Mechanistic Implications of Mg++, Adenine Nucleotide, and Inhibitor Effects on Energy-linked Reactions of Submitochondrial Particles

Abstract

Abstract Oxygen exchange reactions in sonically prepared submitochondrial particles show a nearly absolute dependence upon added adenine nucleotides. The orthophosphate ⇌ adenosine triphosphate and the ATP ⇌ HOH exchange reactions, in contrast to the Pi ⇌ HOH exchange, are favored by presence of ADP rather than ATP, are more sensitive to p-mercuribenzoate and to 2,4-dinitrophenol, and require a higher Mg++ concentration. The marked disparities in exchange rates at low ADP concentrations are theoretically incompatible with a reaction scheme involving only one point of entry of water oxygen. The ATP-driven energy-linked reduction, ATPase activity, and a prominent Pi ⇌ HOH exchange will proceed at much lower Mg++ concentrations than required for the ATP ⇌ HOH and Pi ⇌ ATP exchanges. The Pi ⇌ HOH exchange induced by ATP cleavage occurs principally with Pi of the medium. The ATP ⇌ HOH exchange, but not the Pi ⇌ HOH exchange, appears to occur only in association with oxidative phosphorylation. Reactions separate from the phosphorylation reaction of oxidative phosphorylation appear responsible for ATP-driven energy-linked reduction, ATPase activity, and a prominent Pi ⇌ HOH exchange. These and other results are consistent with a mechanism for oxidative phosphorylation in which a high energy compound or state, independent from concomitant oxidation-reduction, drives water formation directly from an oxygen of Pi, resulting in ATP formation by a concerted reaction or with transient metaphosphate formation. Present data do not, however, eliminate an indirect route of oxygen from Pi to water.