Korkola Natalie C, Stillman Martin J
Department of Chemistry, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 3K7, Canada.
J Am Chem Soc. 2023 Mar 22;145(11):6383-6397. doi: 10.1021/jacs.2c13578. Epub 2023 Mar 13.
Oxidative stress is a state involving an imbalance of reactive oxygen species in a cell and is linked to a variety of diseases. The metal-binding protein metallothionein (MT) may play a role in protection due to its high cysteine content. Many studies have shown that oxidative stress will cause MT to both form disulfide bonds and release bound metals. However, studies on the more biologically relevant partially metalated MTs have been largely neglected. Additionally, most studies to date have used spectroscopic methods that cannot detect specific intermediate species. In this paper, we describe the oxidation and the subsequent metal displacement pathway of fully and partially metalated MTs with hydrogen peroxide. The rates of the reactions were monitored using electrospray ionization mass spectrometry (ESI-MS) techniques, which resolved and characterized the individual intermediate M(SH)MT species. The rate constants were calculated for each species formation. Through ESI-MS and circular dichroism spectroscopy, it was found that the three metals in the β-domain were the first to be released from the fully metalated MTs. The Cd(II) in the partially metalated Cd(II)-bound MTs rearranged to form a protective CdMT cluster structure upon exposure to oxidation. The partially metalated Zn(II)-bound MTs oxidized at a faster rate as the Zn(II) did not rearrange in response to oxidation. Additionally, density functional theory calculations showed that the terminally bound cysteines were more negative and thus more susceptible to oxidation than the bridging cysteines. The results of this study highlight the importance of metal-thiolate structures and metal identity in MT's response to oxidation.
氧化应激是一种细胞内活性氧物种失衡的状态，与多种疾病相关。金属结合蛋白金属硫蛋白（MT）因其高半胱氨酸含量可能在保护过程中发挥作用。许多研究表明，氧化应激会导致MT形成二硫键并释放结合的金属。然而，对生物学相关性更强的部分金属化MT的研究在很大程度上被忽视了。此外，迄今为止大多数研究使用的光谱方法无法检测特定的中间物种。在本文中，我们描述了用过氧化氢对完全金属化和部分金属化MT的氧化及随后的金属置换途径。使用电喷雾电离质谱（ESI-MS）技术监测反应速率，该技术解析并表征了各个中间M(SH)MT物种。计算了每个物种形成的速率常数。通过ESI-MS和圆二色光谱发现，β结构域中的三种金属是首先从完全金属化MT中释放出来的。部分金属化的结合Cd(II)的MT中的Cd(II)在暴露于氧化时重排形成保护性的CdMT簇结构。部分金属化的结合Zn(II)的MT氧化速率更快，因为Zn(II)不会因氧化而重排。此外，密度泛函理论计算表明，末端结合的半胱氨酸比桥连半胱氨酸更负，因此更容易被氧化。本研究结果突出了金属硫醇盐结构和金属特性在MT对氧化反应中的重要性。
