Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged dicopper(I) cluster

John F. Eisses, Jay P. Stasser, Martina Ralle, Jack H. Kaplan, Ninian J. Blackburn

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55 Scopus citations


Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper- histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 Å (2σ2 = 0.011 Å2) and 1.1 N (histidine) at 1.98 Å (2σ2 = 0.005 Å2). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 Å was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2.26 Å. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 Å. The data were best interpreted by a dinuclear μ2- bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.

Original languageEnglish (US)
Pages (from-to)7337-7342
Number of pages6
Issue number25
StatePublished - Jun 27 2000

ASJC Scopus subject areas

  • Biochemistry


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