ATPase/synthase activity of Paracoccus denitrificans Fo·F1 as related to the respiratory control phenomenon

Abstract

The time course of ATP synthesis, oxygen consumption, and change in the membrane potential in Paracoccus denitrificans inside-out plasma membrane vesicles was traced. ATP synthesis initiated by the addition of a limited amount of either ADP or inorganic phosphate proceeded up to very low residual concentrations of the limiting substrate. Accumulated ATP did not decrease the rate of its synthesis initiated by the addition of ADP. The amount of residual ADP determined at State 4 respiration was independent of ten-fold variation of Pi or the presence of ATP. The pH-dependence of Km for Pi could not be fitted to a simple phosphoric acid dissociation curve. Partial inhibition of respiration resulted in a decrease in the rate of ATP synthesis without affecting the ATP/ADP reached at State 4. At pH8.0, hydrolysis of ATP accumulated at State 4 was induced by a low concentration of an uncoupler, whereas complete uncoupling results in rapid inactivation of ATPase. At pH7.0, no reversal of the ATP synthase reaction by the uncoupler was seen. The data show that ATP/ADP×Pi ratio maintained at State 4 is not in equilibrium with respiratory-generated driving force. Possible mechanisms of kinetic control and unidirectional operation of the Fo·F1-ATP synthase are discussed.