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: 10366026
• 负责人: Yilin Hu
• 金额: $ 31.1万
• 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/10366026
• 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; Sulfur; Thinking; base; dimer; exhaustion; in vivo; novel; protein complex; success; 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; 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 氮酶在其M簇位点催化N2到NH3的环境转化。这个反应 代表支持人类存在的氮的可用形式的主要来源 人口。因此，了解这种酶如何影响N2向NH3的环境转化为 与人类健康的重要相关。我们最近对不对称带的结构观察 在两个m-硝基化酶的两个M簇中，具有不同二氮种的硫位移 调用一种新型的N2还原机制，需要动态结构 在催化过程中，M-CLUSTERS的重排。使用遗传，生化， 光谱和结构方法，我们建议研究N2的这种新机制 通过证明通过群集旋转来证明N2降低的逐步机理的减少， 在M-Cluster的三个皮带硫位点分配单个催化事件，并说明 Fe蛋白与两个MOFE蛋白二聚体的替代结合，该二聚体驱动异步 这些二聚体中的群集旋转。通过我们提出的研究，我们希望说明 N2还原期间，莫硝基酶活性位点的动态结构重排和 建立一个框架，以进一步机械探索这种复杂的反应。调查 沿着这条线可以证明（重新）校准的机械思维 氮酶，因为迄今为止的所有机械研究都认为N2还原发生在A 催化过程中结构为“静态”的均匀M簇物种的单位位点。这些 如果成功的话，努力不仅会导致复杂催化的分子描绘 氮酶的机制，但也有助于发展基于氮酶的应用 长期。