Iron-Sulfur Clusters in Biological Radical Generation

描述(申请人提供)：本项目的总体目标是描述Fe/S-S-腺苷蛋氨酸(即所谓的自由基蛋氨酸)超家族酶产生自由基的详细化学机制。这些酶跨越了非常不同的反应范围，似乎代表了整个系统发育王国，发现了数百种自由基SAM酶。这些酶在从细菌到人类的整个生物学中的广泛存在表明了这些酶催化的化学的重要性。在人类中，自由基SAM酶参与硫辛酸的生物合成、血红素的合成和钼蝶呤辅因子的生物合成，以及许多其他基本功能，其中一些功能尚不清楚。尽管催化的反应各不相同，但我们最重要的假设是，依赖腺苷蛋氨酸的铁硫酶都是通过一个共同的机制工作的，在这个机制中，一个还原的簇与S-腺苷蛋氨酸相互作用，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-硫团簇的新化学。为了研究这一新的化学结构，将对丙酮酸甲酸裂解酶(PFL-AE)进行生化、光谱、机理和结构研究。具体目标包括进一步研究PFL-AE的[4Fe-4S]团簇、其独特的铁位置、它与底物的相互作用以及它在催化中的作用。此外，我们还建议探索整个细胞中PFL-AE铁-硫簇的性质。我们还将利用底物类似物作为ES相互作用和PFL-AE催化机理的探针。我们将使用光谱方法，特别是Endor，来探索PFL-AE/ADOMet/PFL络合物的结构和电子特征。我们还将利用冷冻还原和快速冷冻-淬灭光谱方法来鉴定和光谱表征PFL-AE催化的自由基生成反应中的中间体。最后，我们将利用X射线结晶学对PFL-AE及其与PFL和SAM的络合物进行结构表征。

DESCRIPTION (provided by applicant): The overall objective of this project is to delineate the detailed chemical mechanism of radical generation by the Fe/S-S-adenosylmethionine (the so-called radical SAM) superfamily of enzymes. These enzymes span a remarkably diverse range of reactions and appear to be represented across the phylogenetic kingdom, with hundreds of radical SAM enzymes identified. The widespread occurrence of these enzymes throughout biology, from bacteria to humans, is indicative of the significance of the chemistry catalyzed by these enzymes. In humans, radical SAM enzymes are involved in the biosynthesis of lipoic acid, the synthesis of heme, and the biosynthesis of the molybdopterin cofactor, among many other essential functions, some as yet unidentified. Despite the diversity of reactions catalyzed, our overriding hypothesis is that the adenosylmethionine-dependent iron-sulfur enzymes all operate by a common mechanism in which a reduced cluster interacts with S-adenosylmethionine 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) will be pursued. The specific aims include further investigation of the [4Fe-4S] cluster of PFL-AE, its unique iron site, its interactions with substrate, and its role in catalysis. In addition we propose to explore the nature of the PFL-AE iron-sulfur cluster in whole cells. We will also utilize substrate analogs as probes of ES interactions and the PFL-AE catalytic mechanism. We will use spectroscopic approaches, particularly ENDOR, to probe the structural and electronic features of the PFL- AE/AdoMet/PFL complex. We will also utilize cryoreduction and rapid-freeze-quench spectroscopic approaches to identify and spectroscopically characterize intermediates in the radical-generation reaction catalyzed by PFL-AE. Finally, we will pursue structural characterization of PFL-AE and its complexes with PFL and SAM using X-ray crystallography.