Mitochondrial ATP Synthase: Dramatic Mg₂₊-Induced Alterations in the Structure and Function of the F₁-ATPase Moiety

The ATPase activity of the F₁moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 °C in the presence of MgCl₂. The concentration of MgCl₂(130 μM) required to induce half-maximal inactivation is over 30 times higher than the apparent K_m (MgCl₂) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F₁-ATPase activity by Mg₂₊ is accompanied by the dramatic dissociation from the F₁complex of a subunits and part of the 7-subunit population. The latter form a precipitate while the β, δ, and ∊ subunits, and the remaning part of the 7-subunit population, remain soluble. Dissociation is not a sudden “all or none” event but parallels loss of ATPase activity until a subunits have almost completely dissociated together with about 50% of the 7-subunit population. Mg₂₊-induced loss of F₁-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KP_iat high concentration (>200 mM) is effective in partially protecting F₁against inactivation. However, the most effective means of preventing Mg₂₊-induced inactivation of F₁-ATPase activity is to rebind F₁ to its F₀moiety in F₁-depleted particles. When bound to F₀, F₁is protected completely against divalent cation induced inactivation. These results indicate that F₁contains, in addition to the high-affinity divalent cation sites involved in promoting catalysis, additional sites of lower affinity which either are masked in the intact ATP synthase complex (f0f1)or interact with the intact complex with a functional purpose other than inactivation.