SPECTROSCOPIC STUDIES OF COPPER CLUSTERS IN PROTEINS

蛋白质中铜簇的光谱研究

• 批准号: 3230113
• 负责人: EDWARD I SOLOMON
• 金额: $ 21.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-01-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3230113
• 关键词: Raman spectrometry; binding proteins; catalase; circular dichroism; copper; electron density; electron spin resonance spectroscopy; enzyme structure; enzyme substrate complex; ferroxidase; hemocyanin; horseshoe crabs; metalloenzyme; metalloproteins; monophenol monooxygenase; protein structure function; respiratory oxygenation; saltwater environment

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.

许多金属酶的活性中心都含有一个偶联的双核 具有几乎恒定的化学和光谱特性的铜单元 作为其生物功能的一部分，它具有与氧相互作用的特性。 这种铜存在于金属蛋白中，这些金属蛋白在 铜和铁的代谢、氧的活化和电子转移。在……里面 不同的蛋白质，这个位点协同结合氧(血蓝蛋白(HC))， 羟基化单酚(酪氨酸酶)并与其他铜结合 中心，将底物的四个1电子氧化偶联到4电子 将氧气还原为水(漆酶、铜蓝蛋白和抗坏血酸 氧化酶)。这项拟议的研究的目的是详细研究 这个双核位置的化学和光谱来理解它的 独特的光谱特征，因此它的电子和几何结构 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的衍生品，允许活性中心是 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 氧基HC活性中心的模型。我们的研究表明，双核 酪氨酸酶中的铜位置与HC中的铜位置极为相似。但是，在 酪氨酸酶、底物类似物也与铜具有高亲和力。 网站产生不寻常的铜光谱特征。对这些问题的分析 因此，特征导致了对这一机制的重大洞察 分子水平上的酶。双核铜位的研究 漆酶(称为类型3)已经证明有重要的 与HC和酪氨酸酶的差异。特别是， 外源配体不能在类型3位置连接两个铜，但 相反，桥接到额外的铜缆(类型2)中心，从而指示 漆酶中存在三核铜团簇。 我们建议研究这个三核铜位置，它与 外源配体及其与氧的反应及其与氧的相互作用 类型1铜，并确定这些铜之间的相关性 主要集中在漆酶和更复杂的米铜氧化酶中。 此外，由于我们现在已经合理地理解了 O2与HC和酪氨酸酶，我们建议继续进行定向实验 至1)-了解过氧化铜键的性质及其 通向氧化和还原的激活。2)-探测外源和 内源性配体与新近的补体脱氧位点的相互作用 这种形式的结晶学结果，以及3)--使用“光谱探头” HC生物聚合物的衍生物，我们已经准备好用于研究 活跃站点水平上的协作交互的影响。