Studies on a Novel Energy-Coupling Enzyme Family Rnf That Functions at Very Low Redox Potential

荚膜红细菌中的rnf（Rhodobacter nitrogenfixation）操纵子是光下固氮所必需的。这些基因在色素细胞膜上编码一种蛋白复合物，可能作为一种能量消耗型NADH-铁氧还蛋白氧化还原酶发挥功能。1）我们用Ni-NTA亲和柱部分纯化了含有相应修饰的rnfB基因的非极性rnfB突变株KF 111细胞表达的Rnf复合物。2）phoA-融合法分析表明RnfA和RnfE均跨膜6次，但跨膜亚基RnfA、RnfD和RnfE的膜拓扑结构相反。结果表明，RnfA和RnfE以相反的方向跨膜6次，RnfD蛋白跨膜8次，其中心亲水区暴露于周质。3）对已知rnf基因组区域的下游分析表明，存在另一个固氮必需基因（rnfI）。RnfI蛋白与周质黄细胞色素C硫化物脱氢酶的FAD结合亚基相似。

The rnf(rhodobacter nitrogen fixation) operon in Rhodobacter capsulatus is essential for nitrogen fixation under light. The genes encode a protein complex in chromatophore membrane that might function as an energy-consuming NADH-ferredoxin oxidoreductase.1) We have partially purified the Rnf complex using Ni-NTA affinity column with hexahistidine-tagged RnfB expressed the cells of non-polar rnfB mutant strain KF11l containing the corresponding modified rnfB gene. However, the iron-sulfur center in RnfB protein was so unstable that complete purification was unsuccessful.2) Analysis by phoA-fusion method revealed that RnfA and RnfE both span membrane six times with opposite Membrane topology of transmembrane subunits RnfA, RnfD and RnfE was analyzed by the. It was revealed that RnfA and RnfE span membrane 6 times with an opposite orientation and that RnfD protein spans membrane 8 times with its central hydrophilic region exposed to periplasm.3) Downstream analysis of the known rnf genomic region revealed there is another gene (rnfI) that is essential for nitrogen fixation. RnfI protein shows similarity to the FADbinding subunit of periplasmic flavocytochrome c sulfide dehydrogenase.