Membrane Localization, Topology, and Mutual Stabilization of thernfABCGene Products inRhodobacter capsulatusand Implications for a New Family of Energy-Coupling NADH Oxidoreductases

Abstract

The rnf genes in Rhodobacter capsulatus are unique nitrogen fixation genes that encode potential membrane proteins (RnfA, RnfD, and RnfE) and potential iron-sulfur proteins (RnfB and RnfC). In this study, we first analyzed the localization and topology of the RnfA, RnfB, and RnfC proteins. By activity and immunoblot analysis of expression of translational fusions to Escherichia coli alkaline phosphatase, RnfA protein was shown to span the chromatophore membrane with its odd-numbered hydrophilic regions exposed to periplasm. By alkaline treatment of membrane fractions and following immunoblot analysis using antibodies against recombinant proteins expressed in E. coli, both RnfB and RnfC proteins were revealed to situate at the periphery of the chromatophore membranes. Second, mutual interaction of the Rnf proteins was analyzed by immunochemical determinations of RnfB and RnfC proteins in rnf mutants and their complemented derivatives. The contents in cellular fractions indicated that RnfB and RnfC stabilize each other and that the presence of RnfA is necessary for stable existence of both proteins. These results support a hypothesis that the Rnf products are subunits of a membrane complex. Finally, we detected homologs of rnf genes in Haemophilus influenzae and Vibrio alginolyticus by data base searches and in E. coli by cloning of a fragment of an rnfA homolog followed by a data base search. Close comparisons revealed that RnfC has potential binding sites for NADH and FMN which are similar to those found in proton-translocating NADH:quinone oxidoreductases and that RnfA, RnfD, and RnfE show similarity to subunits of sodium-translocating NADH:quinone oxidoreductases. We predict that the putative Rnf complex represents a novel family of energy-coupling NADH oxidoreductases.

Electron Transport in the Pathway of Acetate Conversion to Methane in the Marine Archaeon Methanosarcina acetivorans

“…3A). The Rnf complex was first described in Rhodobacter capsulatus (34), where it is proposed to encode a six-subunit (20) membrane-bound complex (RnfABCDGE) oxidizing NADH and reducing a ferredoxin which then donates electrons to nitrogenase (23,34). It is further proposed that the thermodynamically unfavorable reverse electron transport is driven by an electrochemical gradient (27,33).…”

“…The subunits of the Rnf complex from R. capsulatus (Rc-Rnf) have been previously characterized (20,23), predicting functions for subunits of the M. acetivorans Rnf complex (Ma-Rnf) encoded by MA0658-0665 (Fig. 4).…”

“…The MA0661 product shares identity with the RnfG subunit (Fig. 5C), which extends to a motif (ETPGLGX [37][38][39][40][41][42][43] GAT) that is also conserved with the NqrC subunit of Vibrio species (23) in which the C-terminal threonine of the motif covalently binds FMN (3,14,30). Therefore, the product of MA0661 is hypothesized to contain FMN, accepting electrons from the product of MA0659 (Fig.…”

Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii : Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site

“…RnfA, RnfD, and RnfE are predicted to be transmembrane proteins, whereas RnfC is a soluble protein and RnfB and RnfG are predicted to be associated with the membrane (25). In Rnf complexes indications of flavin mononucleotide and NADH binding sites were found, as were motifs typical of [4Fe-4S]-type ferredoxin-like domains (30). Bioinformatic analyses revealed that Rnf and Na ϩ -pumping NADH:quinone oxidoreductase (Nqr) complexes are similar (25), which leads to the idea that Rnf complexes could function as Na ϩ pumps as well.…”

The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

“…In the phototrophic nonsulfur bacterium Rhodobacter capsulatus a set of nitrogen-regulated genes, the rnf genes, as shown by mutational studies, encode proteins involved in electron transfer to nitrogenase (25,30,42,45). The membrane protein complex encoded by the rnf genes shows similarity to sodium-dependent NADH oxidoreductases and is thought to drive the NADH-dependent reduction of ferredoxin I (the fdxN gene product) by utilizing the PMF derived from the photosynthetic activities of the cell.…”