Iron-Sulfur Clusters in Biological Radical Generation

描述（由申请人提供）：该项目的总体目标是描述Fe/ s - s -腺苷蛋氨酸（所谓的自由基SAM）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，似乎在整个系统发育王国中都有代表，已经确定了数百种自由基SAM酶。这些酶在生物学中的广泛存在，从细菌到人类，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、血红素的合成和钼酸盐辅助因子的生物合成，以及许多其他基本功能，其中一些功能尚未确定。尽管催化的反应多种多样，但我们最重要的假设是，依赖于腺苷蛋氨酸的铁硫酶都通过一种共同的机制运作，即还原簇与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反应。为了研究这种新的丙酮酸甲酸裂解酶激活酶（PFL-AE）的化学、生化、光谱、机理和结构研究将继续进行。具体目标包括进一步研究PFL-AE的[4Fe-4S]簇、其独特的铁位点、与底物的相互作用及其在催化中的作用。此外，我们建议探索全细胞中PFL-AE铁硫团簇的性质。我们还将利用底物类似物作为ES相互作用和PFL-AE催化机制的探针。我们将使用光谱方法，特别是ENDOR，来探测PFL- AE/AdoMet/PFL复合物的结构和电子特征。我们还将利用低温还原和快速冷冻猝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的中间体。最后，我们将利用x射线晶体学对PFL- ae及其与PFL和SAM的配合物进行结构表征。

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.