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 cel l be-type ubiquinol oxidase was replaced with the counterpart of the t hermophilic Bacillus caa(3)-type cytochrome c oxidase containing the C u-A-cytochrome c domain by means of gene engineering techniques, The c himeric terminal oxidase was expressed by a pBR322 derivative in a ter minal 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 p lasmid, The chimeric enzyme showed much higher TMPD (N,N,N',N' -tetram ethyl-p -phenylenediamine) oxidase activity than the wild-type cytochr ome 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 graft ed to this membrane anchor can facilitate electron transfer from reduc ed TMPD to low-spin protoheme b in subunit I.