3.D.6 The Putative Ion (H+ or Na+)-translocating NADH:Ferredoxin Oxidoreductase (NFO or NRF) Family

The NFO family consists of enzyme complexes, each probably consisting of seven distinct subunits that catalyze transfer of electrons from NADH to ferredoxin, an energetically unfavorable reaction. This reaction is probably driven by the flow of H+ or Na+ down the ion electrochemical gradient from the outside to the inside of the bacterial cell. The best characterized system is encoded by the rnfABCDEGH operon found in Rhodobacter capsulatus. The rnf (Rhodobacter nitrogen fixation) genes are essential for normal nitrogen fixation as reduced ferredoxin, generated primarily by the rnf operon gene products, is the normal electron donor to the iron-containing dinitrogenase reductase. Of these proteins, RnfA, D and E are predicted to have a transmembrane topology with RnfA and RnfE, which are 35% identical, having five established TMSs with opposite orientation in the membrane (Sääf et al., 1999). All other proteins of the system (B, C, G and H) are believed to be water soluble.

RnfB and C contain the sequence motif of iron-sulfur centers of the 2[4Fe-4S]-type found in plant-type ferredoxins. These proteins are peripheral proteins of the R. capsulatus chromatophore membrane. RnfC has the putative NADH-binding site of NDHI (complex I; TC #6.1). Ferredoxin I, encoded by the fdxN gene, is the electron acceptor of the Rnf system and the physiological electron donor to nitrogenase. Operons homologous to the rnf operon and with the same gene order are found in E. coli and Haemophilus influenzae except that the rnfE gene is split into two genes encoding two polypeptide chains, the E and F proteins. Only the F proteins of E. coli and H. influenzae are homologous to RnfA.

Similar operons are also found in bacteria including Pseudomonas aeruginosa, Yersinia pestis, Actinobacillus actinomycetemcomitans, Porphyromonas gingvalis, Vibrio cholerae and Thermus thermophilus. A region in the human genome encodes a homologue of the RnfA protein (K. Saeki, personal communication). Thus, NFO family members are widespread in bacteria and possibly in other organisms as well.

The RnfA and E (F part) proteins are homologous to two subunits in the Na+-translocating NADH:quinone dehydrogenases (Na-NDHs; TC #3.D.5) of Vibrio alginolyticus and H. influenzae. The homologous V. alginolyticus subunits are the NqrD and NqrE subunits of the NqrABCDEF complex. The NFO systems can therefore be thought of as hybrid systems with some of the subunits resembling those of H+-translocating NDHs (TC #3.D.1) and other subunits resembling those of Na+-translocating NDHs (TC #3.D.5). It is not known whether H+ or Na+ is transported by the Rnf systems of R. capsulatus.

The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. Ferredoxin:NAD+ oxidoreductase is membrane bound and has subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na+ pump (Imkamp et al., 2007). The following electron transport chain was proposed: H2 → ferredoxin → NAD+ → Etf → caffeyl-CoA reductase. The sodium motive step in the chain is probably the ferredoxin-dependent NAD+ reduction catalyzed by Rnf (Imkamp et al., 2007). Biegel & Müller (2010) further showed that inverted membrane vesicles of A. woodii couple electron transfer from reduced ferredoxin to NAD+ with the transport of Na+ from the outside into the lumen of the vesicles. Na+ transport was electrogenic, and accumulation was inhibited by sodium ionophores but not protonophores, demonstrating a direct coupling of Fno activity to Na+ transport. Results from inhibitor studies are consistent with the hypothesis that Fno activity coupled to Na+ translocation is catalyzed by the Rnf complex, a membrane-bound, iron-sulfur and flavin-containing electron transport complex encoded by many bacterial and some archaeal genomes. Fno is a unique type of primary Na+ pump.

Inverted membrane vesicles of A. woodii couple electron transfer from reduced ferredoxin to NAD+ with the transport of Na+ from the outside into the lumen of the vesicles (Biegel and Müller, 2010). Na+ transport was electrogenic, and accumulation was inhibited by sodium ionophores but not protonophores, demonstrating a direct coupling of a ferredoxin:NAD+ oxidoreductase (Fno) activity to Na+ transport. Results from inhibitor studies are consistent with the hypothesis that Fno activity coupled to Na+ translocation is catalyzed by the Rnf complex, a membrane-bound, iron-sulfur and flavin-containing electron transport complex encoded by many bacterial and some archaeal genomes. Fno is a unique type of primary Na+ pump and represents an early evolutionary mechanism of energy conservation that expands the redox range known to support life. In addition, it explains the lifestyle of many anaerobic bacteria and gives a mechanistic explanation for the enigma of the energetic driving force for the endergonic reduction of ferredoxin with NADH plus H+ as reductant in a number of aerobic bacteria (Biegel and Müller, 2010).

