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.

项目摘要/摘要 该建议旨在阐明细菌金属酶氮酶如何催化 化学上的大气二氮转化为生物利用形式，氨， 以及为什么/如何利用ATP水解来驱动这一反应。是唯一负责的酶 为了减少氮固定，氮酶可以维持约40％的农业/营养需求 人口。除了其全球重要性，氮酶是一个独特的模型系统 与生物氧化还原催化以及ATP/GTP依赖性能量广泛相关 转导过程，既是正确的细胞功能的核心，因此直接 与人类健康有关。 尽管将近五十年的广泛生化，生物物理和结构 表征，关于氮酶机制的两个最重要的问题尚未 详细回答：a）为什么最终将ATP水解用于还原 N2还是替代底物？ b）减少二氮的亲密机制是什么 氮酶活性位点金属簇，Femoco？在 氮酶的研究源于以下事实。 取决于连续的ATP周转率，这导致氧化还原和 氮基酶结合的核苷酸状态很难彼此区分。到 规避了这一挑战，我们启动了一个低温电子研究计划 显微镜（冷冻）以在结构上表征原子氮的动态态 在酶促转移条件下的分辨率。初步实验不仅具有 确定了这种方法的可行性，但也揭示了意外的结构特征 氮酶促进了新的机械假设。在拟议的项目中，我们的目标是 通过a）映射ATP驱动的构象基于这些初步发现 在催化周转条件下以空前的细节，氮酶的景观和b） 阐明原子中的Femoco结构动力学和Feamoco-Small分子相互作用 解决方案，同时也c）有助于发展尖端冷冻方法的发展 用于高度复杂/动态蛋白质组件和 金属生产子。