Defining the structural domain of subunit II of the heme-copper terminal oxidase using chimeric enzymes constructed from the Escherichia coli bo-typeubiquinol oxidase and the thermophilic Bacillus caa(3)-type cytochrome c oxidase

To probe the location of the quinol oxidation site and physical interaction s for intersubunit electron transfer, we constructed and characterized two chimeric oxidases in which subunit II (CyoA) of cytochrome be-type ubiquino l oxidase from Escherichia coli was replaced with the counterpart (CaaA) of caa(3)-type cytochrome c oxidase from thermophilic Bacillus PS3. In pHNchi 5, the C-terminal hydrophilic domain except a connecting region as to trans membrane helix II of CyoA was replaced with the counterpart of CaaA, which carries the Cu-A site and cytochrome c domain. The resultant chimeric oxida se was detected immunochemically and spectroscopically, and the turnover nu mbers for Q(1)H(2) (ubiquinol-l) and TMPD (N,N,N',N'-tetramethyl-p-phenylen ediamine) oxidation were 28 and 8.5 s(-1), respectively. In pHNchi6, the ch imeric oxidase was designed to carry a minimal region of the cupredoxin fol d containing all the Cu-A ligands, and showed enzymatic activities of 65 an d 5.1 s(-1), and an expression level better than that of pHNchi5. Kinetic a nalyses proved that the apparent lower turnover of the chimeric enzyme by p HNchi6 was due to the higher K-m of the enzyme for Q(1)H(2) (220 mu M) than that of cytochrome be (48 mu M), while in the enzyme by pHNchi5, both subs trate-binding and internal electron transfer were purturbed. These results suggest that the connecting region and the C-terminal alpha(1)-alpha(2)- be ta(11)-alpha(3) domain of CyoA are involved in the quinol. oxidation and/or physical interactions for inter-subunit electron transfer, supporting our previous proposal [Sato-Watanabe, M., Mogi, T., Miyoshi, H., and Anraku, Y. (1998) Biochemistry 37, 12744-12752]. The close relationship of E. coli qu inol oxidases to cytochrome c oxidase of Gram-positive bacteria like Bacill us was also indicated.