Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged dicopper(I) cluster
Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged dicopper(I) cluster
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DOI：
10.1021/bi000690j
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发表时间：
2000-06-27
期刊：
影响因子：
2.9
通讯作者：
Blackburn, NJ
中科院分区：
文献类型：
--
作者：
Eisses, JF;Stasser, JP;Blackburn, NJ
用X射线吸收光谱法研究了铜与人铜超氧化物歧化酶（hCCS）分子伴侣的结合。化学计量学测量的透析，作为分离的蛋白质表明，高达3.5铜离子结合每个hCCS分子。用连二亚硫酸钠或二硫苏糖醇还原将铜结合率降低至2个铜/hCCS单体。作为隔离EXAFS数据的分析表明协调铜的S和N的背散射体的混合物，暗示铜的异质性结合之间的结合域I或在和铜-组氨酸SOD 1-样金属结合域II的铜-半胱氨酸结合位点。在2.24埃（2 σ（2）= 0.011埃（2））下使用1.6 Cu-S（半胱氨酸）和在1.98埃（2 σ（2）= 0.005埃（2））下使用1.1 N（组氨酸）获得最佳拟合。在2.7埃处分离的蛋白质的傅立叶变换（FT）中的可变强度的峰提示存在重原子散射体如Cu。连二亚硫酸盐和DTT-还原衍生物的分析表明，铜从组氨酸配位位点丢失，导致在S-仅环境中与铜在2.26埃配位到三个S背散射体。重原子散射体峰现在在FT中是突出的，并且可以通过在2.72埃处的Cu-Cu相互作用很好地拟合。最好的解释数据由双核μ（2）-桥接簇与双桥半胱氨酸配体类似的集群提出存在于细胞色素c氧化酶伴侣COX 17。酵母CCS的一级序列和X-射线结构信息的分析强烈表明，这个簇的桥梁结构域I和III之间的hCCS。对铜离子迁移的机制进行了简要的讨论。
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 In 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 Angstrom (2 sigma(2) = 0.011 Angstrom(2)) and 1.1 N (histidine) at 1.98 Angstrom (2 sigma(2) = 0.005 Angstrom(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 Angstrom 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 Angstrom. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 Angstrom. 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.