SMALL PH GRADIENTS INHIBIT CYTOCHROME-C-OXIDASE - IMPLICATIONS FOR H+ENTRY TO THE BINUCLEAR CENTER

Respiring cytochrome c oxidase proteoliposomes generate internal alkal inity (Delta pH) and a membrane potential (Delta Psi). Valinomycin col lapses Delta Psi, increases Delta pH, and slows steady state respirati on. If Delta pH is heterogeneously expressed trapped probes will under estimate it. Internal pH changes were therefore followed in COV contai ning two buffer systems of differing pKs. The alkalinization rate at p H 7 was unaffected by adding AMPSO (pK 9.0) to the usual internal HEPE S (pK 7.5). At higher pH, AMPSO slowed the approach to steady state. D elta pH inhibition is therefore not due to a large alkalinization in a small COV fraction. The O-2-reducing center may move protons via a lo cal aqueous phase that is near electrical and pH equilibrium with the phase inside the COV. The dielectric in this membrane region can put t he center electrically 'inside' even though it is physically 'outside' . (C) 1995 Academic Press, Inc.