title: Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS.
abstract: The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.
authors.affiliation: Department of Chemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208-3113, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Rae
authors.forename: A S
authors.initials: AS
authors.lastname: Torres
authors.forename: R A
authors.initials: RA
authors.lastname: Pufahl
authors.forename: T V
authors.initials: TV
authors.lastname: O'Halloran
mesh_terms: D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D015003:Yeasts / Q000201:enzymology; D015032:Zinc / Q000737:chemistry
chemical_list: C114678:CCS protein, human; D018832:Molecular Chaperones; C000606290:SOD1 protein, human; D003300:Copper; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D015032:Zinc