A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii

Abstract

The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii.

FIGURE 1.

Na⁺ dependence of PP_i hydrolysis. A, PP_i hydrolysis was determined in membrane vesicles in 25 mm Pipes/KOH buffer (pH 6.8) containing 25 mm MgSO₄, 420 mm sucrose, and varying NaCl concentrations. The effect of NaCl (squares), KCl (diamonds), or LiCl (inverted triangles) on PP_i hydrolysis. B, the corresponding Lineweaver-Burk plot for NaCl is shown. Data from two independent experiments are shown.

FIGURE 2.

Dependence of PP_i hydrolysis and ²²Na⁺ transport on the PP_i concentration. Membrane vesicles (protein concentrations of 1.1 mg/ml in hydrolysis assay and 3.3 mg/ml in transport) in 25 mm Pipes/KOH buffer (pH 6.8) containing 25 mm MgSO₄, 420 mm sucrose, and 1.9 mm NaCl showed PP_i hydrolysis activity (A) and ²²Na⁺ transport depending on the PP_i concentration (B): 0 mm (closed squares), 0.1 mm (triangles), 0.5 mm (inverted triangles), 1 mm (diamonds), 2.5 mm (circles), 5 mm PP_i (open squares). The arrow indicates the addition of PP_i.

FIGURE 3.

Na⁺ dependence of PP_i-dependent transport activity. Membrane vesicles (protein concentration of 3.6 mg/ml) were incubated in 25 mm Pipes/KOH buffer (pH 6.8) containing 25 mm MgSO₄, 420 mm sucrose, and different NaCl concentrations. Transport rates were calculated from the initial slopes and plotted against the Na⁺ concentration in a Michaelis-Menten (A) or Lineweaver-Burk (B) plot. Data from two independent experiments are shown.

FIGURE 4.

²²Na⁺ transport is electrogenic. Membrane vesicles (protein concentration of 2.8 mg/ml) in Pipes/KOH buffer (pH 6.8) containing 25 mm MgSO₄, 420 mm sucrose, 1.9 mm NaCl, 150 mm KCl, and 17 μm valinomycin showed ²²Na⁺ transport upon addition of PP_i (triangles) and no addition of valinomycin (squares). The arrow indicates the addition of PP_i.

FIGURE 5.

²²Na⁺ transport is a primary event. Membrane vesicles (protein concentration of 2.8 mg/ml) in Pipes/KOH buffer (pH 6.8) containing 25 mm MgSO₄, 420 mm sucrose, and 1.9 mm NaCl showed ²²Na⁺ transport upon the addition of 1 mm PP_i (squares). ETH 2120 (100 μm) was added 6 min before (triangles) or 6 min after (inverted triangles) addition of PP_i. SF 6847 (100 μm) (diamonds) was added 6 min before the addition of PP_i. The arrow indicates the addition of PP_i.

FIGURE 6.

Model of caffeate respiration in A. woodii. Flow of electrons from electron donors (fructose or hydrogen) to the acceptor caffeate is shown. The PP_i generated by the caffeyl-CoA synthetase is used by the PPase to pump Na⁺ across the membrane.