Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the Cu-A-cytochrome c domain from the thermophilic Bacillus caa(3)-type cytochrome c oxidase

The C-terminal periplasmic domain of subunit II of the Escherichia cell be-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa(3)-type cytochrome c oxidase containing the Cu-A-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 cell be-type ubiquinol oxidase expressed by the original cytochrome be-expressing plasmid, The chimeric enzyme showed much higher TMPD (N,N,N',N' -tetramethyl-p -phenylenediamine) oxidase activity than the wild-type cytochrome be, but lower activity than the thermophilic Bacillus caa(3)-type cytochrome c oxidase, The chimeric subunit II was confirmed to bind to heme C. These results suggest that the Cu-A-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.