Abstract
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.
Highlights
Ichiro Yamato, and Yasuhiro Anraku of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan
Reduced-minus-oxidized spectra suggest that the cytochrome bsss component is present but that the cytochrome bsea component is not. This heme component is shown to bind to CO, as it does in the intact enzyme
Plasmids expressing portions of cyo were prepared in several ways, and expression of subunits I and II from plasmids was examined immunologically using an E. coli host strain from which cyo was deleted from the chromosome (ST4625)
Summary
Takara Shuzo Co., Kyoto, Japan and New England BioLabs. Bal-31 exonuclease was purchased from Takara Shuio Co., Kyoto, Japan. 0.5% glucose was used for aerobic cell culture and plasmid propagation. In situ colony immunological screening was previously described [22]. TH1559 (recB21, recC22, sbcB15) by double homologous recombination with linearized pHN4001 as described previously [23] and contains no detectable cytochrome o complex immunologically and spectroscopically. To clone the entire cyo region, a DNA library was constructed with Xtir phage vector [27] containing the Sau3A partial digests of E. coli DNA and screened by plaque hybridization with the EcoRI( l)-BglII[1] fragment of pHN3001 as a DNA probe. This plasmid was isolated from cells grown anaerobically, since DHl/pHN3795 failed to grow under aerobic growth condition due to a dosage effect of the TABLE I
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