Kinetics of oxidative phosphorylation catalyzed by inside-out plasma membrane vesicles of Paracoccus denitrificans

Abstract

Coupled inside-out plasma membrane vesicles of Paracoccus denitrificans are capable of proton-motive force (pmf)-required, pmf-generating ATP hydrolysis [1]. The steady-state ATP hydrolysis, catalyzed by P. denitrificans FO·F1 (PFO·F1) proceeds via compulsory sequential mechanism where ADP leaves the enzyme–products (ADP and Pi) complex first followed by irreversible release of Pi [2]. Inasmuch operation of PFO·F1 ATPase (synthase) is macroscopically reversible it seems imperative to reconcile the steady-state kinetics of pmf-generating ATP hydrolysis with that of pmf-utilising ATP synthesis. To reach this goal the dependencies of the initial steadystate rates of oxidative phosphorylation on ADP and Pi concentrations and on pmf were measured. ATP synthesis rate showed simple hyperbolic dependence on either substrate (within the concentration ranges of 1.5–50 μM for ADP and 10–500 μM for Pi) with no mutual dependence of apparent Km values thus suggesting random formation of the enzyme–ADP·Pi complex. When the ‘third substrate’, pmf was varied by limitation of succinate or NADH oxidation rates an apparent ‘ping-pong’ mechanism was evident: a decrease of the maximal rates caused by a decrease of pmf resulted in proportional decrease of apparent Km values for either Pi or ADP. This pattern suggests that an irreversible step, presumably ATP formation at the enzyme active site, precedes the involvement of pmf in the overall reaction (likely at the product release step). Comparison of the steady-state kinetics of ATP hydrolysis [2] and synthesis shows that macroscopic reversibility of the PFO·F1 ATPase (synthase) reaction cannot be ascribed to operation of single microscopically reversible enzyme species. We propose that apparent equilibrium between any given pmf and intracellular (intramitochondrial) phosphoryl potential ([ATP]/[ADP]·[Pi]) is maintained by ‘futile cycle’ of ATP synthesis and hydrolysis catalyzed by kinetically (and structurally) distinct FO·F1 species. Supported by the Russian Foundation for Fundamental Research grant 08-04-00594.