SPECTROSCOPY OF PORPHYRIN AND QUINONE COFACTORS

卟啉和醌辅助因子的光谱学

• 批准号: 3285518
• 负责人: THOMAS M LOEHR
• 金额: $ 18.99万
• 依托单位: OREGON GRADUATE INSTITUTE SCIENCE & TECH
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285518
• 关键词: Raman spectrometry; chemical reaction; chemical structure; chemical synthesis; chlorin; cofactor; cytochrome oxidase; enzyme mechanism; enzyme reconstitution; enzyme substrate analog; heme; metal complex; metalloenzyme; miscellaneous oxidoreductase; oxidation reduction reaction; porphyrins; quinones; site directed mutagenesis

The objectives of this research proposal are the structural identification of the resting and catalytic-intermediate states of organic redox cofactors in proteins. The major focus will be on the heme and chlorin cofactors of cytochrome d oxidase and cytochrome cd1 nitrite reductase. This project builds upon the successful identification of the novel hydroporphyrin cofactors of these two enzymes as 5,6-dihydroxyprotochlorin and 1,3- porphyrindione-6-acrylate, respectively, as well as the total synthesis and metalation of both cofactors. Assisted with these unique model compounds, the exploration of the accessible reaction intermediates of these enzymes will be facilitated, and the characterization of their oxidation, spin, and coordination states by resonance Raman spectroscopy will be carried out. A significant preliminary finding that will be fully explored is the resonance Raman spectroscopic identification of a highly stable oxoferryl intermediate on the chlorin d of cytochrome d oxidase. Studies will be extended to characterize the formation of other possible chlorin d- associated intermediates in the catalytic cycle, such as oxy, peroxo, or ferryl pi-cation radical species. A site-directed mutant lacking heme b558, one of the three porphyrinic cofactors in the enzyme, will be investigated as a trap for these alternate intermediate states. The second aim of this proposal is to conduct a systematic investigation of the two heme cofactors of the dissimilatory nitrite reductase. Through resonance Raman spectroscopy, the coordination chemistry and ligand-binding properties of heme c and the substrate-binding dione will be elucidated. Of particular interest will be the characterization of reaction intermediates of the dioneheme d1 with NOx-type ligands. Research will also be initiated on the novel quinone cofactors pyrroloquinoline quinone of methanol and glucose dehydrogenases, as well as the amino-acid derived quinones tryptophan tryptophylquinone of methylamine dehydrogenase and trihydroxyphenylalanine quinone of amine oxidase. Preliminary studies have demonstrated that resonance Raman spectroscopy successfully differentiates among all three of these redox cofactors in their native, underivatized states. The common reaction mechanisms of quinone cofactors will be investigated using synthetic models, substrate analogs, and enzyme reconstitution.

本研究方案的目标是结构识别 有机氧化还原辅因子的静止态和催化中间态 在蛋白质中。主要的焦点将集中在血红素和氯素的辅助因子上。 细胞色素d氧化酶和细胞色素CD1亚硝酸盐还原酶。这个项目 建立在成功鉴定新型氢卟啉的基础上 这两种酶的辅因子5，6-二羟基原氯和1，3-二羟基原氯 分别合成了卟二酮-6-丙烯酸酯以及总合成和 两种辅因子的金属化。在这些独特的模型化合物的帮助下， 这些酶的可及反应中间体的探索 以及它们的氧化、自旋和 通过共振拉曼光谱进行配位态的计算。 将充分探索的一个重要的初步发现是 一种高稳定氧基铁的共振拉曼光谱鉴定 细胞色素d氧化酶中氯素d的中间体。研究将会是 扩展到表征其他可能的氯-d-的形成 催化循环中的相关中间体，如氧基、过氧基或 铁基聚阳离子自由基物种。缺乏血红素的定点突变体 B558，酶中的三个卟啉辅助因子之一，将是 作为这些交替中间状态的陷阱而被研究的。第二 这项建议的目的是对这两个问题进行系统的调查 异化亚硝酸还原酶的血红素辅助因子。通过共振 拉曼光谱、配位化学和配体结合 血红素c的性质和底物结合的二酮将被阐明。 特别令人感兴趣的是对反应的描述 具有NOx型配体的Dioneheme D1的中间体。研究将会 也是一种新型的喹酮辅因子吡咯喹啉醌 甲醇和葡萄糖脱氢酶，以及衍生的氨基酸 喹酮类色氨酸和甲胺脱氢酶色氨酸对苯二酚 胺氧化酶的三羟基苯丙氨酸对苯二酚。初步研究表明 证明了共振拉曼光谱成功地区分了 在所有这三个氧化还原辅因子中，它们的天然、未充分分化 各州。对苯二酚辅因子的共同反应机理为 使用合成模型、底物类似物和酶进行研究 重建。