LUMINESCENT PROBE OF COPPER-THIOLATE CLUSTER FORMATION WITHIN MAMMALIAN METALLOTHIONEIN
LUMINESCENT PROBE OF COPPER-THIOLATE CLUSTER FORMATION WITHIN MAMMALIAN METALLOTHIONEIN
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DOI：
10.1021/ic00096a046
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发表时间：
1994-08-31
影响因子：
4.6
通讯作者：
STILLMAN, MJ
中科院分区：
文献类型：
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作者：
GREEN, AR;PRESTA, A;STILLMAN, MJ
在一定温度范围内测量了铜(I)在pH值为7时与兔肝锌金属硫蛋白水溶液结合的发射光谱数据。这些数据为金属硫蛋白中铜硫酸盐簇形成的途径提供了一个独特的探针。金属分析表明，在低温（< 15℃）下，Cu(I)作为[Cu(I)]的函数线性取代Zn(II)，直到加入12 Cu(I)，此时所有7 Zn（II）都被取代。在高温下，Zn（II）位移的显著滞后导致6 Cu(I)点处的[Zn(II)] vs [Cu(I)]线出现非线性。当Cu(I)加入到Zn7-MT中时，在600 nm附近的发射带在所有温度下都增强。在低温下（0℃< T < 15℃），当12个Cu(I)结合时，标准化强度（发射强度与Cu(I)结合的函数）随着发射的显著增加大致呈线性增加。在较高温度下（15℃< T < 50℃），Zn7-MT中Cu(I)的摩尔比与发射强度之间存在完全不同的关系。在加入2 ~ 7 Cu(I)之间，归一化发射强度降低。在加入7 ~ 12 Cu(I)之间，发射强度急剧增加。在低温下，12 Cu(I)的发射强度大大超过1 Cu(I)的12倍。在所有温度下，当Cu(I)的加入量为13 ~ 20时，发射强度趋于零。在加入11 Cu(I)之前，600 nm附近的硫酸铜发射带中心大致保持不变，然后在12 Cu(I)处急剧蓝移，然后在13和16 Cu(I)之间显著红移20 nm。在低温和高温之间的温度循环中，辐射强度发生了显著变化。这些变化只能用与蛋白质结合的Cu(I)的迁移率来解释。在6℃条件下，Zn7-MT中加入1 ~ 7 Cu(I)时，发射强度较高。当加热到50℃时，排放强度下降到初始值的10%，冷却到6℃时，仅恢复到原始强度的40%。完全相反，对于含有8 - 12 Cu(I)的溶液，经过相同循环后的强度是原始溶液的两倍。发射强度清楚地探测了金属硫蛋白中多达12个Cu(I)与20个半胱氨酸硫酸盐结合时形成的铜硫酸盐簇的位置和结构特征。通过对这些实验性质的解释，单个Cu(I)原子与Zn7-MT结合的途径可以完全描述如下：(I) α域的Cu(I)硫酸酯簇发出的光是β域Cu(I)的4-10倍。（ii） Cu(I)原子位于α域，可以通过这种高发射强度检测到。（iii） Cu(I)在所有温度下以分布方式在两个结构域上结合。（iv）在高温下Cu(I)重新分布到β结构域，形成特定结构域的产物Cu6(S(cys))9- β，导致发射强度显著降低。(v) Cu(I)不与Zn7-MT协同结合。（vi） Cu12-MT是一种紧密的结构，有效地排除了溶剂对Cu(I)-硫酸酯簇的接触。最后，（vii）对发射强度随温度变化的分析表明，这些数据为溶剂通过金属结合位点的外部结构进入金属-硫代酸酯簇提供了独特而敏感的探针。发射猝灭分析表明，分子模拟技术预测的Cu12-MT结构中的裂纹在Cu12-MT中与Cd5Zn2-MT的结构非常相似。
Emission spectral data measured over a range of temperatures are reported for copper(I) binding to aqueous solutions of rabbit liver zinc metallothionein at pH 7. These data provide a unique probe of the pathways adopted as copper-thiolate clusters form in the metallothionein. Metal analysis shows that, at low temperatures (< 15-degrees-C), Cu(I) displaces the Zn(II) linearly as a function of [Cu(I)] until 12 Cu(I) have been added, at which point all 7 Zn(II) are displaced. At high temperatures, significant hysteresis in the displacement of the Zn(II) results in nonlinearity in the [Zn(II)] vs [Cu(I)] line at the 6 Cu(I) point. As Cu(I) is added to Zn7-MT an emission band near 600 nm intensifies at all temperatures. At low temperatures (0-degree-C < T < 15-degrees-C) the normalized intensity (emission intensity as a function of Cu(I) bound) increases roughly linearly with a significant increase in emission when 12 Cu(I) are bound. At higher temperatures (15-degrees-C < T < 50-degrees-C) a completely different relationship between emission intensity and molar ratio of Cu(I) added to the Zn7-MT is observed. The normalized emission intensity decreases between 2 and 7 Cu(I) added. Between 7 and 12 Cu(I) added there is a dramatic increase in emission intensity. As at low temperatures, the emission intensity for 12 Cu(I) greatly exceeds 12 times the intensity for 1 Cu(I). The emission intensity decreases toward zero as from 13 to 20 Cu(I) are added at all temperatures. The band center of the copper-thiolate emission near 600 nm remains approximately constant until 11 Cu(I) have been added and then blue shifts sharply at the 12 Cu(I) point, before significantly red shifting by 20 nm between 13 and 16 Cu(I). The emission intensity changes dramatically following temperature cycles between low and high temperatures. These changes can only be interpreted in terms of the mobility of the Cu(I) bound to the protein. When between 1 and 7 Cu(I) are added to Zn7-MT at 6-degrees-C, the emission intensity is relatively high. Upon heating to 50-degrees-C, the emission intensity drops to 10% of this initial value and cooling the solution back to 6-degrees-C only recovers 40% of the original intensity. In complete contrast, for solutions containing between 8 and 12 Cu(I), the intensity after this same cycle is twice that of the original solution. The emission intensity clearly probes the location and structural features of the copper-thiolate clusters that form as up to 12 Cu(I) bind to the 20 cysteinyl thiolates in metallothionein. Through intrepretation of these experimental properties, the pathways by which individual Cu(I) atoms bind to Zn7-MT can be completely described on the basis of the following: (i) Cu(I) thiolate clusters in the alpha domain emit 4-10 times the light of Cu(I) in the beta domain. (ii) Cu(I) atoms located in the alpha domain can be detected by this high emission intensity. (iii) Cu(I) binds in a distributed manner statistically across both domains at all temperature. (iv) At high temperatures Cu(I) redistributes to populate the beta domain, forming the domain specific product, Cu6(S(cys))9-beta, which results in significant reduction in the emission intensity.(v) Cu(I) does not bind to Zn7-MT cooperatively. (vi) Cu12-MT is a tight structure, efficiently excluding solvent access to the Cu(I)-thiolate clusters. Finally, (vii) analysis of changes in the emission intensities as a function of temperature shows that these data provide a unique and sensitive probe of solvent access to the metal-thiolate clusters through the outer structure of the metal binding site. Analysis of the emission quenching suggests that the crevices predicted in the structure of Cu12-MT by molecular modeling techniques are present in Cu12-MT much like in the structure of Cd5Zn2-MT.