Understanding the reactivity of the most complex multicopper oxidase ceruloplasmin: insight into the reaction intermediates, mechanism and the roles of the additional type 1 copper sites

项目摘要 多铜氧化酶(MCOs)是一个大而多样的酶家族，它连接一个电子 各种底物的氧化与四电子还原的氧气为水。他们是很重要的 生物过程，并越来越多地被评估为各种生物技术应用，因为其 宽广的衬底范围。人血清铜蓝蛋白(CP)是最复杂的MCO，具有一个三核 铜(TNC)中心和三个1型铜(T1Cu)中心。对CP感兴趣的主要原因之一是 突变性疾病无浆蛋白血症揭示了CP作为一种关键的关键铁氧合酶的作用 铁稳态。对MCOS的催化循环的基本理解是基于 漆树漆酶(RVL)研究然而，CP的催化作用机理尚不清楚。 初步研究表明，CP催化的中间体与RVL的中间体不同，并且CP的功能与RVL不同 Cp的另外两个T1Cu位并没有很好的定义。因此，这项建议侧重于这两个重要的 关于CP机理的问题：1)参与CP催化的独特中间体的性质；2)CP催化 另外两个T1Cu位的作用。所有涉及的中间体或突变体都将进行广泛的分析 与DFT计算相关的光谱方法的范围，以定义相应的电子和 中间体的几何结构，并阐明反应坐标。Cp的相关性 将确定其他MCO中存在的明确定义的中间体的中间体。这些机械式的 对CP的研究将促进对人CP构效关系的基础性认识 为金属介导性疾病的发病机制和机制提供了有价值的见解。 神经毒性。

Project Summary Multicopper oxidases (MCOs) are a large and diverse family of enzymes which couple the one-electron oxidation of various substrates with the four-electron reduction of dioxygen to water. They are important in biological processes and are increasingly evaluated for a variety of biotechnological applications due to their broad substrate range. Human serum ceruloplasmin (Cp) is the most complicated MCO with one trinuclear copper (TNC) center and three type 1 copper (T1Cu) sites. One of the main reasons for the interest in Cp is the mutational disease aceruloplasminemia, which reveals a role for Cp as an essential ferroxidase critical for iron homeostasis. A fundamental understanding of the catalytic cycle of the MCOs has been developed based on the study of Rhus vernicifera laccase (RvL). However, the mechanism of Cp catalysis is still unclear. Preliminary studies reveal that an intermediate of Cp catalysis is different from that of RvL, and the functions of the two additional T1Cu sites of Cp are not well defined. Hence, this proposal focuses on these two important issues regarding the Cp mechanism: 1) The nature of the unique intermediates involved in Cp catalysis; 2) The roles of the two additional T1Cu sites. All the intermediates or mutants involved will be analyzed by a wide range of spectroscopic methods correlated with DFT calculations to define the corresponding electronic and geometric structures of the intermediates and elucidate reaction coordinates. The correlation of Cp intermediates to the well-defined intermediates present in other MCOs will be determined. These mechanistic studies of the Cp will advance the fundamental understanding of the structure-activity relationship of human Cp and provide valuable insight into the pathogenesis of metal-mediated diseases and mechanisms for metal neurotoxicity.