Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the CuA-cytochrome c domain from the thermophilic Bacillus caa3-type cytochrome c oxidase.
The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.
Uchida
A
A
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Fukuoka.
Kusano
T
T
Mogi
T
T
Anraku
Y
Y
Sone
N
N
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003300:Copper; D003573:Cytochrome b Group; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D003580:Cytochromes / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008667:Metalloproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000378:metabolism
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D008667:Metalloproteins; D011993:Recombinant Fusion Proteins; D003300:Copper; C017168:cytochrome bo, E coli; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV