A spectro-electrochemical system is presented in which the proton pumping activity of cytochrome bo(3), 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 bo(3) electrochemically, a maximum pH gradient of 0.54 units is obtained, while the oxygen reducing activity of cytochrome bo(3) is found to increase when the pH inside the vesicle increases from 7.4 to 7.8. (C) 2011 Elsevier Ltd. All rights reserved.