Advanced EPR Studies of Radicals in Biological Catalysis

生物催化中自由基的高级 EPR 研究

• 批准号: 6886760
• 负责人: JOHN L MCCRACKEN
• 金额: $ 26.31万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6886760
• 关键词: active sites; amine oxidase (copper); catalyst; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme activity; free radicals; metalloenzyme; methylamines; oxidoreductase; oxygenases

DESCRIPTION (provided by applicant): The long range goals of this research project are to understand the role played by paramagnetic centers in the catalytic mechanism of several enzymes, to develop correlations between the detailed electronic structures, gained using advanced Electron Paramagnetic Resonance (EPR) methods, and chemical reactivity, and to further the development of advanced EPR methods and data analysis strategies for solving problems in biological systems. The enzymes to be studied are two multicopper oxidases, FET3p and R. vernicifera laccase, and two alpha-ketoglutarate dependent non-heme iron dioxygenases, taurine dioxygenase and AIkB. All of these proteins catalyze reactions that involve paramagnetic intermediate states at some point in their mechanistic cycle. The experiments proposed in this grant will use EPR spectroscopy at both 9 and 95 GHz. Conventional, continuous wave studies will be used along with the "advanced" EPR techniques of cw- and spin echo detected-ENDOR, and ESEEM in 1- and 2-dimensions. These advanced technologies can be used at both conventional, X-band frequencies (9 GHz) and at W-band (95 GHz). The specific aims of the proposal focus on characterizing the detailed electronic structures of the metal ions that form the catalytic sites of the metalloenzymes under study. Rapid Freeze Quench methods will be used to trap key catalytic intermediates. The data will provide information on the electronic structure of these metals, the coordination of ligands and how both electronic structure and ligand bonding change during catalytic turnover. EPR methods are uniquely suited to obtain this information for paramagnetic centers. Of the four enzymes to be studied, two are directly tied to health related issues. The AIkB protein has recently been identified as having the ability to catalyze the repair of methylated adenine and cytosine bases in both single- and double-stranded DNA. The Fet3p enzyme is a multicopper oxidase that shows ferroxidase activity and thus, shares a common reactivity with ceruloplasrnin, a mammalian rnulticopper ferroxidase that is vital to metal ion homeostasis.

描述（由申请人提供）：该研究项目的长期目标是了解顺磁中心在几种酶的催化机制中所发挥的作用，开发使用先进的电子顺磁共振（EPR）方法获得的详细电子结构与化学反应性之间的相关性，并进一步开发先进的EPR方法和数据分析策略来解决生物系统中的问题。要研究的酶是两种多铜氧化酶，FET3p 和 R. vernicifera 漆酶，以及两种 α-酮戊二酸依赖性非血红素铁双加氧酶，牛磺酸双加氧酶和 AIkB。所有这些蛋白质都会在其机械循环的某个时刻催化涉及顺磁中间态的反应。本次资助中提出的实验将使用 9 和 95 GHz 的 EPR 光谱。传统的连续波研究将与连续波和自旋回波检测的“先进”EPR 技术 ENDOR 以及 1 维和 2 维 ESEEM 一起使用。这些先进技术可用于传统的 X 频段频率 (9 GHz) 和 W 频段 (95 GHz)。该提案的具体目标侧重于表征形成所研究的金属酶催化位点的金属离子的详细电子结构。快速冷冻淬灭方法将用于捕获关键催化中间体。这些数据将提供有关这些金属的电子结构、配体的配位以及电子结构和配体键合在催化周转过程中如何变化的信息。 EPR 方法特别适合获取顺磁中心的信息。在要研究的四种酶中，有两种与健康相关问题直接相关。 AIkB蛋白最近被鉴定为具有催化单链和双链DNA中甲基化腺嘌呤和胞嘧啶碱基修复的能力。 Fet3p 酶是一种多铜氧化酶，显示出亚铁氧化酶活性，因此与铜蓝蛋白（一种对金属离子稳态至关重要的哺乳动物多铜亚铁氧化酶）具有共同的反应性。