The overall reaction believed to be catalyzed by NFO family complexes is:

NADH + oxidized ferredoxin + n(H+ or Na+) (out) → NAD+ + reduced ferredoxin + n(H+ or Na+) (in)



This family belongs to the Urea Transporter/Na+ Exporter (UT/RnfD/NqrB) Superfamily.

 

References:

Biegel, E. and V. Müller. (2010). Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase. Proc. Natl. Acad. Sci. USA 107: 18138-18142.

Hess, V., K. Schuchmann, and V. Müller. (2013). The ferredoxin:NAD+ oxidoreductase (Rnf) from the acetogen Acetobacterium woodii requires Na+ and is reversibly coupled to the membrane potential. J. Biol. Chem. 288: 31496-31502.

Imkamp, F., E. Biegel, E. Jayamani, W. Buckel, and V. Müller. (2007). Dissection of the caffeate respiratory chain in the acetogen Acetobacterium woodii: identification of an Rnf-type NADH dehydrogenase as a potential coupling site. J. Bacteriol. 189: 8145-8153.

Jouanneau, Y., H.S. Jeong, N. Hugo, C. Meyer and J.C. Willison (1998). Overexpression in Escherichia coli of the rnf genes from Rhodobacter capsulatus–characterization of two membrane-bound iron-sulfur proteins. Eur. J. Biochem. 251: 54-64.

Kumagai, H., T. Fujiwara, H. Matsubara and K. Saeki (1997). Membrane localization, topology, and mutual stabilization of the rnfABC gene products in Rhodobacter capsulatus and implications for a new family of energy-coupling NADH oxidoreductases. Biochemistry 36: 5509-5521.

Saaf, A., Johansson, M., Wallin, E., and von Heijne, G. (1999). Divergent evolution of membrane protein topology: the Escherichia coli RnfA and RnfE homologues. Proc. Natl. Acad. Sci. USA 96: 8540-8544.

Suharti, S., M. Wang, S. de Vries, and J.G. Ferry. (2014). Characterization of the RnfB and RnfG subunits of the Rnf complex from the archaeon Methanosarcina acetivorans. PLoS One 9: e97966.

Welte C. and Deppenmeier U. (2014). Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens. Biochim Biophys Acta. 1837(7):1130-47.

Yagi, T., T. Yano, S. Di Bernardo, A. Matsuno-Yagi (1998). Procaryotic complex I (NDH-1), an overview. Biochim. Biophys. Acta 1364: 125-133.

Examples:

TC#NameOrganismal TypeExample
3.D.6.1.1

NADH:ferredoxin oxidoreductase

Proteobacteria

RnfABCDEG of Rhodobacter capsulatus

 
3.D.6.1.2

The Rnf-type NADH dehydrogenase, RnfABCDEG (Imkamp et al., 2007). Note: RnfD may be truncated as almost all homologues have 300-330aas with 6-7 TMSs.  This system has been shown to pump Na+ out of the cell when reducing NAD+ with reduced ferredoxin, and dependent on the smf for reduction of ferredoxin with NADH2 (Hess et al. 2013).

Bacteria

RnfABCDEG of Acetobacterium woodii:
RnfA (195 aas) (C4N8U4)
RnfB (333 aas) (C4N8U5)
RnfC (443aas) (C4N8U0)
RnfD (132aas) (C4N8U1)
RnfE (196 aas) (C4N8U3)
RnfG (207 aas) (C4N8U2)

 
3.D.6.1.3

The Rnf-type NADH dehydrogenase, RnfABCDEG/MA0658 (Welte and Deppenmeier 2013). RnfB and RnfC are on the cytoplasmic side of the membrane. Both have 4Fe4S centers (5 and 2, repectively), and RnfB oxidizes reduced ferredoxin (Suharti et al. 2014). The integral membrane proteins are RnfA, D and E, while RnfG and MA0658 are on the outside. RnfE reduces oxidized Mph (Welte and Deppenmeier 2013).

Archaea

RnfABCDEG/MA0658 complex of Methanosarcina acetivorans
RnfA, 199 aas
RnfB, 264 aas
RnfC, 447 aas
RnfD, 288 aas
RnfE, 213 aas
RnfG, 188 aas
MA0658, 500 aas

 
3.D.6.1.4

Rnf-type NADH dehydrogenase, RsxA, RsxD, RsxE, RsxG, RsxB, RsxC of 6, 10, 5, 1, 1 and 1 TMSs, respectively.

Rnf-like system, RsxA, B, C, D, E, G of E. coli