Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase

Abstract

The anaerobic acetogenic bacterium Acetobacterium woodii carries out a unique type of Na(+)-motive, anaerobic respiration with caffeate as electron acceptor, termed "caffeate respiration." Central, and so far the only identified membrane-bound reaction in this respiration pathway, is a ferredoxin:NAD(+) oxidoreductase (Fno) activity. Here we show 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 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.

Fig. 1.

Fno catalyzed ²²Na⁺ transport. Membrane vesicles (protein concentration 3.2 mg/mL) in 50 mM Mops buffer containing 20 mM MgSO₄, 20 mM NaCl, 8 mM DTE, and 2.25 mg/L resazurin translocated ²²Na⁺ upon addition of 7.5 μg ferredoxin, 5 mM titanium citrate, and 3.5 mM NAD⁺ (squares). In control experiments titanium citrate (downward-pointing triangles) or NAD⁺ (upward-pointing triangles) were omitted. Arrow indicates addition of NAD⁺.

Fig. 2.

Na⁺ dependence of transport activity. Membrane vesicles (protein concentration 4 mg/mL) were incubated in 50 mM Mops buffer, 20 mM MgSO₄, 8 mM DTE, 2.25 mg/L resazurin, and varying NaCl concentrations. Transport rates were calculated from the initial slopes and plotted against the Na⁺ concentration.

Fig. 3.

Effect of LiCl on ²²Na⁺ transport and Fno activity. White bars represent the rate of ²²Na⁺ accumulation at IMVs; 100% corresponds to 0.9 nmol ²²Na⁺/mg ⋅ min. Gray bars indicate Fno activity; 100% corresponds to 18 mU/mg.

Fig. 4.

²²Na⁺ transport is a primary event. Membrane vesicles (protein concentration 3.2 mg/mL) in 50 mM Mops buffer (pH 6.0) containing 20 mM MgSO₄, 20 mM NaCl, 8 mM DTE, and 2.25 mg/L resazurin showed ²²Na⁺ transport upon addition of 7.5 μg ferredoxin, 5 mM titanium citrate, and 3.5 mM NAD⁺ (squares). ETH 2120 (100 μM) was added 6 min before (upward-pointing triangles) or 4 min after (downward-pointing triangles) addition of NAD⁺. SF 6847 (100 μM) (diamonds) was added 6 min before addition of NAD⁺. Arrow indicates addition of NAD⁺.

Fig. 5.

Model of caffeate respiration in A. woodii. Flow of electrons from electron donors (fructose or hydrogen) to acceptor caffeate is shown. For explanations see text.