release of the oxygen as water. The enzyme, purified under aerobic conditions, is in the oxidised (Mo(VI)) state. Addition of a large excess of DMS to the oxidised enzyme in solution causes a change in the absorption spectrum of the enzyme. The same reaction occurs within crystals of the enzyme and the crystal structure reveals a complex with DMSO bound to the molybdenum via its oxygen atom. X-ray edge data indicate that the metal is in the Mo(IV) state. The DMSO ligand replaces one of the two oxo groups which ligate the oxidised form of the enzyme, suggesting very strongly that this is the oxygen which is transferred during catalysis. Residues 384 to 390, disordered in the oxidised enzyme, are now clearly seen in the cleft leading to the active site. The side-chain of Trp388 forms a lid trapping the substrate/product.

Analysis of dimethylsulfoxide reductase from Rhodobacter capsulatus showed that it contained 1 mol Mo and 2 mol GMP. This indicates that the molybdenum cofactor in dimethylsulfoxide reductase is bis(molybdopterin guanine dinucleotide) molybdenum. The absorption spectrum of the molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase after denaturation of the holoenzyme was compared with those of pterin standards of known redox state. The spectra were most similar to pterin standards in the dihydro state and oxidised state. The reduction of 2,6-dichloroindophenol by molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase and by pterin standards was also measured and approximately 2 mol electrons/2 mol molybdopterin guanine dinucleotide were found to reduce 2,6-dichloroindophenol. These results are consistent with the presence of one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a dihydropteridine and one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a fully aromatic pteridine. It is suggested that the pyrazine ring of Q-molybdopterin guanine dinucleotide is fully aromatic and contains a 5,6 double bond.

The dimethylsulfoxide reductase structural gene (dorA) of Rhodobacter capsulatus was cloned from a lambda expression library. The nucleotide sequence of the dorA gene was determined and it was found to encode a protein of 825 amino acids. Comparison of the deduced amino-acid sequence of DorA with N-terminal sequence of purified dimethylsulfoxide reductase from Rhodobacter capsulatus showed that the pre-protein possesses a 41-amino-acid N-terminal signal polypeptide. All of the conserved segments which have been described in bacterial enzymes which bind molybdopterin guanine dinucleotide (Berks, B.C., Ferguson, S.J., Moir, J.W.B. and Richardson, D.J. (1995) Biochim, Biophys. Acta 1232, 97-173) were identified in Rhodobacter capsulatus dimethylsulfoxide reductase.

Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides.

The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with gav 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with gav values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co-ordinating molybdenum via O or N, for a cysteine co-ordinating via S. Since the two signals with gav 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.

Dimethylsulphoxide (DMSO) reductase from R. capsulatus contains a molybdenum-pterin cofactor at its active site. As prepared the molybdenum is in the 6+ oxidation state, devoid of EPR signals. Stepwise reduction generates an EPR signal characteristic of Mo(V) having hyperfine coupling to a single proton and integrating to less than 25% of the total molybdenum. The low temperature MCD spectrum shows oppositely signed bands between approximately 550-700 nm. These bands are assigned as dithiolene-to-Mo(V) charge transitions. A simple theoretical model can satisfactorily account for the bands in the MCD spectrum. No evidence is found for cysteine coordination to Mo(V).

Dimethyl sulphoxide reductase was purified from the photosynthetic bacterium Rhodobacter capsulatus. The enzyme is composed of a single polypeptide of Mr 82,000 and contains a pterin-type molybdenum cofactor as the only detectable prosthetic group. The oxidized molybdenum cofactor of dimethyl sulphoxide reductase is a weak chromophore and exhibits broad absorption bands in the u.v.-visible-absorption spectral region. A distinct spectrum was generated upon addition of dithionite.