Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase
Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase
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DOI：
10.1042/bj20040360
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发表时间：
2004-08-15
影响因子：
4.1
通讯作者：
Sillard, R
中科院分区：
文献类型：
--
作者：
Palumaa, P;Kangur, L;Sillard, R
Cox 17是细胞色素c氧化酶的铜分子伴侣，是一种重要的高度保守蛋白。Cox 17的结构和功能机制尚不清楚，甚至其金属结合化学计量也是难以捉摸的。在本研究中，我们证明，使用电喷雾电离-MS，猪Cox 17结合合作四个Cu+离子。Cu（4）Cox 17在pH值大于3时稳定，荧光光谱表明存在溶剂屏蔽的多核Cu（I）簇。结合我们的结果与早期EXAFS结果酵母CuCox 17，我们建议，Cu（4）Cox 17包含一个Cu 4S 6型集群。在超毫摩尔浓度下，二硫苏糖醇从Cu（4）Cox 17中萃取金属，根据这些结果计算出表观铜解离常数K-Cu = 13 fM。不同形式Cox 17的电荷态分布表明，第一个Cu+离子与考克斯17的结合导致构象从开放状态到紧凑状态的变化，这可能是形成Cu（4）Cox 17的限速步骤。Cox 17非合作地结合两个Zn 2+离子，但不结合Ag+离子，这突出了其极高的金属结合特异性。我们进一步证明，猪Cox 17也可以部分氧化（两个二硫桥）和完全氧化（三个二硫桥）的形式存在。部分氧化的Cox 17可以结合一个Cu+或Zn+离子，而完全氧化的Cox 17不结合金属。Cox 17的金属结合特性意味着，与其他铜分子伴侣相比，Cox 17被设计用于同时转移多达四个铜离子到伴侣蛋白。金属可以通过非氧化和氧化机制从Cox 17中释放。
Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein. The structure and mechanism of functioning of Cox17 are unknown, and even its metal-binding stoichiometry is elusive. In the present study, we demonstrate, using electrospray ionization-MS, that porcine Cox17 binds co-operatively four Cu+ ions. Cu(4)Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster. Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu(4)Cox17 contains a Cu4S6-type cluster. At supramillimolar concentrations, dithiothreitol extracts metals from Cu(4)Cox17, and an apparent copper dissociation constant K-Cu = 13 fM was calculated from these results. Charge-state distributions of different Cox17 forms suggest that binding of the first Cu+ ion to Cox 17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu(4)Cox17. Cox17 binds non-co-operatively two Zn2+ ions, but does not bind Ag+ ions, which highlights its extremely high metal-binding specificity. We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms. Partly oxidized Cox17 can bind one Cu+ or Zn+ ion, whereas fully oxidized Cox17 does not bind metals. The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins. Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms.