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 O(2).
Kim
Seong K
SK
Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
Stark
Benjamin C
BC
Webster
Dale A
DA
D019943:Amino Acid Substitution; D001693:Biological Transport, Active / Q000502:physiology; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial; D020589:Vitreoscilla / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D012964:Sodium; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D000254:Sodium-Potassium-Exchanging ATPase