Identification of the electron transfers in cytochrome oxidase that are coupled to proton-pumping

Abstract

MITOCHONDRIAL cytochrome oxidase is a functionally complex, membrane-bound respiratory enzyme which catalyses both the reduction of O₂ to water and proton-pumping. During respiration, an exogenous donor, cytochromec, donates four electrons to O₂ bound at the bimetallic haem a₃ Fe–Cu centre within the enzyme. These four electron transfers are mediated by the enzyme's haem a and Cu_A redox centres and result in the translocation of four protons across the inner mitochondria! membrane.¹ The molecular mechanism of proton translocation has not yet been delineated, however, and in the absence of direct experimental evidence all four electron transfers have been assumed to couple equally to proton-pumping. Here, I report the effects of proton-motive force and membrane potential on two equilibria involving intermediates of the bimetallic centre at different levels of O₂ reduction.^2–7 The results show that only two of the electron transfers, to the 'peroxy' and 'oxyferryl' intermediates of the bimetallic centre, are linked to proton translocation, a finding which strongly constrains candidate mechanisms for proton-pumping.