Studies of the Energy‐Transfer System of Submitochondrial Particles

Abstract

Added in appropriate concentrations oligomycin stimulates oxidative phosphorylation and its reversal as well as P_i‐ATP exchange in submitochondrial particles derived from beef heart mitochondria by sonication in the presence of EDTA. The optimal oligomycin concentration for these effects is in the range of 0.2–0.4 μg/mg protein, above which concentration oligomycin is inhibitory. The optimal oligomycin concentration inhibits the ATPase reaction catalyzed by the same particles only by 20–25%. The maximal P/O ratio observed in the presence of oligomycin is approximately 1 with NADH, 0.8 with succinate, and 0.2 with ascorbate plus tetramethyl‐p‐phenylene diamine as substrate. Mg⁺⁺, which is obligatory for the phosphorylation, lowers the P/O ratio when used in concentrations exceeding 2 mM. Oligomycin‐stimulated reversal of oxidative phosphorylation also is Mg⁺⁺ dependent, but insensitive to high Mg⁺⁺ concentrations. Oligomycins A, B, C, and D (rutamycin) are equally effective in exhibiting these effects, whereas aurovertin is ineffective and even abolishes the effects of oligomycin. Oligomycin does not stimulate phosphorylation in intact mitochondria or in submitochondrial particles prepared in the presence of ATP and Mg⁺⁺. Optimal response with EDTA particles is obtained when these are sonicated in the presence of 2 mM EDTA at pH 8.5–8.7. Oligomycin also induces a respiratory inhibition in EDTA particles which is relieved by uncouplers. The inhibition is 60–70% in the case of NADH, and 40–50% in the case of succinate as substrate. Little or no oligomycin‐induced “respiratory control” is observed with ascorbate plus tetramethyl‐p‐phenylenediamine as substrate. The oligomycin‐induced respiratory control does not require Mg⁺⁺ and is diminished by the latter and by other divalent cations as well as certain anions. Aurovertin does not duplicate the effect of oligomycin in inducing respiratory control, and has no effect on the oligomycin‐induced respiratory control or its relief by uncouplers. The “coupling factors” F₁, F₄, and F₀, alone or in combination, do not duplicate the effect of oligomycin in inducing “respiratory control” in these particles. Oligomycin and aurovertin inhibit oxidative phosphorylation and P_i‐ATP exchange in phosphorylating submitochondrial particles prepared in the presence of ATP and Mg⁺⁺, and the effects of the two antibiotics on the P/O ratio are additive. In contrast, aurovertin fails to inhibit the reversal of oxidative phosphorylation measured as ATP‐supported succinate‐linked NAD⁺ reduction, in concentrations at which oligomycin is completely inhibitory. At much higher concentrations aurovertin does inhibit the latter reaction, but the degree of inhibition decreases with time of incubation in the presence of ATP and Mg⁺⁺. It is concluded that oligomycin acts on the respiratory chain‐linked energy‐transfer system of EDTA particles in two ways: at low concentrations, it inhibits the hydrolytic cleavage of a nonphosphorylated high‐energy intermediate, which is common for the 3 energy‐coupling sites of the respiratory chain, and in higher concentrations it inhibits the interaction of the same intermediate with P_i and ADP to form ATP. The available evidence suggests that aurovertin acts on the phosphorylation sequence by a mechanism or at a site different from the mechanism or site of action of oligomycin.