Mechanism of Energy Transduction and Substrate Activation in Biological Nitrogen Fixation

生物固氮中的能量转换和底物激活机制

• 批准号: 10795182
• 负责人: Faik Akif Tezcan
• 金额: $ 5.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10795182
• 关键词: ATP Hydrolysis; Active Sites; Agriculture; Ammonia; Atmosphere; Binding; Biochemical; Biological; Biological Availability; Biological Models; Biological Products; Biophysics; Catalysis; Cell physiology; Chemicals; Complex; Couples; Cryoelectron Microscopy; Development; Enzymes; Guanosine Triphosphate; Health; Human; Investigation; Maps; Metabolism; Metals; Methodology; Molecular Conformation; Nitrogen Fixation; Nitrogenase; Nucleotides; Nutritional; Oxidation-Reduction; Persons; Population; Process; Protein Dynamics; Reaction; Research; Resolution; enzyme model; experimental study; metalloenzyme; programs; small molecule; welfare

PROJECT SUMMARY/ABSTRACT This proposal aims to elucidate how the bacterial metalloenzyme nitrogenase catalyzes the chemically difficult transformation of atmospheric dinitrogen into a bioavailable form, ammonia, and why/how it utilizes ATP hydrolysis to drive this reaction. Being the only enzyme responsible for reductive nitrogen fixation, nitrogenase sustains the agricultural/nutritional needs of ~40% of the human population. Aside from its global importance, nitrogenase is a unique model system with broad relevance to biological redox catalysis as well as ATP/GTP-dependent energy transduction processes, which are both central to proper cellular functioning and thus directly relevant to human health. Despite nearly five decades of extensive biochemical, biophysical, and structural characterization, the two most important questions about nitrogenase mechanism have not been answered in detail: a) Why and how ATP hydrolysis is ultimately utilized for the reduction of N2 or alternative substrates? b) What is the intimate mechanism of dinitrogen reduction on the nitrogenase active site metal cluster, FeMoco? The major experimental challenge in the investigations of nitrogenase arises from the fact that the catalytic activity of nitrogenase depends on continuous ATP turnover, which leads to a heterogeneous mixture of redox and nucleotide-bound states of nitrogenase that are difficult to distinguish from one another. To circumvent this challenge, we have initiated a research program in cryogenic electron microscopy (cryoEM) to structurally characterize dynamic states of nitrogenase at atomic resolution under enzymatic turnover conditions. Preliminary experiments have not only established the feasibility of this approach but also revealed unexpected structural features of nitrogenase which have fueled new mechanistic hypotheses. In the proposed project, we aim to build upon on these preliminary findings by a) mapping the ATP-driven conformational landscape of nitrogenase in unprecedented detail under catalytic turnover conditions and b) elucidating FeMoco structural dynamics and FeMoco-small molecule interactions in atomic resolution, while also c) contributing to the development of cutting-edge cryoEM methodologies for the structural interrogation of highly complex/dynamic protein assemblies and metallocofactors.

项目总结/摘要 该建议旨在阐明细菌金属酶固氮酶如何催化 化学上很难将大气中的二氮转化为生物可利用的形式，氨， 以及它为什么/如何利用ATP水解来驱动这个反应。作为唯一的酶 对于还原性固氮，固氮酶维持约40%的农业/营养需求。 人类人口。除了它的全球重要性，固氮酶是一个独特的模式系统 与生物氧化还原催化以及ATP/GTP依赖的能量具有广泛的相关性 转导过程，这两者都是中央适当的细胞功能，从而直接 与人类健康有关。 尽管近50年来广泛的生物化学，生物物理和结构， 表征，关于固氮酶机制的两个最重要的问题还没有 详细回答：a）为什么以及如何ATP水解最终用于还原 N2或替代底物？B）在反应过程中， 固氮酶活性中心金属簇，FeMoco？实验中的主要挑战是 固氮酶的研究源于固氮酶的催化活性 依赖于连续的ATP周转，这导致了氧化还原和 固氮酶的核苷酸结合状态，难以彼此区分。到 为了克服这一挑战，我们已经启动了一项低温电子研究计划， cryoEM），以在原子水平上表征固氮酶的动态结构 在酶促周转条件下拆分。初步实验不仅 建立了这种方法的可行性，但也揭示了意想不到的结构特征， 固氮酶，这激发了新的机制假说。在建议的项目中，我们的目标是 在这些初步发现的基础上，通过a）绘制ATP驱动的构象 在催化转化条件下固氮酶前所未有细节景观和B） 阐明FeMoco结构动力学和原子中FeMoco-小分子相互作用 分辨率，同时c）有助于开发尖端的cryoEM方法 用于高度复杂/动态蛋白质组装的结构询问， 金属辅因子