Analysis of novel electron-transporting system in DMSO respiration of a photosynthetic bacterium

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A photodenitrifier, Rhodobacter sphaeroides f.sp. denitrifican, can grow under anaerobic-light condition with dimethyl sulfoxide (DMSO) as the terminal electron acceptor in DMSO respiration. The terminal enzyme of this respiration, dimethyl su1foxide redoctase (DMSOR), was recently seqoenced by our team and analyzed crystallographically by U.S.researchers. Attention was focussed worldwide on the relation between roles of molybdenum-cofactor in active site of the enzyme and this novel DMSO respiration system. This research was therefore organized to clarify the details of this respiration system.In the upstream of the gene involving DMSO reductase (dmsA), there was two open reading frames composed of dmsB and cmsC.DmsB was suggested to be low molecular weight membrane protein and dmsC was also membraneous and contains pentaheme with molecular weight of 45000. From the comparison of tor operon in E.coil which has high homology with dms operon, dmsC was deduced as a direct electron-transporter to DMSOR.Many trials were done to extress dmsC in E.coli using varieties of operons, dmsA, dmsB, dmsC, dmsBC, and dmsABC.DmsC was expressed as an inclusion body in the cells but no activity was confirmed. The fact that penetaheme was not introduced in the protein molecule seemed to be the limiting factor to express matured proteins.To elucidate the actual electron flow from dmaC to dmsA, isolation of dmsC from the Rhodobacter cells was performed After many steps of chromatographic purification, dmsC was purified to a single band in SDS-PAGE.Preliminary in-vitro reconstitution experiment, however, showed no activity, indicating that further studies were needed to establish the reconstitution conditions.