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 Blackburn

Research output: Contribution to journalArticle

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Abstract

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
JournalBiochemistry
Volume39
Issue number25
DOIs
StatePublished - Jun 27 2000

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Superoxide Dismutase
Cysteine
Copper
Dithionite
Fourier Analysis
Histidine
Fourier transforms
X-Ray Absorption Spectroscopy
Binding Sites
Atoms
X ray absorption spectroscopy
Dithiothreitol
Electron Transport Complex IV
Stoichiometry
Yeast
Sequence Analysis
Proteins
Yeasts
Monomers
Metals

ASJC Scopus subject areas

  • Biochemistry

Cite this

Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged dicopper(I) cluster. / Eisses, John F.; Stasser, Jay P.; Ralle, Martina; Kaplan, Jack H.; Blackburn, Ninian.

In: Biochemistry, Vol. 39, No. 25, 27.06.2000, p. 7337-7342.

Research output: Contribution to journalArticle

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title = "Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged dicopper(I) cluster",
abstract = "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 {\AA} (2σ2 = 0.011 {\AA}2) and 1.1 N (histidine) at 1.98 {\AA} (2σ2 = 0.005 {\AA}2). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 {\AA} 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 {\AA}. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 {\AA}. 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.",
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T1 - Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged dicopper(I) cluster

AU - Eisses, John F.

AU - Stasser, Jay P.

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AU - Kaplan, Jack H.

AU - Blackburn, Ninian

PY - 2000/6/27

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N2 - 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.

AB - 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.

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