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J Mol Biol. 2002 Sep 13;322(2):369-81.

Molecular mechanism of the ATP synthase's F(o) motor probed by mutational analyses of subunit a.

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Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstr. 7, CH-8092 Zürich, Switzerland.


The most prominent residue of subunit a of the F(1)F(o) 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 F(o) 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, F(o) with the aR227A mutation did not catalyse 22Na(+)(out)/Na(+)(in) exchange at high internal sodium concentrations, and (ii) at a constant (Delta)pNa(+), 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.

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