The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

• Published in: The Journal of biological chemistry Vol. 288; no. 44; pp. 31496 - 31502
• Main Authors: Hess, Verena, Schuchmann, Kai, Müller, Volker
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Acetobacterium - enzymology; Bacterial Proteins - metabolism; Bioenergetics; Bioenergetics/Electron Transfer Complex; Carbon Monoxide - metabolism; Electron Transfer; Energy Metabolism; Enzyme Kinetics; Ferredoxin-NADP Reductase - metabolism; Hydrogen-Ion Concentration; Lithium - metabolism; Membrane Energetics; Membrane Potentials - physiology; Membrane Transport; Oxidation-Reduction; Physiology; Rnf; Sodium - metabolism; Sodium Transport; Sodium Transport; Energy Metabolism; Oxidation-Reduction; Electron Transfer; Bioenergetics/Electron Transfer Complex; Membrane Transport; Physiology; Enzyme Kinetics; Rnf; Membrane Energetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.510255

The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na+-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD+ with the generation of a primary electrochemical Na+ potential across its cytoplasmic membrane. In previous assays in which Ti3+ was used to reduce ferredoxin, Na+ transport was observed, but not a Na+ dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD+ reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na+ dependence of this electron transfer reaction. The Km was 0.2 mm. Na+ could be partly substituted by Li+. Na+ dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na+ as a coupling ion. Electron transport from reduced ferredoxin to NAD+ was coupled to electrogenic Na+ transport, indicating the generation of Δμ̃Na+. Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of Δμ̃Na+, and was accompanied by Na+ efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na+ gradient. The physiological significance of this finding is discussed. Background: Ferredoxin:NAD+-oxidoreductases (Rnf) found in many bacteria are novel ion-translocating electron transport chains. Results: A Na+ requirement for the reaction and its reversible coupling to the transmembrane Na+ gradient are demonstrated. Conclusion: Na+ is the coupling ion. Rnf not only generates a Na+ potential but also uses it to drive the reverse reaction. Significance: Evidence for a function of Rnf in ferredoxin reduction is provided.