title: Molecular mechanism of the ATP synthase's F(o) motor probed by mutational analyses of subunit a.
abstract: 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.
authors.affiliation: Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstr. 7, CH-8092 Zürich, Switzerland.
authors.forename: Franziska
authors.initials: F
authors.lastname: Wehrle
authors.forename: Georg
authors.initials: G
authors.lastname: Kaim
authors.forename: Peter
authors.initials: P
authors.lastname: Dimroth
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005673:Fusobacterium / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D021122:Protein Subunits; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D021122:Protein Subunits; D011510:Proteolipids; C020485:proteoliposomes; D006639:Histidine; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D012964:Sodium; D006180:Proton-Translocating ATPases; D008239:Lysine