Efficient ATP synthesis by thermophilic Bacillus F(o)F(1)-ATP synthase

F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in the F(1) portion when protons flow through F(o) to rotate the shaft common to F(1) and F(o). Rotary synthesis in isolated F(1) alone has been shown by applying external torque to F(1) of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 F(o)F(1) (TF(o)F(1)), however, has so far been poor in vitro, of the order of 1 s(−1) or less, hampering reliable characterization. Here, by using a mutant TF(o)F(1) lacking an inhibitory segment of the ε-subunit, we have developed highly reproducible, simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis, which was driven by acid–base transition and K(+)-diffusion potential. The synthesis activity reached ∼ 16 s(−1) at 30 °C with a Q(10) temperature coefficient of 3–4 between 10 and 30 °C, suggesting a high level of activity at the physiological temperature of ∼ 60 °C. The Michaelis–Menten constants for the substrates ADP and inorganic phosphate were 13 μm and 0.55 mm, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis.