Molecular Mechanism of the ATP Synthaseʹs Fo Motor Probed by Mutational Analyses of Subunit a

The most prominent residue of subunit a of the F1Fo ATP synthase is a universally conserved arginine (aR227 in Propionigenium modestum), which was reported to permit no substitution with retention of ATP synthesis or H+-coupled ATP hydrolysis activity. We show here that ATP synthases with R227K or R227H mutations in the P. modestum a subunit catalyse ATP-driven Na+ transport above or below pH 8.0, respectively. Reconstituted Fo with either mutation catalysed 22Na+out/Na+in exchange with similar pH profiles as found in ATP-driven Na+ transport. ATP synthase with an aR227A substitution catalysed Na+-dependent ATP hydrolysis, which was completely inhibited by dicyclohexylcarbodiimide, but not coupled to Na+ transport. This suggests that in the mutant the dissociation of Na+ becomes more difficult and that the alkali ions remain therefore permanently bound to the c subunit sites. The reconstituted mutant enzyme was also able to synthesise ATP in the presence of a membrane potential, which stopped at elevated external Na+ concentrations. These observations reinforce the importance of aR227 to facilitate the dissociation of Na+ from approaching rotor sites. This task of aR227 was corroborated by other results with the aR227A mutant: (i) after reconstitution into liposomes, Fo with the aR227A mutation did not catalyse 22Na+out/Na+in exchange at high internal sodium concentrations, and (ii) at a constant ΔpNa+, 22Na+ uptake was inhibited at elevated internal Na+ concentrations. Hence, in mutant aR227A, sodium ions can only dissociate from their rotor sites into a reservoir of low sodium ion concentration, whereas in the wild-type the positively charged aR227 allows the dissociation of Na+ even into compartments of high Na+ concentration.