Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster
| Autor: | Jay P. Stasser, John F. Eisses, Martina Ralle, Jack H. Kaplan, Ninian J. Blackburn |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
biology
Superoxide Dismutase Electron Spin Resonance Spectroscopy Protozoan Proteins chemistry.chemical_element HCCS Dithionite Biochemistry Copper Copper chaperone for superoxide dismutase Crystallography chemistry.chemical_compound Chloroplast Proteins chemistry COX17 Humans biology.gene Binding site Histidine Cysteine Electron Probe Microanalysis Plant Proteins Protein Binding |
| Zdroj: | Biochemistry. 39(25) |
| ISSN: | 0006-2960 |
| Popis: | 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 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A 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 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-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. |
| Databáze: | OpenAIRE |
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