Abstract
FoF1-ATP synthase (FoF1) synthesizes ATP in the F1 portion when protons flow through Fo to rotate the shaft common to F1 and Fo. Rotary synthesis in isolated F1 alone has been shown by applying external torque to F1 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 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 Q10 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.
Highlights
FoF1-ATP synthase (FoF1) synthesizes the majority of cellular ATP from ADP and Pi in respiratory and photosynthetic organisms [1,2,3,4]
ATP synthesis by mutant Bacillus PS3 FoF1-ATPase (TFoF1) lacking the C-terminal domain of the e-subunit (TFoFe1Dc) reconstituted into liposomes
In the absence of a nucleotide in the medium, TFoF1 is resting in a state inhibited by the e-subunit [25], and recent studies suggest the possibility that activation of such TFoF1 to initiate ATP synthesis requires an extra proton motive force (PMF) in addition to the thermodynamically required magnitude of PMF [26,27]
Summary
FoF1-ATP synthase (FoF1) synthesizes the majority of cellular ATP from ADP and Pi in respiratory and photosynthetic organisms [1,2,3,4]. In the simplest version of bacterial FoF1, the subunit compositions are ab2c10–15 (Fo) and a3b3cde (F1) Both Fo and F1 are rotary motors, Fo being driven by proton flow and F1 by ATP hydrolysis. When the proton motive force (PMF) is greater than the free energy drop in ATP hydrolysis, Fo wins and lets F1 rotate in its reverse direction. Because the thermophilic enzyme is robust and suited to single-molecule studies [3,5,6,7,8,10,11], we investigated whether TFoF1 with high synthesis activity can be prepared. The activity at room temperature (25 °C) of 10 s) suggests, on the basis of three ATPs per revolution [24], a rotary rate of 3 revolutions s), which should be readily detected in single-molecule studies under a microscope
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