SPECTROSCOPIC STUDIES OF COPPER CLUSTERS IN PROTEINS

许多金属酶的活性中心含有一个偶联的双核 铜单位，其化学和光谱性质几乎恒定 作为其生物学功能的一部分，其特性和与氧的相互作用。 这种铜位点存在于金属蛋白中， 铜和铁代谢、氧活化和电子转移。 在 不同的蛋白质，该位点协同结合氧（血蓝蛋白（Hc））， 羟基化单酚（酪氨酸酶），并与其他铜 中心，将底物的四个1-电子氧化耦合到4-电子 将分子氧还原为水（漆酶、血浆铜蓝蛋白和抗坏血酸 氧化酶）。 这项研究的目的是详细研究 这个双核位点的化学和光谱学， 独特的光谱特征，以及电子和几何结构 以及这些特征与蛋白质功能的相关性。 我们已经生成了一系列衍生物，这些衍生物允许活性位点被 系统地变化和研究，通过各种适当的 光谱方法 这些研究导致了一种 oxy Hc活性中心的模型。 我们的研究表明， 酪氨酸酶中的铜位点与Hc中的铜位点极其相似。 但在 酪氨酸酶、底物类似物也以高亲和力与铜结合 现场生产不寻常的铜光谱特征。 分析这些 因此，这些特征导致了对这一机制的深刻理解。 在分子水平上。 双核铜中心的研究 漆酶（称为3型）已经证明， 相对于Hc和酪氨酸酶的差异。 特别是， 外源性配体不能在3型位点桥接两个铜，但 而是桥接到附加的铜质（类型2）中心，从而指示 漆酶中存在三核铜簇。 我们建议调查这个三核铜网站，其结合 外源配体，其与氧的反应及其与 1型铜，并确定这些铜之间的相关性 中心的漆酶和那些在更复杂的多铜氧化酶。 此外，由于我们现在对 O2与HC和酪氨酸酶，我们建议进行实验指导 1）-理解过氧化铜键的性质及其 氧化和还原的活化。 2)-探索外源性和 内源性配体与脱氧位点的相互作用， 这种形式的晶体学结果，和3）-使用“光谱探针” Hc生物聚合物的衍生物，我们已经准备研究 在活性位点水平上的协同作用的影响。

The active sites of a number of metalloenzymes contain a coupled binuclear copper unit which exhibits nearly constant chemical and spectroscopic properties and interacts with oxygen as part of its biological function. This copper site is found in metalloproteins which perform vital roles in copper and iron metabolism, oxygen activation, and electron transfer. In different proteins, this site cooperatively binds oxygen (hemocyanin (Hc)), hydroxylates monophenols (tyrosinase) and in conjunction with other copper centers, couples four 1-electron oxidations of substrate to the 4-electron reduction of dioxygen to water (laccase, ceruloplasmin and ascorbic acid oxidase). The objective of this proposed research is to study in detail the chemistry and spectroscopy of this binuclear site to understand its unique spectral features and thus its electronic and geometric structure and the correlation of these features with protein function. We have generated a series of derivatives which allow the active site to be systematically varied and studied through a variety of appropriate spectroscopic methods. These studies have led to the development of a model of the oxy Hc active site. Our studies have shown that the binuclear copper site in tyrosinase is extremely similar to that in Hc. However, in tyrosinase, substrate analogues also bind with high affinity to the copper site producing unusual copper spectral features. Analysis of these features has thus led to significant insight into the mechanism of this enzyme on a molecular level. Studies on the binuclear copper site in laccase (called Type 3) have demonstrated that there are important differences relative to that in Hc and tyrosinase. In particular, exogenous ligands cannot bridge the two coppers at the Type 3 site, but instead bridge to an additional copper (Type 2) center, thus indicating the presence of a trinuclear copper cluster in laccase. We propose to investigate this trinuclear copper site, its binding of exogenous ligands, its reaction with oxygen and its interaction with the Type 1 copper, and to determine the correlations between these copper centers in laccase and those in the more complicated milticopper oxidases. Further, since we now have a reasonable understanding of the interaction of O2 with Hc and tyrosinase, we propose to proceed with experiments directed to 1)- understanding the nature of the peroxide copper bond and its activation toward oxygenation and reduction. 2)- probing exogenous and endogenous ligand interactions with the deoxy site in complement to recent crystallographic results on this form, and 3)- using a "spectral probe" derivative of the Hc biopolymer, which we have prepared to study the effects of cooperative interactions at an active site level.