Spectroscopic and Computational Investigation of Copper Monooxygenases

描述(由申请人提供)：双核铜酶为神经肽和激素的单氧合作用执行O2激活，这是所有高等真核生物神经化学所必需的反应。根据两个铜中心之间的磁相互作用，双核铜单异构酶可分为偶联和非偶联。偶联双核酶(例如酪氨酸酶(Ty)和邻苯二酚氧化酶(CaO))有两个强偶联的铜位，它们将O2还原为影响亲电芳香取代(EAS)的过氧化二铜中间体。非偶联的双核铜单加氧酶(氨基甘氨酸羟化单加氧酶(PHM)、多巴胺单加氧酶(D？M)和酪氨酸单加氧酶(T？M))具有两个相距很远的铜中心，不存在磁交换作用。这些非偶联酶产生一种反应性的单铜-O2物种，通过氢原子提取(HAA)进行反应。然而，电子结构和交换耦合如何影响双核铜位对EAS或HAA反应活性的影响尚不清楚。与研究较多的偶联双核酶相比，非偶联双核铜单加氧酶家族中反应中间产物的直接光谱探针不能提供足够的信息来解释HAA和随后的羟化机制。PHM和T？M表达系统的最新进展为首次制备活性位点突变体提供了机会，这种突变体具有动力学捕获关键反应中间体用于光谱分析的潜力。这些研究需要应用先进的光谱学方法，如共振拉曼光谱、电子顺磁共振、磁圆二色谱和基于同步辐射的方法，并结合计算方法，特别是密度泛函理论，这些结果将与酶催化有关，并为非偶联双核铜加氧酶的活性中心结构元素提供理论基础。结合以前在偶联双核铜酶家族中的研究结果，我们将建立一个电子结构/功能模型来解释偶联和非偶联双核铜酶活性的差异。这些研究将提供有关铜酶激活O2的新细节，这有助于在分子水平上理解铜的生物化学。

DESCRIPTION (provided by applicant): Binuclear copper enzymes perform O2 activation for the monooxygenation of neuropeptides and hormones, reactions that are essential for neurochemistry for all higher eukaryotes. Binuclear copper monoogygenases can be classified into "coupled" and "non-coupled" based on the magnetic interaction between the two copper centers. The coupled binuclear enzymes (for example, tyrosinase (Ty) and catechol oxidase (CaO)) have two strongly coupled copper sites that reduce O2 to a dicopper peroxide intermediate that effects electrophilic aromatic substitution (EAS). Non-coupled binuclear copper monooxygenases (peptidylglycine ¿-hydroxylating monooxygenase (PHM), dopamine ¿-monooxygenase (D¿M), and tyramine ¿-monooxygenase (T¿M)) feature two copper centers that are distant, and exhibit no magnetic exchange interaction. These non-coupled enzymes generate a reactive monocopper-O2 species that reacts through hydrogen-atom abstraction (HAA). However, the means by which electronic structure and exchange coupling influence binuclear copper sites towards either EAS or HAA reactivity are not known. In contrast to the more well-studied coupled binuclear enzymes, direct spectroscopic probes of reaction intermediates in the non-coupled binuclear copper monooxygenase family have not provided sufficient information to explain the HAA and subsequent hydroxylation mechanisms. Recent advances in PHM and T¿M expression systems provide the opportunity to prepare active-site mutants for the first time which have the potential to allow kinetic trapping of key reaction intermediates for spectroscopic analysis. These studies require application of advanced spectroscopies such as resonance Raman, electron paramagnetic resonance, magnetic circular dichroism, and synchrotron-based methods, and in combination with computational methods, particularly density functional theory, the results will be related to enzymatic catalysis and provide a rationale for active site structural elements in the non-coupled binuclear copper oxygenases. Combined with previous results in the coupled binuclear family, an electronic structure/function model will be developed to explain the differences in reactivity between coupled and non- coupled binuclear copper enzymes. These studies will yield new details concerning the activation of O2 by copper enzymes, insight that is useful for understanding the biochemistry of copper on a molecular level.