ATP synthesis in an ancient ATP synthase at low driving forces

Abstract

Hyperthermophilic archaea are close to the origin of life. Some hyperthermophilic anaerobic archaea live under strong energy limitation and have to make a living near thermodynamic equilibrium. Obviously, this requires adaptations of the energy-conserving machinery to harness small energy increments. Their ATP synthases often have an unusual motor subunit c that is predicted to prevent ATP synthesis. We have purified and reconstituted into liposomes such an archaeal ATP synthase found in a mesophilic bacterium. The enzyme indeed synthesized ATP at physiological membrane potentials, despite its unusual c subunit, but the minimal driving force for ATP synthesis was found to be even lower than in ATP synthases with usual c subunits. These data not only reveal an intermediate in the transition from ATP hydrolases to ATP synthases but also give a rationale for a bioenergetic adaptation of microbial growth near the thermodynamic equilibrium.

Keywords: ATP synthesis; acetogenic bacteria; archaea; bioenergetics; driving forces.

Fig. 1.

ΔµNa⁺-driven ATP synthesis of the A₁A_O ATP synthase from E. callanderi. (A) Scheme of artificial, ΔµNa⁺-driven ATP synthesis in proteoliposomes. ΔµNa⁺ is composed of ΔpNa and Δψ. ΔpNa is generated by an Na⁺ gradient (Na⁺_inside: high; Na⁺_outside: low) and Δψ by a K⁺ gradient (K⁺_inside: low; K⁺_outside: high). Δψ is induced by addition of valinomycin (Val), as K⁺ enter the lumen of the vesicles, while Cl⁻ are retained. (B) Inhibition of ΔµNa⁺-driven ATP synthesis by ionophores. Proteoliposomes containing the reconstituted ATP synthase were preincubated for 10 min at room temperature in the presence of 30 µM of the ionophores indicated (▴TCS, ▪ ETH 2120), before ATP synthesis was started by addition of 0.5 mM ADP and 2 µM valinomycin (final concentration each). In ♦, ADP was omitted. ● contained the solvent (0.1% ethanol) only. A ΔµNa⁺ of 230 mV was applied for each experiment. All data points are mean ± SEM; n = 3 independent experiments.

Fig. 2.

Dependence of Δψ-driven ATP synthesis on the Na⁺ concentration. (A) Time dependence of ATP formation. Δψ was applied to the proteoliposomes containing 100 µg of the reconstituted A₁A_O ATP synthase from E. callanderi as described in Materials and Methods. Δψ was kept constant at 120 mV. The internal and external NaCl concentration were kept the same ranging from 0 to 20 mM (as indicated). The contaminating amount of Na⁺ was 81 μM. ATP synthesis was started by addition of 2 µM valinomycin and 0.5 mM ADP (final concentration each). (B) Na⁺-dependence of ATP synthesis. The rates were calculated from A. All data points are mean ± SEM; n = 3 independent experiments.

Fig. 3.

Inhibition of Δψ-driven ATP synthesis of the A₁A_O ATP synthase from E. callanderi by DCCD and its relief by Na⁺. Before reconstitution into proteoliposomes, the purified ATP synthase was incubated for 30 min at room temperature with 0.3 mM DCCD (▪), 0.3 mM DCCD and 200 mM NaCl (●) or only 200 mM NaCl (▴). ATP synthesis was measured at a constant Δψ of 120 mV as described in Materials and Methods. The reaction was started by addition of 0.5 mM ADP and 2 µM valinomycin (final concentration each). All data points are mean ± SEM; n = 3 independent experiments.

Fig. 4.

Impact of different driving forces on ATP synthesis by the A₁A_O ATP synthase from E. callanderi. (A) Impact of Δψ (▪), ΔpNa (●), and µNa⁺ (▴) on ATP synthesis. ΔµNa⁺ was applied by keeping Δψ constant at 60 mV and varying ΔpNa. (B) Impact of ΔμNa⁺-driven ATP synthesis. ΔμNa⁺ was applied by different combinations of constant Δψ (30 [▪], 45 [▴], and 60 mV [●]) and varying ΔpNa. The driving forces were applied as described in Materials and Methods. All data points are mean ± SEM; n = 3 independent experiments.

Fig. 5.

Impact of different driving forces on ATP synthesis by the F₁F_O ATP synthases from A. woodii, P. modestum, and E. coli. ΔµNa⁺ was applied by keeping Δψ constant at 60 mV and varying ΔpNa. (A) Impact of Δψ (▪), ΔpNa (●), and µNa⁺ (▴) on ATP synthesis by the ATP synthase from A. woodii. (B) Impact of Δψ (▪), ΔpNa (●), and ΔµNa⁺ (▴) on ATP synthesis by the ATP synthase from P. modestum. (C) Impact of Δψ (▪), ΔpH (●), and ΔµH⁺ (▴) on ATP synthesis by the ATP synthase from E. coli. ΔµH⁺ was applied by keeping Δψ constant at 120 mV and varying ΔpH. The driving forces were applied as described in Materials and Methods. All data points are mean ± SEM; n = 3 independent experiments.