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: 10566582
• 负责人: Faik Akif Tezcan
• 金额: $ 28.32万
• 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/10566582
• 关键词: ATP Hydrolysis; Active Sites; Agriculture; Ammonia; Atmosphere; Binding; Binding Sites; Biochemical; Biological; Biological Availability; Biological Models; Biological Products; Biophysics; Catalysis; Cell physiology; Chemicals; Complex; Couples; Cryoelectron Microscopy; Dependence; Development; Electron Transport; Electrons; Enzymes; Goals; Guanosine Triphosphate; Health; Human; Investigation; Location; Maps; Mechanics; Mediating; Metabolism; Metals; Methodology; Molecular Conformation; Molybdoferredoxin; Mutagenesis; Nature; Nitrogen Fixation; Nitrogenase; Nucleotides; Nutritional; Oxidation-Reduction; Persons; Population; Process; Productivity; Protein Dynamics; Proteins; Protons; Reaction; Research; Research Project Grants; Resolution; Roentgen Rays; Structure; Substrate Interaction; Testing; Work; enzyme model; experimental study; inhibitor; innovation; insight; metalloenzyme; particle; programs; small molecule; spectroscopic survey; stoichiometry; 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 single-particle 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.

项目概要/摘要 该提案旨在阐明细菌金属酶固氮酶如何催化 将大气中的二氮转化为生物可利用的形式氨，在化学上是困难的 以及为什么/如何利用 ATP 水解来驱动该反应。是唯一负责的酶 对于还原性固氮，固氮酶可维持约 40% 的农业/营养需求 人口。除了其全球重要性之外，固氮酶还是一个独特的模型系统 与生物氧化还原催化以及 ATP/GTP 依赖性能量具有广泛相关性 转导过程，对于正常的细胞功能来说都是核心，因此直接 与人类健康相关。 尽管近五年来广泛的生物化学、生物物理和结构研究 表征中，关于固氮酶机制的两个最重要的问题还没有 已详细回答：a) 为什么以及如何最终利用 ATP 水解来进行还原 N2 或替代底物？ b) 二氮还原的内在机制是什么？ 固氮酶活性位点金属簇，FeMoco？主要的实验挑战 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，这会导致氧化还原和 固氮酶的核苷酸结合状态很难相互区分。到 为了规避这一挑战，我们启动了一项单粒子低温研究计划 电子显微镜 (cryoEM) 在结构上表征固氮酶的动态 酶促周转条件下的原子分辨率。初步实验不仅 证实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，这推动了新的机制假设。在拟议的项目中，我们的目标是 基于这些初步发现，a) 绘制 ATP 驱动的构象图 在催化周转条件下以前所未有的细节呈现固氮酶的景观，b) 阐明 FeMoco 结构动力学和 FeMoco-小分子原子相互作用 分辨率，同时c) 为尖端冷冻电镜方法的开发做出贡献 用于高度复杂/动态蛋白质组装体的结构询问和 金属辅因子。