摘要：The cytochrome bo 3 quinol oxidase from Vitreoscilla (vbo 3 ) catalyses oxidation of ubiquinol and reduction of O 2 to H 2 O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo 3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na + on the oxidase activity of purified vbo 3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo 3 does not pump Na + ions, but H + across the membrane, and that H + -pumping is not influenced by the presence of Na + . (iii) Using an oxygen pulse method, it was found that 2 H + /e - are ejected from proteoliposomes, in agreement with the values found for the H + -pumping bo 3 oxidase of Escherichia coli (ecbo 3 ). This coincides with the interpretation that 1 H + /e - is pumped across the membrane and 1 H + /e - is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo 3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na + . Overall the data show that vbo 3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo 3 quinol oxidase.
