Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Evidence for linkage of the energy-yielding and energy-consuming steps by freely diffusible intermediates and for an allosteric mechanism of respiratory control at coupling site 2

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Abstract
The three coupling segments of the respiratory chain of bovine heart mito-chondria were examined individually by steady-state kinetic methods to determine whether or not freely diffusible intermediates occur between the energy-yielding and energy-consuming steps involved in the oxidative phosphorylation of extramitochondrial ADP. The principal method employed was the dual inhibitor technique, for which an appropriate model is provided. The results indicate that in accordance with the chemiosmotic theory the intermediate reactants that link the energy-yielding rotenone-sensitive (Site 1), cytochromebc 1 (Site 2), and cytochromeaa 3 (Site 3) reactions of the respiratory chain to the energy-consuming ATP synthetase, AdN transport, and Pi transport reactions are freely diffusible (delocalized). Site 2 was found to differ from the others in regard to the mechanism by which the energy-linked respiratory chain reaction is controlled by the energy-consuming steps. Whereas the Site 1 and Site 3 respiratory chain reactions are controlled primarily by the thermodynamic mechanism of reaction reversal, the Site 2 respiratory reaction is controlled primarily by a kinetic mechanism in which an intermediate that links it to the energy-consuming steps inhibits it allosterically. From the effects of nigericin and valinomycin the allosteric intermediate appears to be the electrical component of the protonmotive force.