Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

The operon (cyo) encoding the Na+-pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H+ transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na+-pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na+ against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na+ through the D-channel to the exterior and transports four H+ through the K-channel for the reduction of each O2.