Sakamoto K, Mogi T, Noguchi S, Sone N
Department of Biochemical Engineering and Science, Kyusyu Institute of Technology, Iizuka, Fukuoka, 820-8502, Japan.
J Biochem. 1999 Nov;126(5):934-9. doi: 10.1093/oxfordjournals.jbchem.a022537.
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.
为了探究喹啉氧化位点的位置以及亚基间电子传递的物理相互作用，我们构建并表征了两种嵌合氧化酶，其中来自大肠杆菌的细胞色素 bo 型泛醇氧化酶的亚基 II（CyoA）被嗜热芽孢杆菌 PS3 的 caa(3) 型细胞色素 c 氧化酶的对应亚基（CaaA）所取代。在 pHNchi5 中，CyoA 除跨膜螺旋 II 的连接区域外的 C 末端亲水结构域被携带 Cu(A) 位点和细胞色素 c 结构域的 CaaA 对应结构域所取代。通过免疫化学和光谱学方法检测到了所得的嵌合氧化酶，其对 Q(1)H(2)（泛醇 -1）和 TMPD（N,N,N',N'-四甲基 -p -苯二胺）氧化的周转数分别为 28 和 8.5 s⁻¹。在 pHNchi6 中，设计该嵌合氧化酶携带包含所有 Cu(A) 配体的铜蓝蛋白折叠的最小区域，其酶活性分别为 65 和 5.1 s⁻¹，且表达水平优于 pHNchi5。动力学分析证明，pHNchi6 嵌合酶明显较低的周转是由于该酶对 Q(1)H(2) 的 Kₘ（220 μM）高于细胞色素 bo（48 μM），而在 pHNchi5 的酶中，底物结合和内部电子传递均受到干扰。这些结果表明，CyoA 的连接区域和 C 末端α(1)-α(2)-β(11)-α(3) 结构域参与了泛醇氧化和/或亚基间电子传递的物理相互作用，支持了我们之前的提议 [佐藤 - 渡边，M.，茂木，T.，三好，H.，和荒乐，Y.（1998 年）生物化学 37，12744 - 12752]。还表明了大肠杆菌泛醇氧化酶与芽孢杆菌等革兰氏阳性细菌的细胞色素 c 氧化酶之间的密切关系。
