The properties of enzymes involved in energy transduction from a mesophilic (Bacillus subtilis) and a thermophilic (B. stearothermophilus) bacterium were compared. Membrane preparations of the two organisms contained dehydrogenases for NADH, succinate, L-ac-glycerophosphate, and L-lactate. Maximum NADH and cytochrome c oxidation rates were obtained at the respective growth temperatures of the two bacteria. The enzymes involved in the oxidation reactions in membranes of the thermophilic species were more thermostable than those of the mesophilic species. The apparent microviscosities of the two membrane preparations were studied at different temperatures. At the respective optimal growth temperatures, the apparent microviscosities of the membranes of the two organisms were remarkably similar. The transition from the gel to the liquid-crystalline state occurred at different temperatures in the two species. In the two species, the oxidation of physiological (NADH) and nonphysiological (N,N,N',N'-tetramethyl-p-phenylenediamine or phenazine methosulfate) electron donors led to generation of a proton motive force which varied strongly with temperature. At increasing temperatures, the efficiency of energy transduction declined because of increasing H+ permeability. At the growth temperature, the efficiency of energy transduction was lower in B. stearothermophilus than in the mesophilic species. Extremely high respiratory activities enabled B. stearothermophilus to maintain a high proton motive force at elevated temperatures. The pH dependence of proton motive force generation appeared to be similar in the two membrane preparations. The highest proton motive forces were generated at low external pH, mainly because of a high pH gradient. At increasing external pH, the proton motive force declined.

Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy‐transducing properties were studied. The reconstitution procedure used included Ca2+‐induced fusion of pre‐formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N′,N′‐tetramethyl‐p‐phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef‐heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110–140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18–23%, indicating that the oxidase is an efficient proton‐motive‐force‐generating system.

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