Mechanism of Copper Incorporation in Subunit II of Cytochrome c Oxidase from Thermus thermophilus: Identification of Intermediate Species

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• 作者：Manas Kumar Ghosh ; Priyanka Basak ; Shyamalava Mazumdar
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：July 9, 2013
• 年：2013
• 卷：52
• 期：27
• 页码：4620-4635
• 全文大小：603K
• 年卷期：v.52,no.27(July 9, 2013)
• ISSN：1520-4995

文摘

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 (TtCu_A) 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 Cu_A. 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 TtCu_A. 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 K_d for WT TtCu_A 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 Cu_A center in cytochrome c oxidase.