Defining the Structural Domain of Subunit II of the Heme-Copper Terminal Oxidase Using Chimeric Enzymes Constructed from the Escherichia coli bo-Type Ubiquinol Oxidase and the Thermophilic Bacillus caa3-Type Cytochrome c Oxidase1.

To probe the location of the quinol oxidation site and physical interactions for inter-subunit electron transfer, we constructed and characterized two chimerlc oxidases in which subunit II (CyoA) of cytochrome bo-type ubiquinol oxidase from Escherichia coli was replaced with the counterpart (CaaA) of caa3 -type cytochrome c oxidase from thermo-philic Bacillus PS3. In pHNchi5, the C-terminal hydrophilic domain except a connecting region as to transmembrane helix II of CyoA was replaced with the counterpart of CaaA, which carries the CuA site and cytochrome c domain. The resultant chimeric oxidase was detected immunochemically and spectroscopically, and the turnover numbers for Q1H2 (ubiquinol-1) and TMPD ( N, N, N', N'-tetramethyl-p-phenylenediamine) oxidation were 28 and 8.5 s-1, respectively. In pHNchi6, the chimeric oxidase was designed to carry a minimal region of the cupredoxin fold containing all the CuA ligands, and showed enzymatic activities of 65 and 5.1 s-1 and an expression level better than that of pHNchi6. Kinetic analyses proved that the apparent lower turnover of the chimeric enzyme by pHNchiB was due to the higher Km of the enzyme for Q1H2 (220 μM) than that of cytochrome bo (48 μM), while in the enzyme by pHNchi5, both substrate-binding and internal electron transfer were purturbed. These results suggest that the connecting region and the C-terminal a1-a2-β11 - a3 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 quinol oxidases to cytochrome c oxidase of Gram-positive bacteria like Bacillus was also indicated. [ABSTRACT FROM AUTHOR]