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

光反硝化细菌球形红杆菌(Rhodbacter sphaeroides f.sp.)反硝化细菌能在厌氧光照条件下生长，以二甲基亚砜(DMSO)作为DMSO呼吸作用的末端电子受体。这一呼吸作用的末端酶--二甲基亚甲氧基还原酶(DMSOR)最近被我们的团队测得，并由美国研究人员进行了结晶学分析。这种新型的DMSO呼吸系统与该酶活性部位的钼辅助因子的作用关系引起了世界各国的广泛关注。在涉及DMSO还原酶(Dmsa)的基因上游有两个由dmsB和cmsC组成的开放阅读框，dmsB是低分子量膜蛋白，dmsc也是膜蛋白，含有分子量为45000的五铁血红素。通过与DMS操纵子的同源性比较，推测DMSc是DMSOR的直接电子转运子。利用DMSA、DMSB、DMSc、dmsBC和dmsABC等多种操纵子在大肠杆菌中表达DMSc的实验表明，DmsC在细胞中以包涵体形式表达，但没有证实其活性。蛋白质分子中没有引入穿透血红素似乎是表达成熟蛋白的限制因素。为了阐明Dmac到DMSA的实际电子流动，在经过多次层析纯化后，从红杆菌细胞中分离出DMSc，在SDS-PAGE中纯化为单一条带，然而，初步的体外重组实验没有显示出活性，这表明需要进一步研究重组条件。

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.