Advanced EPR Studies of Radicals in Biological Catalysis

描述(申请人提供)：这项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起的作用，发展利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之间的关联，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。被研究的酶是两种多铜加氧酶FET3p和R.vernicifera漆酶，以及两种α-酮戊二酸依赖的非血红素铁双加氧酶牛磺酸双加氧酶和AIkB。所有这些蛋白质在其机械循环的某个时刻催化涉及顺磁中间态的反应。这项拨款中提议的实验将使用9 GHz和95 GHz的EPR光谱。常规的连续波研究将与1维和2维的连续波和自旋回波探测Endor以及ESEEM的“先进”EPR技术一起使用。这些先进技术既可以在传统的X频段(9 GHz)使用，也可以在W频段(95 GHz)使用。该提案的具体目标侧重于描述构成所研究的金属酶的催化部位的金属离子的详细电子结构。将使用快速冷冻冷冻法来捕获关键的催化中间体。这些数据将提供关于这些金属的电子结构、配体的配位以及在催化周转过程中电子结构和配体成键如何变化的信息。EPR方法特别适合于获得顺磁中心的这一信息。在将要研究的四种酶中，有两种直接与健康相关的问题。AIkB蛋白最近被证实具有催化单链和双链DNA中甲基化的腺嘌呤和胞嘧啶碱基修复的能力。Fet3p酶是一种多铜氧化酶，具有铁氧合酶活性，因此与铜蓝蛋白有共同的反应活性，铜蓝蛋白是哺乳动物的一种铁氧合酶，对金属离子的动态平衡至关重要。

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.