Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, USA.
J Am Chem Soc. 2011 Aug 10;133(31):12229-37. doi: 10.1021/ja2045259. Epub 2011 Jul 18.
The prion protein (PrP) takes up 4-6 equiv of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60-91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc. 2005, 127, 12647-12656). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens, et al. PLoS Pathog.2009, 5 (4), e1000390). A current working hypothesis is that the high occupancy binding mode quenches copper's inherent redox cycling, thus, protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrP's putative role in cellular signaling.
朊病毒蛋白（PrP）在其伸展的 N 端结构域中结合 4-6 个当量的铜，该结构域由八重复（OR）片段（人序列残基 60-91）和两个单核结合位点（位于 His96 和 His111；也称为非 OR 区）组成。OR 片段在低铜水平下以多 His 模式，在高铜水平下以单 His、酰胺氮模式响应特定的铜浓度（Chattopadhyay 等人，J. Am. Chem. Soc. 2005, 127, 12647-12656）。PrP 在健康组织中的具体功能尚不清楚，但有大量报道将铜摄取与调节细胞应激的神经保护作用联系起来（Stevens 等人，PLoS Pathog.2009, 5（4），e1000390）。目前的工作假说是，高占据结合模式使铜固有的氧化还原循环猝灭，从而防止不受调节的芬顿型反应产生的活性氧物质。在这里，我们通过对低和高占据铜结合模式进行详细的 pH 依赖电化学测量，直接测试了这一假说。与目前的观点相反，我们发现低占据模式完全猝灭氧化还原循环，但高占据模式通过复合物促进的氧气催化还原导致温和的过氧化氢产生。这些电化学发现得到了独立的动力学测量的支持，这些测量可探测抗坏血酸的使用情况和过氧化物的产生。还观察到非 OR 区对应的片段产生过氧化氢。总的来说，这些结果推翻了当前的工作假说，并表明，高占据模式下与 PrP 结合的铜的氧化还原循环没有被猝灭，而是被调节。观察到的过氧化氢产生表明了一种机制，该机制可以解释 PrP 在细胞信号中的假定作用。
