title: Role of the surface-exposed leucine 155 in the metal ion binding loop of the CuA domain of cytochrome c oxidase from Thermus thermophilus on the function and stability of the protein.
abstract: The role of Leu155 in the metal ion binding loop in the soluble CuA binding domain of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) was investigated by site-specific mutations of this residue to arginine (L155R) and glutamic acid (L155E). The UV-visible absorption and electron paramagnetic resonance spectra suggested that the Cu(2)S(2) core of TtCuA was almost unchanged by the mutations. The redox potential of the metal center in the L155R mutant was ~20 mV higher than that in the WT protein, while that of the L155E mutant was almost the same as that of the wild type (WT-TtCuA). The rate of transfer of an electron from cytochrome c(552) to the L155E mutant was much lower than that of transfer to the WT protein, while that for transfer to the L155R mutant was similar to that of WT-TtCuA. The total reorganization energy was increased for both the mutant proteins compared to WT-TtCuA. The results suggest that the presence of a negatively charged residue at the site of Leu155 in TtCuA possibly disfavors the protein-protein interaction between the two redox partners. The mutation also affected the equilibrium pH dependence of the protein. The thermal and thermodynamic stability of TtCuA was drastically decreased upon the mutation, which is most prominent in the L155R mutant. These studies indicate that the hydrophobic patch at the surface of TtCuA consisting of Leu155 is important for the transfer of an electron between cytochrome c(552) and TtCuA.
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: Jitumani
authors.initials: J
authors.lastname: Rajbongshi
authors.forename: Debajani
authors.initials: D
authors.lastname: Basumatary
authors.forename: Shyamalava
authors.initials: S
authors.lastname: Mazumdar
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004563:Electrochemistry; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D008670:Metals; D003576:Electron Transport Complex IV; D007930:Leucine