Iron Sulfur Clusters in Biological Radical Generation

这个项目的总体目标是描述铁/S-S-腺苷蛋氨酸(ADOMet)家族酶产生自由基的详细化学机制。假设：依赖腺苷蛋氨酸的铁硫酶都通过一个共同的机制工作，在这个机制中，一个还原的簇与ADOMet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-硫团簇的新化学。为了研究这一新的化学结构，我们将对丙酮酸甲酸裂解酶(PFL-AE)及其相关酶进行生化、光谱、机理和结构研究。我们还将探讨铁依赖的黏附蛋白猝灭PFL自由基的机制。本研究的具体目的如下：1.研究Fe-S/ADOMet酶家族中铁-硫簇的结构和性质，及其与ADOMet的相互作用。我们的工作将主要集中在PFL-AE上，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理的抑制剂作为ES相互作用和PFL-AE催化机理的探针。3.利用快速冷冻猝灭光谱对PFL-AE催化的自由基生成反应中的中间体进行鉴定和光谱表征。4.利用X射线结晶学对PFL-AE的结构进行表征。到目前为止，还没有关于FE-S/ADOMet酶的任何详细的结构信息，这些结构信息可以极大地提高机械实验的设计和解释。5.表征ADHE的金属结合部位，以及该金属在甘氨酸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.