Expression of cyoA and cyoB demonstrates that the CO-binding heme component of the Escherichia coli cytochrome o complex is in subunit I.
The cytochrome o complex of the Escherichia coli aerobic respiratory chain is a ubiquinol oxidase. The enzyme consists of at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and contains two heme b prosthetic groups (b555 and b562) plus copper. The sequence of the cyo operon, encoding the subunits of the oxidase, reveals five open reading frames, cyoABCDE. This paper describes results obtained by expressing independently cyoA and cyoB in the absence of the other subunits of the complex. Polyclonal antibodies which react with subunits I and II of the purified oxidase demonstrate that cyoA and cyoB correspond to subunit II and subunit I, respectively, of the complex. These subunits are stably inserted into the membrane when expressed. Furthermore, expression of cyoB (subunit I) results in elevated heme levels in the membrane. Reduced-minus-oxidized spectra suggest that the cytochrome b555 component is present but that the cytochrome b562 component is not. This heme component is shown to bind to CO, as it does in the intact enzyme. Hence, subunit I alone is sufficient for the assembly of the stable CO-binding heme component of this oxidase.
Nakamura
H
H
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Yamato
I
I
Anraku
Y
Y
Lemieux
L
L
Gennis
R B
RB
D015153:Blotting, Western; D002248:Carbon Monoxide / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000032:analysis; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006418:Heme / Q000032:analysis* / Q000378:metabolism; D046911:Macromolecular Substances; D010957:Plasmids; D015183:Restriction Mapping; D013053:Spectrophotometry
D003573:Cytochrome b Group; D046911:Macromolecular Substances; D006418:Heme; C048756:cytochrome b 555; D003300:Copper; D002248:Carbon Monoxide; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV