title: Two distinct proton binding sites in the ATP synthase family.
abstract: The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes.
authors.affiliation: Institut für Mikrobiologie, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: von Ballmoos
authors.forename: Peter
authors.initials: P
authors.lastname: Dimroth
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D045182:Fusobacteria / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D019617:Halobacterium salinarum / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008929:Mitochondria, Heart / Q000201:enzymology; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012399:Rotation; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018724:Spinacia oleracea / Q000166:cytology; D013379:Substrate Specificity
chemical_list: D021122:Protein Subunits; D011522:Protons; D011994:Recombinant Proteins; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D012964:Sodium; D025243:Bacterial Proton-Translocating ATPases