The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain.
The aerobic respiratory chain of Escherichia coli is branched. In aerobically grown cells harvested in midexponential phase, a respiratory chain containing only b-type cytochromes is predominant. This chain contains a terminal oxidase which is a b-type cytochrome, referred to as cytochrome o. However, when the bacteria are grown under conditions of oxygen limitation, additional components of the respiratory chain are induced, as evidenced by the appearance of new spectroscopic species. These include a new b-type cytochrome, cytochrome b558, as well as cytochrome a1 and cytochrome d. In this paper, a purification protocol and the initial characterization of the terminal oxidase complex containing cytochrome d are reported. Solubilization of the membrane is effected by Zwittergent 3-12, and purification is accomplished by chromatography with DEAE-Sepharose CL-6B and hydroxyapatite. The complex contains cytochrome b558, a1, and d. Analysis by sodium dodecyl sulfate-polyacrylamide gels indicates that the complex contains only two types of polypeptides with the molecular weights estimated to be 57,000 and 43,000. The purified complex has oxidase activity in the presence of detergents, utilizing substrates including ubinquinol-1, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,3,5,6-tetramethyl-p-phenylenediamine. The cytochrome d complex contains protoheme IX and iron, but does not contain nonheme iron or copper. Approximately half of the cytochromes which are thought to participate in E. coli aerobic respiration are accounted for by this single complex. These results suggest that the E. coli aerobic respiratory chain is organized around a relatively small number of cytochrome-containing complexes.
D003573:Cytochrome b Group / Q000302:isolation & purification; D017937:Cytochrome d Group; D003580:Cytochromes / Q000302:isolation & purification*; D045263:Cytochromes a1; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008970:Molecular Weight; D019255:NADPH Oxidases; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D045263:Cytochromes a1; D017937:Cytochrome d Group; C017168:cytochrome bo, E coli; C036888:cytochrome b558; D019255:NADPH Oxidases