Biophysical Studies of Metalloenzymes

电子顺磁共振波谱学一直是确定金属酶活性位点结构和功能的重要工具。该项目将继续开发独特的方法，在X（9 GHz）和Q（35 GHz）波段以及W波段（95 GHz）微波频率下进行CW/脉冲ENDOR和ESEEM实验。这种星座的方法，结合新的程序开发的冷冻溶液ENDOR和ESEEM光谱的分析，提供了一个强大的，选择性的，和灵敏的工具，表征金属酶活性位点及其与底物/抑制剂/产品的相互作用，无论是在静止状态和关键中间体。本项目将重点研究以下三个重要课题：（I）固氮酶MoFe蛋白的铁-铁辅因子上底物/抑制剂结合的定位及底物还原过程中形成的中间态的表征;（II）"自由基S-腺苷甲硫氨酸"超家族的三个成员的酶促机制，它们都利用[4Fe-4S]簇和S-腺苷甲硫氨酸（S-adenosylmethionine，S-adenosylmethionine，S-Met）产生催化必需的自由基。(III)活性中间体和底物/抑制剂与钼和钨氧代转移酶（α-dopterins）的相互作用。更广泛的影响：该项目将开发复杂的分析程序和算法，用于确定完整的配位几何形状和金属中心的键合。这些能力通过网络、正式合作和非正式磋商传播给金属生物化学研究人员。除了这些研究贡献外，该项目还将为本科院校的一名教员提供研究机会。此外，该项目将涉及研究生和博士后培训，该项目由分子生物化学和分子生物物理学联合资助。

Electron paramagnetic resonance spectroscopy has long been a central tool for determining the structure and function of metalloenzyme active sites. This project will continue to develop unique methods to perform CW/pulsed ENDOR and ESEEM experiments at X (9 GHz) and Q (35 GHz) bands, and W band (95 GHz) microwave frequencies. This constellation of approaches, combined with new procedures developed for the analysis of frozen-solution ENDOR and ESEEM spectra, provides a powerful, selective, and sensitive tool for the characterization of metalloenzyme active sites and their interactions with substrates/inhibitors/products, both in the resting state and in key intermediates. This project will emphasize three important topics:(I) Localization of substrate/inhibitor binding at the molybdenum-iron cofactor of the nitrogenase MoFe protein and characterization of intermediate states formed during substrate reduction;(II) The enzymic mechanism of three members of "radical S-adenosylmethionine" superfamily, which all use [4Fe-4S] clusters and S-adenosylmethionine (AdoMet) to generate catalytically essential radicals.(III) Reactive intermediates and substrate/inhibitor interactions with molybdenum and tungsten oxotransferases (molybdopterins).Broader Impact: This project will develop sophisticated analytical procedures and algorithms for determining the complete coordination geometry and bonding at a metal center. These capabilities disseminated to the metallobiochemistry researchers via web, through formal collaborations and by informal consultation. Besides these research contributions, the project will provide research opportunities for a faculty member from an undergraduate institution. In addition, the project will involve training at the graduate and postdoctoral level.This project is funded jointly by Molecular Biochemistry and Molecular Biophysics.