Interaction of Mg2+ with F0·F1 mitochondrial ATPase as related to its slow active/inactive transition

Abstract

Bovine heart submitochondrial particles incubated with a low concentration of ADP in the presence of Mg2+ and passed through a Sephadex column equilibrated with EDTA exhibit sensitivity of their initial ATPase activity to preincubation with Mg2+. By using particles thus prepared, several characteristics of a Mg2+-specific inhibitory site on F0·F1 ATPase were studied. The inhibition was shown to be both time- and Mg2+-concentration-dependent, with an equilibrium constant (at infinite time) of 2 x 10-6 M (25°C, pH 7.5). The dependence of the pseudo-first-order rate constant for the inhibition process on Mg2+ concentration suggests the presence of a single Mg2+-binding site with K(s) = 1.1 x 10-4 M. The data obtained are consistent with a two-step mechanism of Mg2+-F0·F1 interaction which results in a loss of the ATPase activity; it includes rapid pH-dependent binding of Mg2+ at the site with K(s) = 1.1 x 10-4 M, followed by a slow interconversion of the Mg2+-F1 complex into inactive ATPase (k(in.) = 0.65 min-1, k(act.) = 0.01 min-1). The Mg2+-inhibited ATPase is very slowly (t( 1/2 ) ~ 90 min) re-activated in the presence of EDTA. The rate of EDTA-induced re-activation is pH-independent and can be dramatically increased by added ATP, P(i) and sulphite. The dissociation constants for free ATP and P(i) (5 x 10-7 M and 1 x 10-3 M respectively) and the maximal activation rates were determined by measuring the hyperbolic dependencies of the EDTA-induced re-activation of Mg2+-de-activated ATPase on the concentrations of the accelerating ligands. Taken together, the data obtained show two functionally detectable free nucleotide-specific binding sites, on site for P(i) and one Mg2+-specific ATPase-inhibitory site on the F0·F1 mitochondrial ATP synthase complex.