Impact of pH-gradients on the proton uptake rate of cytochrome c oxidase

Cytochrome c oxidase (CcO) is a transmembrane protein and terminal oxidase in the respiratory electron transfer chain of many bacteria and in the mitochondria of eukaryotic cells. In this position, CcO receives electrons, which were generated during cellular respiration, and uses these electrons to reduce dioxygen to water, thereby acting as an electron sink in the respiratory chain. As electrons and protons are taken up from opposite sides of the membrane, the enzymatic activity of CcO contributes to the generation of an electrochemical gradient acting across the membrane hosting the respiratory chain proteins, which drives the synthesis of ATP. Due to its importance in bioenergetics, CcO is subject of intense studies for decades. Nevertheless, hardly any information is currently available regarding the proton uptake rate of CcO, especially regarding the impact of the electrochemical gradient on this rate. In this study, a single-proteoliposome assay is employed to follow proton uptake of individual CcOs from Rhodobacter sphaeroides operating against the action of a well-defined pH gradient. Our measurements reveal that proton uptake is not affected by small pH gradients (< 0.7 pH units) and decays exponentially for larger pH gradients. Furthermore, a linear dependence of substrate concentration on the proton uptake rate is observed over more than 3 orders of magnitude. The obtained scaling laws are surprisingly simple, considering the fact that 4 protons have to be taken up to reduce dioxygen to water, and are discussed in terms of the sequential uptake of protons and electrons occurring during CcOs catalytic cycle.