SPECTROSCOPIC STUDIES OF COPPER CLUSTERS IN PROTEINS

蛋白质中铜簇的光谱研究

• 批准号: 2138634
• 负责人: EDWARD I SOLOMON
• 金额: $ 34.71万
• 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 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2138634
• 关键词: Raman spectrometry; albinism; allosteric site; binding proteins; catalase; copper; electron density; electron spin resonance spectroscopy; enzyme structure; enzyme substrate complex; ferroxidase; hemocyanin; horseshoe crabs; metalloenzyme; metalloproteins; monophenol monooxygenase; protein structure; protein structure function

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.

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