Metallothionein proteins are essential for Cu(I) and Zn(II) homeostasis as well as heavy metal detoxification. The metallation properties of MT2 are of great interest due to their wide patterns of expression and correlation with multiple diseases including cancers, neurological disorders, and respiratory diseases. Use of isotopically pure 63Cu(I) and 68Zn(II) eliminates the complexity of the Cu, Zn-MT2 mass spectral peaks due to significant overlap of naturally abundant isotopes. This allows for the resolution of the precise Cu(I) and Zn(II) stoichiometries when both Cu(I) and Zn(II) are bound to MT2 at physiological pH as expected in vivo. Exact Cu: Zn ratios were determined from mass spectral simulations carried out for every point in the titration. We report that Cu(I) metallation of Zn7-MT2 can only be understood in terms of two pathways occurring in parallel with pathway ① resulting in Cu5Zn5-MT2 and Cu9Zn3-MT2. Pathway ② results in Cu6Zn4-MT2 and Cu10Zn2-MT2, which are the major products of the reaction. From the electrospray ionization (ESI)-mass spectral data we report a series of formation constants (KF) for species starting from Zn7-MT2 up to Cu11Zn2-MT2. Room temperature phosphorescence and circular dichroism (CD) spectra were measured in parallel with the ESI-mass spectrometry data allowing for the assignment of specific species to specific spectral bands. Through analysis of the CD spectral bands, we propose that Cu(I) binds to the β domain first to form a Cu5Zn1 cluster or Cu6 cluster with emission at 670 and 750 nm, respectively, leaving the Zn4 cluster in the α domain.

中文翻译：

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Cu(I) 通过两条平行途径与 Zn7-MT2 结合。

金属硫蛋白对于 Cu(I) 和 Zn(II) 稳态以及重金属解毒至关重要。MT2 的金属化特性因其广泛的表达模式以及与多种疾病（包括癌症、神经系统疾病和呼吸系统疾病）的相关性而备受关注。使用同位素纯 63Cu(I) 和 68Zn(II) 消除了由于天然丰富的同位素显着重叠而导致的 Cu、Zn-MT2 质谱峰的复杂性。当 Cu(I) 和 Zn(II) 在体内预期的生理 pH 下与 MT2 结合时，这可以解析精确的 Cu(I) 和 Zn(II) 化学计量。准确的 Cu: Zn 比率是通过对滴定中每个点进行的质谱模拟来确定的。我们报告说，Zn7-MT2 的 Cu(I) 金属化只能通过与途径 ① 平行发生的两条途径来理解，从而产生 Cu5Zn5-MT2 和 Cu9Zn3-MT2。途径②产生Cu6Zn4-MT2和Cu10Zn2-MT2，它们是反应的主要产物。根据电喷雾电离 (ESI)-质谱数据，我们报告了从 Zn7-MT2 到 Cu11Zn2-MT2 的一系列物质的形成常数 (KF)。室温磷光和圆二色性 (CD) 光谱与 ESI 质谱数据并行测量，从而将特定物种分配到特定光谱带。通过CD光谱带的分析，我们提出Cu(I)首先与β结构域结合形成Cu5Zn1簇或Cu6簇，分别在670 nm和750 nm处发射，而将Zn4簇留在α结构域中。