Proton pumping by cytochrome oxidase as studied by time-resolved stopped-flow spectrophotometry

The H+/e- stoichiometry for the proton pump of cytochrome c oxidase reportedly varies between 0 and 1, depending on experimental conditions. In this paper, we report the results obtained by a combination of transient optical spectroscopy with a time resolution of 10 ms and a singular value decomposition analysis to follow the kinetics, separate the observed spectral components, and quantitate the stoichiometry of the pump. By using cytochrome oxidase reconstituted into small unilamellar vesicles, we show that the time courses of ferrocytochrome c oxidation and phenol red acidification or alkalinization fit a simple kinetic scheme. The fitting procedure leads to unbiased and objective determination of the H+/e- ratio under various experimental conditions. The proton-pumping stoichiometry was found to be 1.01 +/- 0.10, independent of the number of turnovers, proton back-leak rate, or type of experiment (oxidant or reductant pulse).