title: Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants.
abstract: Cu-metallothionein was purified from Saccharomyces cerevisiae harboring plasmids containing mutated CUP1 metallothionein genes resulting in deletions at the carboxy-terminal end of the polypeptide. The truncated polypeptides are recovered as polypeptides of 35 and 48 residues in length. The Cu-S cluster in the wild-type metallothionein and the two truncates were characterized. The truncated proteins, designated T35 and T48, contain 4 and 2 fewer cysteinyl residues, respectively, compared to the 12 cysteines in wild-type metallothionein; yet the mutant molecules bind Cu(I) ions in a stoichiometry comparable to the wild-type protein, i.e. 7-8 mol eq. The Cu(I) ions bound to T48 are as tenaciously bound as those bound to the wild-type molecule. The electronic transitions in the ultraviolet are similar for Cu-T48 and the wild-type protein. Both mutants and wild-type Cu-protein exhibit luminescence. The corrected emission maxima occurs at 609 nm with a corrected excitation peak near 277 nm. The luminescence quantum yield and lifetime of fluorescence decay of Cu-T48 and wild-type Cu-metallothionein are similar. The absolute quantum yield of the wild-type Cu-protein luminescence is 0.0058 and has a 440-ns lifetime. The similar fluorescence rate constant in the two molecules suggests they possess a similar chromophore. The Cu-T35 protein is more labile than Cu-T48 or the wild-type protein in the association of Cu(I) ions and the air sensitivity of the electronic transitions and luminescence. Although T48 lacks 2 of the 12 cysteines in the wild-type protein, we are unable to detect any differences in the properties of the native metal clusters in the two molecules; T35 lacking 4 cysteinyl residues forms a Cu(I) cluster with properties significantly different from the wild-type molecule. Properties of the Cu-thiolate cluster were also studied in Cu(I)-reconstituted samples. The cluster in wild-type metallothionein forms in all-or-nothing fashion. This conclusion is based on copper binding stoichiometry and luminescence studies. The relative quantum yield of samples with intermediate Cu(I) levels was constant, consistent with all-or-none cluster formation.
authors.affiliation: Department of Medicine, University of Utah Medical Center, Salt Lake City 84132.
authors.forename: J
authors.initials: J
authors.lastname: Byrd
authors.forename: R M
authors.initials: RM
authors.lastname: Berger
authors.forename: D R
authors.initials: DR
authors.lastname: McMillin
authors.forename: C F
authors.initials: CF
authors.lastname: Wright
authors.forename: D
authors.initials: D
authors.lastname: Hamer
authors.forename: D R
authors.initials: DR
authors.lastname: Winge
mesh_terms: D000596:Amino Acids / Q000032:analysis; D003300:Copper / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008163:Luminescent Measurements; D008668:Metallothionein / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013455:Sulfur / Q000032:analysis
chemical_list: D000596:Amino Acids; D013438:Sulfhydryl Compounds; D013455:Sulfur; D003300:Copper; D008668:Metallothionein