Probing a novel reaction mechanism of nitrogenase with dynamic active-site rearrangements

探讨固氮酶动态活性位点重排的新反应机制

• 批准号: 10177516
• 负责人: Yilin Hu
• 金额: $ 35.12万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10177516
• 关键词: Active Sites; Binding; Biochemical; Calibration; Catalysis; Complex; Cryoelectron Microscopy; Development; Electrons; Enzymes; Event; Genetic; Health; Human; Individual; Investigation; Lead; Light; Modeling; Molecular; Molecular Conformation; Molybdoferredoxin; Mutation; Nitrogen; Nitrogenase; Population; Proteins; Reaction; Research; Rotation; Site; Source; Structure; Sulfur; Thinking; base; dimer; exhaustion; in vivo; novel; protein complex; success

PROJECT SUMMARY Nitrogenase catalyzes the ambient conversion of N2 to NH3 at its M-cluster site. This reaction represents a major source of the usable form of nitrogen that supports the existence of human population. As such, understanding how this enzyme effects ambient conversion of N2 to NH3 is of significant relevance to human health. Our recent structural observation of asymmetric belt sulfur displacements with distinct dinitrogen species in the two M-clusters of Mo-nitrogenase invokes a novel mechanism of N2 reduction that necessitates dynamic structural rearrangements of the M-clusters during catalysis. Using combined genetic, biochemical, spectroscopic and structural approaches, we propose to investigate this novel mechanism of N2 reduction by demonstrating the stepwise mechanism of N2 reduction via cluster rotation, assigning individual catalytic events at the three belt-sulfur sites of the M-cluster, and illustrating the alternate binding of Fe protein to the two MoFe protein dimers that drives the asynchronous cluster rotation in these dimers. Through our proposed studies, we hope to illustrate the dynamic structural rearrangements of the active site of Mo-nitrogenase during N2 reduction and establish a framework for further mechanistic exploration of this intricate reaction. Investigations along this line could prove instrumental in (re)calibrating the mechanistic thinking of nitrogenase, as all mechanistic studies to date have assumed that N2 reduction occurs at a single site of a uniform M-cluster species that is structurally `static' during catalysis. These efforts, if successful, will not only lead to a molecular depiction of the intricate catalytic mechanism of nitrogenase, but also facilitate development of nitrogenase-based applications in the long run.

项目摘要 固氮酶在其M簇位点催化N2到NH3的环境转化。该反应 是维持人类生存的氮的主要来源 人口因此，了解这种酶如何影响N2到NH3的环境转化是非常重要的。 对人类健康有重大意义。我们对不对称带的最新构造观测 钼固氮酶两个M簇中不同双氮物种的硫置换 调用了一种新的N2还原机制， 在催化过程中的M-簇的重排。利用基因，生化， 光谱和结构的方法，我们建议调查这一新的机制N2 通过展示经由簇旋转的N2还原的逐步机制来还原， 在M-簇的三个带硫位点分配单独的催化事件，并说明 Fe蛋白与两个MoFe蛋白二聚体的交替结合， 在这些二聚体中的簇旋转。通过我们的研究，我们希望说明 氮还原过程中钼固氮酶活性中心的动态结构重排， 为进一步探索这一复杂反应机理建立了一个框架。调查 沿着这条线可以证明有助于（重新）校准机械思维， 固氮酶，因为迄今为止的所有机理研究都假设N2还原发生在 在催化过程中结构上“静态”的均匀M簇物种的单一位点。这些 如果成功的话，这些努力不仅会导致对复杂的催化作用的分子描述， 固氮酶的作用机理，也有利于开发固氮酶基础应用， 长远来看