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.

项目概要 固氮酶在其 M 簇位点催化环境中 N2 转化为 NH3。这个反应 是支持人类生存的可用氮的主要来源 人口。因此，了解这种酶如何影响环境中 N2 到 NH3 的转化是非常重要的 与人类健康有着重要的关系。我们最近对不对称带的结构观察 Mo-固氮酶的两个 M 簇中不同二氮物种的硫置换 调用了一种新颖的 N2 还原机制，需要动态结构 催化过程中 M 团簇的重排。结合遗传、生化、 光谱和结构方法，我们建议研究 N2 的这种新机制 通过簇旋转演示 N2 还原的逐步机制， 在 M 簇的三个带硫位点分配各个催化事件，并说明 Fe 蛋白与两个 MoFe 蛋白二聚体交替结合，驱动异步 这些二聚体中的簇旋转。通过我们提出的研究，我们希望阐明 N2还原过程中Mo固氮酶活性位点的动态结构重排 建立一个框架，进一步探索这一复杂反应的机制。调查 沿着这条线可能有助于（重新）校准机械思维 固氮酶，因为迄今为止所有的机制研究都假设 N2 还原发生在 均匀 M 簇物种的单个位点，在催化过程中结构“静态”。这些 如果成功，我们的努力将不仅能够对复杂的催化过程进行分子描述 固氮酶机制，同时也促进了基于固氮酶的应用的开发 从长远来看。