title: Characterization of the redox and metal binding activity of BsSco, a protein implicated in the assembly of cytochrome c oxidase.
abstract: Members of the Sco protein family are implicated in the assembly of the respiratory complex cytochrome c oxidase. Several possible roles have been proposed for Sco: a copper delivery agent, a site-specific thiol reductase, and an indicator of cellular redox status. Two cysteine residues (C45 and C49) in the sequence CXXXCP and a histidine (H135) approximately 90 residues toward the C-terminus are conserved in Sco from bacteria, yeast, and humans. The soluble domain of Sco has a thioredoxin fold that is suggestive of redox activity for this protein. We have characterized the soluble domain of the Sco protein from Bacillus subtilis (i.e., sBsSco) for its redox reactivity and metal binding capacity. In oxidized sBsSco, the cysteines are present as an intramolecular disulfide. Oxidized sBsSco does not bind metal, but can be reduced in vitro to a metal-binding form. Reduction of the disulfide in sBsSco is accompanied by increased intrinsic fluorescence. The reducibility of the cystine is unchanged when the conserved histidine is mutated to alanine. Tight binding by reduced sBsSco is observed for Cu(II) by electronic absorption, intrinsic fluorescence, and EPR spectroscopies, and isothermal titration calorimetry with an observed stoichiometry of one Cu(II) ion per sBsSco and a KD of approximately 50 nM. Tight binding of Cu(I) and Ag(I) is observed by quenching of intrinsic tryptophan fluorescence. Cobalt(II) exhibits weak binding, whereas Ni(II) and Zn(II) do not appear to bind. The high-affinity binding of metals by BsSco is triggered by its redox state, and this property could be important for its function in vivo.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6 Canada.
authors.forename: Iveta
authors.initials: I
authors.lastname: Imriskova-Sosova
authors.forename: Diann
authors.initials: D
authors.lastname: Andrews
authors.forename: Katherine
authors.initials: K
authors.lastname: Yam
authors.forename: David
authors.initials: D
authors.lastname: Davidson
authors.forename: Brahm
authors.initials: B
authors.lastname: Yachnin
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Hill
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003300:Copper; D003545:Cysteine; D003553:Cystine; D004220:Disulfides; D004229:Dithiothreitol; D003576:Electron Transport Complex IV; D008565:Membrane Proteins; D008670:Metals; D010720:Phosphines; D011725:Pyridines; D011994:Recombinant Proteins; C482909:Sco protein, Bacillus subtilis; C080938:tris(2-carboxyethyl)phosphine; C007542:4,4'-dipyridyl disulfide; C028419:cuprous chloride