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 Oxidase

Abstract

To probe the location of the quinol oxidation site and physical interactions for inter-subunit electron transfer, we constructed and characterized two chimeric oxidases in which subunit II (CyoA) of cytochrome bo-type ubiquinol oxidase from Escherichia coli was replaced with the counterpart (CaaA) of caa(3)-type cytochrome c oxidase from thermophilic 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 Cu(A) site and cytochrome c domain. The resultant chimeric oxidase was detected immunochemically and spectroscopically, and the turnover numbers for Q(1)H(2) (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 Cu(A) ligands, and showed enzymatic activities of 65 and 5.1 s(-1), and an expression level better than that of pHNchi5. Kinetic analyses proved that the apparent lower turnover of the chimeric enzyme by pHNchi6 was due to the higher K(m) of the enzyme for Q(1)H(2) (220 microM) than that of cytochrome bo (48 microM), while in the enzyme by pHNchi5, both substrate-binding and internal electron transfer were perturbed. These results suggest that the connecting region and the C-terminal alpha(1)-alpha(2)-beta(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 quinol oxidases to cytochrome c oxidase of Gram-positive bacteria like Bacillus was also indicated.

Transient Formation of Ubisemiquinone Radical and Subsequent Electron Transfer Process in theEscherichia coliCytochromebo

“…Bacterial quinol oxidases except cytochrome bd are members of the heme-copper terminal oxidases, and have evolved from cytochrome c oxidase of a Gram-positive bacterium ( 1−3 , 42 ). Structure−function studies on the quinone/quinol redox sites in the E. coli cytochrome bo revealed the presence and properties of the Q L and Q H sites in bacterial quinol oxidases ( 5−8 , 23−29 , − , instead of Cu A , an electron accepting site in subunit II of cytochrome c oxidases ( 2 , − . During the catalytic cycle, the bound Q 8 at the Q H site can be stabilized as ubisemiquinone radical and could undergo the two-electron reduction followed by protonation to yield Q 8 H 2 ( , ).…”

Elucidating mechanisms in haemcopperoxidases: The high-affinity QHbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

“…The location of the two proposed quinone-binding sites within QOX is not clear and, indeed, direct unambiguous evidence that there are two different sites is lacking. [6][7][8][9] In contrast to the deduced low-affinity quinol binding site, the presence of a tightly bound quinone has become clearer.…”

Identification of the Residues Involved in Stabilization of the Semiquinone Radical in the High-Affinity Ubiquinone Binding Site in Cytochrome bo3 from Escherichia coli by Site-Directed Mutagenesis and EPR Spectroscopy

“…The low-affinity site that binds the substrate ubiquinol was suggested to be located at least in part within subunit II of the enzyme on the basis of site-directed mutagenesis, photoaffinity labeling, and inhibitor-resistant mutants (5)(6)(7)(8). Recently, a chimeric construct was investigated, in which parts of subunit II of cytochrome bo 3 from E. coli were replaced by their corresponding parts, the cytochrome c binding and/or Cu A sites, of cytochrome c oxidase (9). This construct still exhibits ubiquinol oxidase activity, and this in fact would exclude these regions of subunit II of E. coli enzyme from being involved in ubiquinol binding.…”