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.

Four genes, dorC, dorD, dorB and dorR of the DMSO respiratory gene cluster of Rhodobacter capsulatus have been identified and sequenced. dorC encodes a pentahaem c-type cytochrome of the NirT class and the derived DorC protein sequence shows highest similarity to TorC from the Escherichia coli trimethylamine-N-oxide (TMAO) respiratory system. Mutagenesis of dorC resulted in the loss of a 46 kDa haem-staining polypeptide from membranes of R. capsulatus. dorD encodes a protein with highest sequence similarity to TorD from the E. coli TMAO respiratory system. DMSO reductase polypeptide (DorA) could not be detected in cell-free extracts of a dorD mutant and it is suggested that DorD has a role in stabilizing the DorA apo-protein prior to insertion of the pterin molybdenum cofactor. dorB encodes a protein with highest sequence similarity to NapD of Paracoccus denitrificans. Mutagenesis of dorB reduced the activity of DMSO reductase and led to the accumulation of a larger form of the enzyme that is presumed to represent a cytoplasmic precursor polypeptide. It is suggested that DorB has a role in the biogenesis of DMSO reductase prior to its secretion into the periplasm. dorR is transcribed in the opposite direction to dorC. The derived amino acid sequence of DorR indicates that it is a response regulator and mutation of dorR shows that it is essential for expression of the dorCDA operon. Expression of a chromosomal dorA::lacZ fusion was also dependent on the transcriptional regulator Fnr. The intergenic region between dorR and dorC contains four putative binding sites for DorR but no binding site for Fnr was identified.

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 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 1.82-Å X-ray crystal structure of the oxidised (Mo(VI)) form of the enzyme dimethylsulfoxide reductase (DMSOR) isolated from Rhodobacter capsulatus is presented. The structure has been determined by building a partial model into a multiple isomorphous replacement map and fitting the crystal structure of DMSOR from Rhodobacter sphaeroides to the partial model. The enzyme structure has been refined, at 1.82-Å resolution, to an R factor of 14.8% (Rfree = 18.4%). The molybdenum is coordinated by seven ligands: four dithiolene sulfurs, Oγ of Ser147 and two oxo groups. The four sulfur ligands, at a metal-sulfur distance of 2.4 Å or 2.5 Å, are contributed by the two molybdopterin guanine dinucleotide (MGD) cofactors. The coordination sphere of the molybdenum is different from that in previously reported structures of DMSOR from R. sphaeroides and R. capsulatus. The 2.8-Å structure of DMSOR, reduced by addition of sodium dithionite, is also described and differs from the structure of the oxidised enzyme by the removal of a single oxo ligand from the molybdenum coordination sphere. A structure, at 2.5-Å resolution, has also been obtained from crystals soaked in mother liquor buffered at pH 7.0. No differences are observed in the structure at pH 7 when compared with the native crystal structure at pH 5.5.

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.