The Proton Pump of Cytochrome c Oxidase and Its Stoichiometry

Abstract

The operation of cytochrome c oxidase with ascorbate/N,N,N'',N''-tetramethyl-p-phenylenediamine as substrate in antimycin-A-inhibited rat liver mitochondria is coupled to proton ejection. Measurements of the initial rate of valinomycin-dependent K+ uptake showed that nearly 4 K+ are taken up as 2 electrons are transferred from cytochrome c to O2. This proves directly that a charge separation of nearly 4 occurs across the inner mitochondrial membrane each time 2 electrons are transferred to O2. Measurements of the initial rate of proton movement after addition of the reductant show that about 1.6 protons are released by the mitochondria as 2 electrons are transferred from cytochrome c to O2. The data support the suggestion of a proton pump coupled to the operation of cytochrome c oxidase.