Evidence for energy-dependent change in phosphate binding for mitochondrial oxidative phosphorylation based on measurements of medium and intermediate phosphate-water exchanges.
Characteristics of the exchange reactions catalyzed by beef heart submitochondrial particles give new insight into energy transducing steps of oxidative phosphorylation. The uncoupler-insensitive portion of the total Pi in equilibrium  HOH exchange in presence of ATP, ADP, and Pi is the intermediate Pi in equilibrium HOH exchange, that is the exchange occurring with Pi formed by hydrolysis of ATP prior to release of Pi from the catalytic site. The exchange of medium Pi with HOH is as sensitive to uncouplers as the Pi in equilibrium ATP exchange and net oxidative phosphorylation, demonstrating a requirement of an uncoupler-sensitive energized state, probably a transmembrane potential or proton gradient, for bringing medium Pi to the reactive state. The covalent bond forming and breaking step at the catalytic site (ADP + Pi in equilibrium ATP + HOH) appears relatively insensitive to uncouplers. Thus to the extent that uncouplers dissipate transmembrane proton-motive force, it is unlikely that such a force is used to drive ATP formation by direct protonations of Pi oxygens. When only Pi and ADP are added and formation of ATP from added ADP by adenylate kinase and subsequent ATP hydrolysis are adequately blocked, no Pi in equilibrium HOH exchange can be observed, demonstrating a requirement of energization by ATP binding and cleavage for such an exchange. This uncoupler-insensitive energization is suggested to represent a conformationally energized state that can be used reversibly to develop a transmembrane protonmotive force accompanying ADP and Pi release. Rates of various exchanges as estimated by improved procedures are compatible with all oxygen exchanges occurring by dynamic reversal of ATP hydrolysis at the catalytic site.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004140:Dinitrophenols / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006593:Hexokinase / Q000494:pharmacology; D007700:Kinetics; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D010710:Phosphates / Q000378:metabolism*; D051381:Rats
D004140:Dinitrophenols; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase; D000251:Adenosine Triphosphatases; D005947:Glucose