Purification and properties of a cytochrome b560-d complex, a terminal oxidase of the aerobic respiratory chain of Photobacterium phosphoreum.
A cytochrome b560-d complex, a terminal oxidase in the respiratory chain of Photobacterium phosphoreum grown under aerobic conditions, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides with molecular weights of 41,000 and 54,000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 10.2 nmol of protoheme and 22.5 nmol of iron/mg of protein. The enzyme is a "cytochrome bd-type oxidase," showing absorption peaks at 560 and 625 nm in its reduced minus oxidized difference spectrum at 77K. This oxidase combined with CO, and its CO difference spectrum at room temperature in the Soret region showed a peak at 418 nm and a trough at 434 nm. In addition, a trough at 560 nm (cytochrome b), and a trough at 620 nm and a peak at 639 nm (cytochrome d) were observed in the CO-binding spectrum. This cytochrome b560-d complex catalyzed the oxidation of ubiquinol-1 and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride or phenazine methosulfate. The oxidase activity required phospholipids and was inhibited by the respiratory inhibitors, KCN and NaN3, and the divalent cation, ZnSO4. Formation of a membrane potential by the cytochrome b560-d complex reconstituted into liposomes was observed with the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide, on the addition of ubiquinol-1, showing that the enzyme provided a coupling site for oxidative phosphorylation.
D000596:Amino Acids / Q000032:analysis; D001420:Bacteria, Aerobic; D003486:Cyanides / Q000494:pharmacology; D003573:Cytochrome b Group / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000502:physiology; D017937:Cytochrome d Group; D003580:Cytochromes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000502:physiology; D042963:Electron Transport Complex II; D007700:Kinetics; D008081:Liposomes / Q000032:analysis; D008564:Membrane Potentials; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification*; D010455:Peptides / Q000032:analysis; D010776:Photobacterium / Q000201:enzymology*; D013053:Spectrophotometry; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D000596:Amino Acids; D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D008081:Liposomes; D010455:Peptides; D014451:Ubiquinone; C025355:cytochrome b560; D017937:Cytochrome d Group; D010088:Oxidoreductases; D042963:Electron Transport Complex II; C003741:ubiquinol