The Gluconobacter suboxydans respiratory chain has a cytochrome o as a terminal oxidase. The cytochrome o-type oxidase was solubilized with octyl glucoside after washing the membranes with Triton X-100, and was purified by one-step ion-exchange chromatography. The purified oxidase contains four polypeptides, two b-type cytochromes ( b-558 and b-562), and 2 mol of heme/mol of enzyme. The oxidase was shown to be a typical cytochrome o and to have two CO-binding sites in the molecule. The enzyme catalyzes the oxidation of ubiquinol, and the activity is inhibited with KCN or quinone analogues. The purified cytochrome o can be reconstituted with phospholipids prepared from G. suboxydans into proteoliposomes by octyl glucoside dilution. The proteoliposome generates a proton electrochemical gradient (inside negative and alkaline) of about −140 mV during ubiquinol oxidation. The generation of membrane potential and pH gradient was determined by fluorometric methods using carbocyanine and dansylglycine, respectively. Thus, cytochrome o of G. suboxydans was shown to be an ubiquinol oxidase functioning as an energy-generator.