Control of Proton Conduction by the H⁺‐ATPase in the Inner Mitochondrial Membrane

Abstract

Proton conduction in submitochondrial particles with various degrees of resolution of the H + ‐ATPase has been studied. Proton conduction was analyzed by following the kinetics of the anaerobic decay of protons taken up by submitochondrial particles during respiratory pulses. In EDTA submitochondrial particles proton release exhibited biphasic kinetics; both phases were depressed by collapsing aerobic Δφ. The slow phase of proton diffusion was inhibited by oligomycin and N,N′ ‐dicyclohexylcarbodiimide as well as by F 1 ligands like adenylyl 5′‐imidophosphate and alkyl cations. Inhibition of proton conduction by F 1 ligands exhibited a sigmoidal titration curve and was synergistic with inhibition by oligomycin. Removal of the ATPase inhibitor markedly enhanced the kinetic constant of the slow proton diffusion process. The initial rapid phase of proton diffusion was inhibited by N,N′ ‐dicyclohexylcarbodiimide and oligomycin but was unaffected by F 1 ligands. When the F 1 moiety was removed from the particles, proton conduction became monophasic and was not any more inhibited by F 1 ligands. It is concluded that the H + ‐ATPase contributes to both phases of proton conduction and that energy‐linked cooperative interaction of the F 1 with the F 0 moiety controls proton conductivity. Evidence is also providëd for involvement of negatively charged groups in hydrophobic environments of the F 1 moiety in proton translocation by the H + ‐ATPase.