Chemiosmotic energy conservation with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by Acetobacterium woodii

Abstract

Cell suspensions of Acetobacterium woodii prepared from cultures grown on fructose plus caffeate catalyzed caffeate reduction with electrons derived from molecular hydrogen. Hydrogen-dependent caffeate reduction was strictly Na(+) dependent with a K(m) for Na(+) of 0.38 mM; Li(+) could substitute for Na(+). The sodium ionophore ETH2120, but not protonophores, stimulated hydrogen-dependent caffeate reduction by 280%, indicating that caffeate reduction is coupled to the buildup of a membrane potential generated by primary Na(+) extrusion. Caffeate reduction was coupled to the synthesis of ATP, and again, ATP synthesis coupled to hydrogen-dependent caffeate reduction was strictly Na(+) dependent and abolished by ETH2120, but not by protonophores, indicating the involvement of a transmembrane Na(+) gradient in ATP synthesis. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) abolished ATP synthesis, and at the same time, hydrogen-dependent caffeate reduction was inhibited. This inhibition could be relieved by ETH2120. These experiments are fully compatible with a chemiosmotic mechanism of ATP synthesis with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by A. woodii.

FIG. 1.

Reduction of caffeate to hydrocaffeate as carried out by A. woodii.

FIG. 2.

Na⁺ dependence of hydrogen-dependent caffeate reduction by A. woodii. (A) Cell suspensions of A. woodii (1.54 mg of protein/ml) grown on fructose plus caffeate were prepared and incubated under an atmosphere of hydrogen at 30°C in a shaking water bath in a buffer containing NaCl as indicated. After preincubation for 30 min, caffeate was added from a stock solution. At the time points indicated, samples were withdrawn and analyzed for caffeate as described in Materials and Methods. Panel B displays the caffeate reduction rates as a function of the external Na⁺ concentration.

FIG. 3.

Stimulation of hydrogen-dependent caffeate reduction by sodium ionophores. Cell suspensions (1.25 mg of protein/ml) were treated as described in the legend to Fig. 2. At the time indicated by the arrow, one cell suspension received the protonophore SF6847 (○) (final concentration, 27 μM), and another received the sodium ionophore ETH2120 (□) (final concentration, 27 μM). The control (•) received the solvent only.

FIG. 4.

Na⁺ dependence of ATP synthesis coupled to hydrogen-dependent caffeate reduction. Cell suspensions of A. woodii (1.33 mg of protein/ml) were incubated in buffer in the absence of supplemental Na⁺ (•) or in the presence of 10 mM Na⁺ (○). Caffeate was added to a final concentration of 10 mM at the time indicated by the arrow. At time points indicated, samples were withdrawn and analyzed for cellular ATP content as described in Materials and Methods.

FIG. 5.

Inhibition of ATP synthesis coupled to hydrogen-dependent caffeate reduction by the sodium ionophore ETH2120. Cell suspensions of A. woodii (1.54 mg of protein/ml) were preincubated under a hydrogen atmosphere in the presence of 10 mM Na⁺ and 20 μM TCS (□), 20 μM SF6847 (•), or 20 μM ETH2120 (▪). A control received the solvent only (○). Caffeate was added to a final concentration of 10 mM at the time point indicated by the arrow. At time points indicated, samples were withdrawn and analyzed for cellular ATP content as described in Materials and Methods.

FIG. 6.

Inhibition of ATP synthesis and hydrogen-dependent caffeate reduction by the ATPase inhibitor DCCD. Cell suspensions of A. woodii (1.54 mg of protein/ml) were preincubated under a hydrogen atmosphere in the presence of 3 mM Na⁺ and absence (•) or presence (○) of 100 μM DCCD for 30 min. Caffeate was added at the time point indicated by the arrow (A) or at zero time to a final concentration of 10 mM (B). At time points indicated, samples were withdrawn and analyzed for cellular ATP content (A) or caffeate concentration (B) as described in Materials and Methods.

FIG. 7.

Inhibition of hydrogen-dependent caffeate reduction by the ATPase inhibitor DCCD and relief of DCCD inhibition by the sodium ionophore ETH2120. Cell suspensions of A. woodii (1.54 mg of protein/ml) were preincubated under a hydrogen atmosphere in the presence of 3 mM Na⁺ and absence (□) or presence (○ and •) of 100 μM DCCD for 30 min. Caffeate was added to a final concentration of 10 mM at zero time. At the time point indicated by the arrow one suspension received the sodium ionophore ETH2120 (○) at a final concentration of 36 μM. Samples were withdrawn and analyzed for caffeate concentration as described in Materials and Methods.