SPECTROSCOPIC STUDIES OF COPPER CLUSTERS IN PROTEINS

偶联双核铜中心存在于各种不同的 参与结合、激活和多电子的蛋白质和酶 将氧气还原为水。这些物质包括血蓝蛋白、酪氨酸酶 和多铜氧化物酶。在多铜氧化物酶中，偶联 双核中心已被确定为三核铜的一部分 集群站点--O2所需的最小结构单元 还原。与大多数中心相比，这些中心显示出独特的光谱特征 小分子铜络合物。这项研究的总体目标是 计划一直在了解这些光谱特征的起源 几何结构和电子结构的术语，以定义 与差异相关的几何和电子结构；在 功能，并评估电子结构对 这些位点在生物学上的反应性。越来越明显的是，这些 独特的光谱特征反映了新的活性中心电子结构 它可以对催化做出重大贡献。一种组合 光谱分析和理论分析相结合的方法被用于研究原生生物和 被扰动的蛋白质位点和模拟特定蛋白质的无机复合体 活动站点的功能。这项建议的具体目的是：L )完成电子设备的实验和理论描述 氧合血蓝蛋白/酪氨酸氧合酶的结构和定义 节肢动物和软体动物血蓝蛋白的定量差异和 与反应性差异相关的酪氨酸酶。2)定义 端面键合过氧基铜(II)络合物的电子结构 是多铜氧化酶中过氧化水平中间体的模型。 为了确定电子贡献促进了 过氧化氢不可逆转地还原为水。3)推广化学和 真菌酪氨酸酶对哺乳动物酶及其活性的光谱研究 与底物的相互作用，并定义由 与酪氨酸酶相关的眼皮肤白化病相关的突变。 4)确定活性中心和氧中间体之间的差异 酪氨酸酶和非偶联双核铜酶多巴胺-β- 羟基酶催化多巴胺转化为 神经递质和激素去甲肾上腺素。5)定义几何和 四电子中两个中间体的电子结构 多聚铜氧化物酶中O2还原为H2O获得分子 对催化机理的层次洞察。6)关联详细光谱 漆酶中三核铜团簇位置对细菌生长的影响 抗坏血酸氧化酶中的结晶学定义的三核位置，以及 将这些光谱研究扩展到三核团簇位置，可能是 存在于铜蓝蛋白和铜甲烷单加氧酶中。7)定义 金属-配体成键相互作用约为12.5埃 连接蓝铜和铜的电子转移途径(Cys-His) 三核铜在多铜氧化酶中的中心和测定 这些中心之间的变构相互作用及其对 分子内电子转移。8)将这些研究扩展到铜蓝蛋白 它在铜和铁的新陈代谢中起着关键作用，为了定义 在场的其他警察的作用，与 底物，以及非常大的基点间的性质和意义 这种多中心酶中存在相互作用。

Coupled binuclear copper centers are found in a variety of different proteins and enzymes involved in the binding, activation and multielectron reduction of dioxygen to water. These include the hemocyanins, tyrosinases and the multicopper oxidases. In the multicopper oxidases, the coupled binuclear center has been determined to be part of a trinuclear copper cluster site which is the minimum structural unit required for O2 reduction. These centers exhibit unique spectral features compared to most small molecule copper complexes. The general goals of this research program have been to understand the origin of these spectral features in terms of geometric and electronic structure, to define differences in geometric and electronic structure which correlate with differences; in function and to evaluate electronic structural contributions to the reactivity of these sites in biology. It is becoming clear that these unique spectral features reflect novel active site electronic structures which can make significant contributions to catalysis. A combination of spectral and theoretical methods are applied to the study of native and perturbed protein sites and to inorganic complexes which model specific features of the active site. The specific aims of this proposal are to: l )Complete the experimental and theoretical description of the electronic structure of the oxyhemocyanin/oxytyrosinase site and to define quantitative differences over the arthropod and mollusc hemocyanins and tyrosinase which correlate to differences in reactivity. 2)Define the electronic structure of the end-on bound hydroperoxo-Cu(II) complex which is a model for the peroxide level intermediate in the multicopper oxidases in order to determine electronic contributions which promote the irreversible reduction of peroxide to water. 3)Extend chemical and spectral studies of fungal tyrosinase to the mammalian enzyme and its interaction with substrate, and define active site perturbations caused by mutations associated with tyrosinase related oculocutaneous albinism. 4)Define active site and oxygen intermediate differences between tyrosinase and the non-coupled binuclear copper enzyme dopamine beta- hydroxylase which catalyzes the conversion of dopamine to the neurotransmitter and hormone noradrenalin. 5)Define the geometric and electronic structure of the two intermediates present in the four electron reduction of O2 to H2O in the multicopper oxidases to obtain molecular level insight into the catalytic mechanism. 6)Correlate detailed spectral studies on the trinuclear copper cluster site in laccase to the crystallographically defined trinuclear site in ascorbate oxidase, and extend these spectral studies to the trinuclear cluster sites which may be present in ceruloplasmin and copper methane monooxygenase. 7)Define the metal-ligand bonding interactions with the approximate 12.5 Angstrom electron transfer pathway (Cys-His) connecting the blue copper and the trinuclear copper centers in the multi-copper oxidases and determine the allosteric interactions between these centers and their effects on intramolecular electron transfer. 8)Extend these studies to ceruloplasmin which plays a key role in copper and iron metabolism, to define the function of the additional coppers present, the interaction with substrate, and the nature and significance of the very large intersite interactions present in this multicenter enzyme.