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: 10557174
• 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/10557174
• 关键词: Active Sites; Binding; Biochemical; Calibration; Catalysis; Complex; Cryoelectron Microscopy; Crystallography; Development; Electrons; Enzymes; Event; Genetic; Health; Human; Individual; Investigation; Lead; Modeling; Molecular; Molecular Conformation; Molybdoferredoxin; Mutation; Nitrogen; Nitrogenase; Population; Proteins; Reaction; Research; Rotation; Site; Source; Sulfur; Thinking; dimer; exhaustion; in vivo; novel; protein complex; success; usability

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.

项目总结 固氮酶催化N_2在其M-簇位上转化为NH_3。这种反应 代表了支持人类生存的可用形态氮的主要来源 人口。因此，了解这种酶如何影响环境中氮气到氨的转化是 对人类健康有重大影响。我们最近对不对称带的结构观察 钼固氮酶两个M-簇中不同氮源物种的硫置换 调用一种新的氮气还原机制，该机制需要动态结构 M-团簇在催化过程中的重排。结合遗传，生化， 通过光谱和结构手段，我们建议研究氮气的这种新机制。 通过演示通过团簇旋转的氮气还原的逐步机制来进行还原， 指定了M-团簇的三个带硫位置上的单独催化事件，并举例说明 铁蛋白与驱动异步化的两个MoFe蛋白二聚体的交替结合 这些二聚体中的团簇旋转。通过我们建议的研究，我们希望说明 氮气还原过程中钼固氮酶活性中心的动态结构重排 为进一步从机制上探索这种复杂的反应建立一个框架。调查 沿着这条线可以证明在(重新)校准机械思维方面是有帮助的 固氮酶，因为迄今为止所有的机理研究都假设氮气还原发生在 均一的M-簇物种的单一位置，在催化过程中在结构上是‘静态的’。这些 如果成功，这些努力不仅将导致对复杂的催化反应的分子描述 固氮酶的作用机理，也促进了固氮酶在 从长远来看。