title: Mechanism of copper incorporation in subunit II of cytochrome C oxidase from Thermus thermophilus: identification of intermediate species.
abstract: 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.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research , Colaba, Mumbai 400005, India.
authors.forename: Manas Kumar
authors.initials: MK
authors.lastname: Ghosh
authors.forename: Priyanka
authors.initials: P
authors.lastname: Basak
authors.forename: Shyamalava
authors.initials: S
authors.lastname: Mazumdar
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013056:Spectrophotometry, Ultraviolet; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D003300:Copper; D003576:Electron Transport Complex IV