Inhibition of respiration in submitochondrial particles by uncouplers of oxidative phosphorylation.

High concentrations of uncouplers of oxidative phosphorylation inhibit the oxidation of succinate and NADH catalyzed by phosphorylating beef heart submitochondrial particles. Inhibition is reversed by bovine serum albumin but not by ATP. ATP does not influence either the stimulation of respiration at low uncoupler concentrations or the inhibition of respiration at high uncoupler concentrations. The cytochromes of the electron transfer chain appear to be in the oxidized state during the oxidation of NADH catalyzed by submitochondrial particles partially inhibited by uncoupler, indicating that the inhibitory site resides at, or prior to, the substrate dehydrogenase. These data are consistent with a recent hypothesis which attempts to account for uncoupling action on the basis of competition between uncoupler and substrate molecules for an energy-requiring transport system in the mitochondrial membrane. However, not supportive of such a hypothesis are data indicating that in submitochondrial particles the inhibition does not appear to be of the competitive type and, furthermore, agents and procedures which destroy permeability barriers in mitochondria, and preparations of “open” submitochondrial particles, do not interfere with the uncoupler-mediated inhibition of substrate oxidation by the submitochondrial particle. It is suggested that high uncoupler concentrations interfere with the function of the nonheme iron moieties of the succinic and NADH dehydrogenases. Consistent with this suggestion are the findings that dithiothreitol partially reverses the uncoupler-mediated inhibition of substrate oxidations in submitochondrial particles and also protects against the inhibitory effects of uncoupler. In addition, the nonheme iron components of the succinic and NADH dehydrogenases were reducible by their respective substrates, while the characteristic electron paramagnetic resonance signal of the succinic dehydrogenase nonheme iron moiety was not observed in the uncoupler-inhibited submitochondrial particle upon the addition of NADH.