The proton pumping bo oxidase from Vitreoscilla

The cytochrome bo₃ quinol oxidase from Vitreoscilla (vbo₃) catalyses oxidation of ubiquinol and reduction of O₂ to H₂O. 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 vbo₃ 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 vbo₃ solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo₃ 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 bo₃ oxidase of Escherichia coli (ecbo₃). 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 vbo₃ 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 vbo₃ is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo₃ quinol oxidase.