A STUDY ON A NOVEL ENERGY-COUPLING PROTEIN COMPLEX THAT REDUCE FERREDOXIN BY NADH

包囊红杆菌（Rhodobacter capsutatus）的rnf操纵子对光下固氮至关重要。我们发现rnf操纵子的产物在染色质膜上构成一个蛋白质复合体，并提出该复合体具有两个亚复合体的嵌合结构，一个跨膜亚复合体类似于Na^+易位NADH醌氧化还原酶，另一个亚复合体类似于H^+易位NADH醌氧化还原酶。其功能应为：耗能的nadh -铁氧还蛋白氧化还原酶。1)我们发现，在暗厌氧条件下，以二甲亚砜为终端电子受体的Rnf复合物也需要固氮，这证实了Rnf复合物的功能与光合反应中心没有直接联系。2)通过EPR分析发现Rnf复合物中至少存在一个Fe-S中心，聚类显示出一个新的信号g=1。84，在10K时，氧化还原电位极低。由于琥珀酸-醌氧化还原酶的其他强信号干扰了详细的分析，我们目前正在寻找克服这一困难的条件和突变体。3)大肠杆菌和其他4种非重氮营养细菌具有rnf操纵子的同源性。在e.c oli中过度表达E.coil同源物似乎是致命的。该大肠杆菌的操纵子不能与荚膜胞杆菌的rnf-null突变体互补。4)化学交联研究表明，该复合物的RnfC亚基与一个20K的道尔顿肽密切相互作用，可能是RnfB或RnfA肽。

The rnf(rbodobacter nitrogen fixation)operon in Rhodobacter capsutatus is essential for nitrogen fixation under light. We showed that products of the rnf operon constitute a protein complex in chromatophore membrane and proposed that the complex has a chimerie construct of two subcomplexes, one transmembrane subcomplex similar to Na^+-translocating NADH quinone oxidoreductase and the other peripheral subcomptex similar to H^+-translocating NADH quinone oxidoreductase. Its function should : be energy-consuming NADH-ferredoxin oxidoreductase. 1) We revealed that the complex is also required for nitrogen fixation under dark anaerobic conditions using dimethylsulfoxide as a terminal electron acceptor this agrees that the Rnf complex functions without direct link to photosynthetic reaction center. 2) Presence of at least one Fe-S center in Rnf complex was identified by EPR analysis The cluster showed a novel signal, g=1 .84, at 10K with extremely low redox potential. Since other strong signals from succinate-quinone oxidoreductase interfered detailed analysis, we are currently seeking conditions and mutants to overcome this difficulty. 3) Escheriehia coil and four other non-diazotrophic bacteria possess homologous of the rnf operon. Over expression of the E.coil homologue in E.ccli seemed lethal. This E.coli operon did not complemented the rnf-null mutants of R.capsulatus. 4) Chemical crosslinking studies showed that RnfC subunit of the complex interacts closely with a 20K Dalton peptide, which is possibly RnfB or RnfA peptide.