Rossi M, Pollock W B, Reij M W, Keon R G, Fu R, Voordouw G
Department of Biological Sciences, University of Calgary, Alberta, Canada.
J Bacteriol. 1993 Aug;175(15):4699-711. doi: 10.1128/jb.175.15.4699-4711.1993.
The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.
普通脱硫弧菌希登伯勒亚种hmc操纵子的核苷酸序列表明存在八个开放阅读框，编码蛋白质Orf1至Orf6、Rrf1和Rrf2。Orf1是周质高分子量细胞色素（Hmc），含有16个c型血红素，之前已有描述（W. B. R. Pollock、M. Loutfi、M. Bruschi、B. J. Rapp-Giles、J. D. Wall和G. Voordouw，《细菌学杂志》173:220 - 228，1991年）。Orf2是一种具有四个铁硫簇的跨膜氧化还原蛋白，这从其与大肠杆菌DmsB的相似性可以看出。Orf3、Orf4和Orf5都是高度疏水的整合膜蛋白，与NADH脱氢酶或细胞色素c还原酶的亚基相似。Orf6是一种含有两个铁硫簇的细胞质氧化还原蛋白，这从其与脱硫弧菌属[Fe]氢化酶铁氧化还原蛋白结构域的相似性可以看出。Rrf1属于应答调节蛋白家族，而Rrf2的功能无法从基因序列推导得出。利用T7系统在大肠杆菌中对各个基因的表达进行了验证，确定了Orf2、Orf6和Rrf1的开放阅读框。在脱硫脱硫弧菌G200中删除orf1上游0.4 kb的片段会消除Hmc的表达，表明该区域包含hmc操纵子启动子。在脱硫脱硫弧菌G200中表达两个截短的hmc基因产生了稳定的周质c型细胞色素，证实了Hmc的结构域结构。我们提出，Hmc和Orf2至Orf6形成一个跨膜蛋白复合物，使电子能够从周质氢化酶流向催化硫酸盐还原的细胞质酶。Hmc的结构域结构可能是与多种氢化酶相互作用所必需的。
