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化反应执行O2活化，这些反应对于所有高级真核生物的神经化学至关重要。双核铜单合酶根据两个铜中心之间的磁相互作用可分为“偶联”和“非偶联”两类。偶联的双核酶（例如，酪氨酸酶（Ty）和儿茶酚氧化酶（CaO））具有两个强偶联的铜位点，可以将O2还原为过氧化铜中间体，从而产生亲电芳香取代（EAS）。非偶联双核铜单加氧酶（肽基甘氨酸-羟基化单加氧酶（PHM）、多巴胺-单加氧酶（D¿M）和酪胺-单加氧酶（T¿M））具有两个铜中心相距较远的特点，并且没有磁交换相互作用。这些非偶联酶产生反应性的单铜- o2物种，通过氢原子抽象（HAA）反应。然而，电子结构和交换耦合影响双核铜位对EAS或HAA反应性的方式尚不清楚。与更深入研究的偶联双核酶相比，非偶联双核铜单加氧酶家族反应中间体的直接光谱探针还没有提供足够的信息来解释HAA和随后的羟基化机制。PHM和T¿M表达系统的最新进展首次为制备活性位点突变体提供了机会，这些突变体有可能允许关键反应中间体的动力学捕获用于光谱分析。这些研究需要应用先进的光谱技术，如共振拉曼、电子顺磁共振、磁圆二色性和基于同步加速器的方法，并结合计算方法，特别是密度泛函理论，结果将与酶催化有关，并为非偶联双核铜加氧酶的活性位点结构元素提供理论基础。结合先前在偶联双核家族中的研究结果，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之间的反应性差异。这些研究将提供有关铜酶活化O2的新细节，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.