Department of Chemical Sciences, Tata Institute of Fundamental Research , Colaba, Mumbai 400005, India.
Biochemistry. 2013 Jul 9;52(27):4620-35. doi: 10.1021/bi400101g. Epub 2013 Jun 26.
Detailed spectroscopic and kinetic studies of incorporation of copper ion in the wild type (WT) and the D111AA (AA = K, N, or E) mutants of the metal ion binding site of the soluble fragment of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) showed the formation of at least two distinct intermediates. The global analyses of the multiwavelength kinetic results suggested a four-step reaction scheme involving two distinct intermediates in the pathway of incorporation of copper ions into the apoprotein forming the purple dinuclear CuA. An early intermediate similar to the red copper binding proteins was detected in the WT as well as in all the mutants. The second intermediate was a green copper species in the case of WT TtCuA. Mutation of Asp111, however, formed a second intermediate that is distinctly different from that formed in the case of the WT protein, suggesting that mutants follow pathways of copper ion incorporation different from that in the WT protein. The electrostatic interaction between Asp111 and the coordinating His114 possibly plays a subtle role in the mechanism of incorporation of metal ion into the protein. The overall Kd for WT TtCuA was found to be ~8 nM, which changed with mutation of the Asp111 residue. The activation and thermodynamic parameters were also determined from the temperature- and pH-dependent multiwavelength kinetics, and the results are discussed to unravel the role of Asp111 in the mechanism of formation of the dinuclear CuA center in cytochrome c oxidase.
详细的光谱和动力学研究表明，铜离子在来自嗜热脂肪芽孢杆菌（Thermus thermophilus）可溶性亚基 II 片段金属离子结合位点的野生型（WT）和 D111AA（AA = K、N 或 E）突变体中至少形成了两种不同的中间体。多波长动力学结果的全局分析表明，铜离子进入脱辅基蛋白形成紫色双核 CuA 的途径涉及四个反应步骤，涉及两种不同的中间体。在 WT 以及所有突变体中都检测到类似于红色铜结合蛋白的早期中间体。然而，在 WT TtCuA 的情况下，第二个中间体是绿色铜物种。然而，天冬氨酸 111 的突变形成了与 WT 蛋白形成的中间体明显不同的第二个中间体，这表明突变体遵循的铜离子掺入途径与 WT 蛋白不同。天冬氨酸 111 和配位组氨酸 114 之间的静电相互作用可能在金属离子掺入蛋白的机制中发挥微妙作用。WT TtCuA 的整体 Kd 约为 8 nM，这一数值随天冬氨酸 111 残基的突变而改变。还从温度和 pH 依赖的多波长动力学确定了激活和热力学参数，并对结果进行了讨论，以揭示天冬氨酸 111 在细胞色素 c 氧化酶中二核 CuA 中心形成机制中的作用。
