title: The role of the Cys-X-X-X-Cys motif on the kinetics of cupric ion loading to the Streptomyces lividans Sco protein.
abstract: The mechanisms and spectroscopic properties generated by intermediate states upon cupric ion binding to flexible peptide motifs in proteins are of considerable interest. One such motif is the Cys-X-X-X-Cys motif characteristic to members of the Sco family of proteins. In the antibiotic producing bacterium, Streptomyces lividans, a role for its Sco protein (Sco(Sl)) as a cupric metallochaperone to the extracytoplasmic CuA domain of cytochrome c oxidase has been revealed. Stopped-flow kinetic studies have revealed a mechanism of cupric ion capture by Sco(Sl), which passes through a monothiolate intermediate, with distinct spectral features. In the present study we have used two site directed mutants of Sco(Sl), C86A and C90A, to determine which Cys in the CXXXC motif acts as the capture ligand. Comparison of kinetic and thermodynamic parameters obtained from cupric ion binding to the C86A and C90A mutants clearly indicate that Cys86 is the capture ligand and this finding can be reconciled with structural data. At subsaturating levels of cupric ions both mutants bind copper rapidly, but the absorbance properties are distinctly different from wild type Sco(Sl). This is most extreme for the C86A mutant where the Cys90 thiolate bond is considered to be weaker than the Cys86 thiolate bond in the C90A mutant. We put forward an explanation for this behaviour whereby we propose that the cupric ion is moving to a second site with no thiolate coordination.
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK.
authors.forename: Katie L I M
authors.initials: KL
authors.lastname: Blundell
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
authors.forename: Erik
authors.initials: E
authors.lastname: Vijgenboom
authors.forename: Jonathan A R
authors.initials: JA
authors.lastname: Worrall
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D048170:Streptomyces lividans / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D003300:Copper; D003576:Electron Transport Complex IV