Vesicle-modified electrodes to study proton-pumping by membrane proteins

A spectro-electrochemical system is presented in which the proton pumping activity of cytochrome bo3, an ubiquinol oxidase from Escherichia coli, is measured simultaneously with its oxygen reducing activity. Proteoliposomes are loaded with a pH-sensitive fluorescent probe and adsorbed on a gold electrode. The electrode is modified such that the vesicles retain their integrity while the lipophilic ubiquinone in the vesicles is in electric contact with the electrode. After activating cytochrome bo3 electrochemically, a maximum pH gradient of 0.54 units is obtained, while the oxygen reducing activity of cytochrome bo3 is found to increase when the pH inside the vesicle increases from 7.4 to 7.8.