Iron Sulfur Clusters in Biological Radical Generation

该项目的总体目标是描绘由Fe/S-S-腺苷甲硫氨酸（NAMMet）酶家族产生自由基的详细化学机制。假设：腺苷甲硫氨酸依赖性铁硫酶都通过一种共同的机制运行，其中还原簇与腺苷甲硫氨酸相互作用，产生直接参与催化的腺苷自由基中间体。 这些反应代表了铁硫簇合物的新化学。 为了研究这种新的化学，生化，光谱，机制，和结构的丙酮酸甲酸裂解酶激活酶（PFL-AE）和相关酶的研究将继续进行。我们还将研究铁依赖性AdhE蛋白淬灭PFL自由基的机制。 本提案的具体目标如下：1.研究Fe-S/蛋氨酸铁硫簇合物的结构、性质及其与蛋氨酸铁硫簇合物的相互作用。 我们的工作将主要集中在PFL-AE，并将包括光谱（EPR，ENDOR，ESEEM，穆斯堡尔，共振拉曼），电化学和动力学研究。2.利用底物类似物和基于机制的抑制剂作为ES相互作用和PFL-AE催化机制的探针。3.使用快速冷冻淬灭光谱法鉴别和光谱表征PFL-AE催化的自由基生成反应中的中间体。4.使用X射线晶体学对PFL-AE进行结构表征。 到目前为止，没有详细的结构信息是可用于任何FE-S/METASMet酶，这样的结构信息可以大大提高设计和解释的机制实验。5. 研究AdhE的金属结合位点及其在甘氨酰自由基猝灭中的作用。

The overall objective of this project is to delineate the detailed chemical mechanism of radical generation by the Fe/S-S-adenosylmethionine (AdoMet) family of enzymes. Hypothesis: The adenosylmethionine-dependent iron-sulfur enzymes all operate by a common mechanism in which a reduced cluster interacts with AdoMet to generate an adenosyl radical intermediate which is directly involved in catalysis. These reactions represent novel chemistry for iron-sulfur clusters. To investigate this novel chemistry, biochemical, spectroscopic, mechanistic,, and structural studies of pyruvate formate-lyase activating enzyme (PFL-AE) and related enzymes will be pursued. We will also investigate the mechanism of PFL radical quenching by the iron-dependent AdhE protein. The specific aims of this proposal are as follows: 1. To investigate the structures and properties of iron-sulfur clusters in the Fe-S/AdoMet family of enzymes, and the interactions of the clusters with AdoMet. Our work will focus primarily on PFL-AE, and will include spectroscopic (EPR, ENDOR, ESEEM, Mossbauer, resonance Raman), lectrochemical, and kinetic studies. 2. To utilize substrate analogs and mechanism-based inhibitors as probes of ES interactions and the PFL-AE catalytic mechanism.. 3. To identify and spectroscopically characterize intermediates in the radical-generation reaction catalyzed by PFL-AE by using rapid-freeze quench spectroscopy. 4. To pursue structural characterization of PFL-AE using X-ray crystallography. To date, no detailed structural information is available for any of the FE-S/AdoMet enzymes, and such structural information could greatly enhance the design and interpretation of mechanistic experiments. 5. To characterize the metal binding site of AdhE, and the role of the metal in glycyl radical quenching.