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Two Distinct Proton Binding Sites in the ATP Synthase Family†
journal contribution
posted on 2007-10-23, 00:00 authored by Christoph von Ballmoos, Peter DimrothThe 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.
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Distinct Proton Binding SitesATP hydrolysis pH profilespH rangearchaeum Halobacterium salinariumDCCDinhibitor dicyclohexylcarbodiimidebinding sitestitration curveproton bindingATP synthase family1Fsigmoidal pH profilecarboxylate protonationion binding modesbinding site residuesCurrent modelsEscherichia coliADPATP synthasescovalent modificationelectrochemical gradientIlyobacter tartaricusproton translocation mechanismhydronium ionacid sequenceacidic c ring residuespH profilec ring
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