Small pH Gradients inhibit cytochrome C oxidase: Implications for H⁺ entry to the binuclear center

Respiring cytochrome c oxidase proteoliposomes generate internal alkalinity (ΔpH) and a membrane potential (ΔΨ). Valinomycin collapses ΔΨ, increases ΔpH, and slows steady state respiration. If ΔpH is heterogeneously expressed trapped probes will underestimate it. Internal pH changes were therefore followed in COV containing two buffer systems of differing pKs. The alkalinization rate at pH 7 was unaffected by adding AMPSO (pK 9.0) to the usual internal HEPES (pK 7.5). At higher pH, AMPSO slowed the approach to steady state. ΔpH inhibition is therefore not due to a large alkalinization in a small COV fraction. The O₂-reducing center may move protons via a local aqueous phase that is near electrical and pH equilibrium with the phase inside the COV. The dielectric in this membrane region can put the center electrically 'inside' even though it is physically 'outside'.