Nucleotide/H+‐dependent change in Mg2+ affinity at the ATPase inhibitory site of the mitochondrial F1‐F0 ATP synthase

Abstract

The interactions between ADP and Mg²⁺ that result in the slowly reversible inhibition of the mitochondrial F₁-F₀ ATPase were studied. The K _i for the inhibitory Mg²⁺ is shown to be strongly dependent on the occupation of the nucleotide-binding sites. The inhibitory binding site for Mg²⁺ is not seen unless a stoichiometric amount of ADP is added [Biochem. J. 276 (1991) 149-156]; it appears (K _i = 2.10⁻⁶ M) in the presence of stoichiometric ADP and the affinity for inhibitory Mg²⁺ decreases to a K _i, value of 7.10⁻⁵ M when the second nueleotide binding site with k _d = 5.10⁻⁶ M is loaded with ADP. The binding of the inhibitory Mg²⁺ is competitively inhibited by H⁺ ions within the pH interval 6.8–8.2. The nucleotide-dependent affinity transition of the Mg²⁺-specific site suggests that H⁺/Mg²⁺ exchange may play an important role in the catalytic mechanism of ATP synthesis/hydrolysis at the active site(s) of F₁-F₀ ATP synthase.