Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase

FoF1-ATP synthase (FoF1) 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 FoF1 (TFoF1), 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 TFoF1 lacking an inhibitory segment of the epsilon-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 similar to 16 s(-1) at 30 degrees C with a Q(10) temperature coefficient of 3-4 between 10 and 30 degrees C, suggesting a high level of activity at the physiological temperature of similar to 60 degrees C. The Michaelis-Menten constants for the substrates ADP and inorganic phosphate were 13 mu M and 0.55 mM, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis.