Small pH Gradients Inhibit Cytochrome c Oxidase: Implications for H+ entry to the Binuclear Center

Martyn A. Sharpe, John M. Wrigglesworth, Jerome Louewen and Peter Nicholls Biochem. Biophys. Res Comm. 216, 931-938, 1995.

SUMMARY: Respiring cytochrome c oxidae proteoliposomes (COV) generate internal alkalinity (Delta-pH) and a membrane potential (Delta-psi). Valinomycin collapses Delta-psi, increases Delta-pH and slows steady-state respiration. If Delta-pH is heterogeneously expressed trappd probes will underestimate it. Internal pH changes were therefore followed in COV containing 2 buffer systems of differing pK. The alkalinization rate at pH 7 was unaffected by added AMPSO (pK 9.0) to the usual internal HEPES (pK 7.5) At higher pH, AMPSO slowed the approach to steady-state. Delta-pH inhibition is therefore not due to a large alkalinization in a small COV fraction. The oxygen 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 can put the the center electrically "inside" even though it is physically "outside".