Purified cbb 3-type oxidase of Bradyrhizobium japonicum was reconstituted into phospholipid vesicles. Tight vesicles were obtained as shown by the disturbance of ΔpH with CCCP and the membrane potential with valinomycin, which led to a six-fold increase in cytochrome c oxidase activity. The vesicles were thus suitable for proton translocation experiments. In the presence of valinomycin, a pulse with reduced cytochrome c caused an acidification with a subsequent alkalinization, whereas the same pulse caused only an alkalinization in the presence of valinomycin plus CCCP. We conclude that the cbb 3-type oxidase of B. japonicum is a proton pump.
