Purification and partial characterization of the membrane-bound cytochrome o(561,564) from Vitreoscilla.
Cytochrome o(561,564) terminal oxidase was solubilized from the membrane fraction of the bacterium Vitreoscilla sp., strain C1, and purified by differential pH dialysis, gel filtration chromatography, and ion-exchange chromatography. Subunit molecular weights, determined on sodium dodecyl sulfate-polyacrylamide gels by the Ferguson plot method, were 49,500 and 23,500. There were two protohemes IX, two coppers, and 45 mol of phosphorus per mole of protomer (73,000). The molecular weight of the cytochrome o complex estimated by chromatography on Sephacryl-400 in deoxycholate was 265,000, which is consistent with the enzyme complex under these conditions being a dimer (146,000) with the remaining molecular weight contribution arising from bound phospholipid, deoxycholate, and possibly other, smaller subunits. Difference spectra of the dithionite-reduced enzyme have split alpha absorption maxima at 561 and 564 nm at room temperature and 558 and 561 nm at 77 K. The CO difference spectrum at room temperature has absorption maxima at 570, 534, and 416 nm. Dissociation constants for CO and cyanide binding to the reduced and oxidized forms of the oxidase are 5.2 microM and 3.5 mM, respectively. The hemes in the cytochrome are one electron accepting centers, both with midpoint potentials around +165 mV at pH 7.0. The enzyme is highly autoxidizable, and its menadiol oxidizing activity is stimulated by phospholipids.
Georgiou
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CD
Department of Biology, Illinois Institute of Technology, Chicago 60616.
Webster
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DA
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003573:Cytochrome b Group; D003580:Cytochromes / Q000302:isolation & purification* / Q000378:metabolism; D041964:Cytophagaceae / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C017168:cytochrome bo, E coli