Atomic Models of Nitrogen Fixation by Nitrogenase from CryoEM Structures

CryoEM 结构中固氮酶固氮的原子模型

• 批准号: 10313954
• 负责人: Rebeccah Warmack
• 金额: $ 6.64万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313954
• 关键词: Active Sites; Air; Ammonia; Anaerobic Bacteria; Binding; Bioavailable; Biological; California; Chemistry; Complex; Cryoelectron Microscopy; Crystallization; Data; Data Collection; Disease; Electron Beam; Electron Microscopy; Electron Transport; Electrons; Enzymes; Error Sources; Evolution; Eye; Food production; Foundations; Freezing; Goals; Health; Human; Industrialization; Institutes; Kinetics; Knowledge; Life; Light; Metabolic; Methodology; Methods; Micro Electron Diffraction; Microscope; Modeling; Molybdoferredoxin; Multienzyme Complexes; Nature; Nitrogen; Nitrogen Fixation; Nitrogenase; Oxidoreductase; Pathway interactions; Physiological; Planet Earth; Play; Preparation; Procedures; Production; Proteins; Radial; Radiation induced damage; Reaction; Research; Resolution; Rest; Role; Sample Size; Sampling; Seminal; Source; Structure; System; Techniques; Technology; Temperature; Training; Visualization; Water; Work; cofactor; cryogenics; data structure; design; electron radiation; experimental study; flexibility; frontier; homocitrate; innovation; insight; kinetic model; metalloenzyme; method development; microorganism; nitrogenase reductase; particle; pressure; protein structure; time use

PROJECT SUMMARY: The nitrogenase enzyme complex is the only biological pathway for producing metabolically useful forms of nitrogen from atmospheric dinitrogen. This two component protein system made up of an obligate reductase, Fe-protein, and the catalytic protein, MoFe-protein, is only present in a small number of microorganisms, but produces nearly 50% of all bioavailable nitrogen. Thus, the biological nitrogen fixation mechanism has extremely important effects on global crop production and human health. Seminal works in the past half century have not only revealed an intricate kinetic pathway for nitrogenase, but also the most complex metallocluster found in nature within the MoFe-protein active site. This cluster, known as the FeMo-cofactor, has the composition [7Fe:9S:1C:1Mo]-R-homocitrate and is only coordinated by two residues within the active site. Electrons are shuttled through a [4Fe:4S] cluster in the Fe-protein to an [8Fe:7S] center in the MoFe-protein, known as the P- cluster. These reducing equivalents are then delivered to the active site FeMo-cofactor where substrate reduction occurs. Four consecutive cycles of electron transfer are required to bind dinitrogen, and four more are required to fully convert one dinitrogen molecule to two ammonia molecules. It is unknown how the FeMo-cofactor is primed for substrate binding, how the FeMo-cofactor binds or shuttles electrons to the substrate, or what role the surrounding active site residues play in substrate reduction. The primary hypothesis of this proposal is that physical rearrangements of the atoms in the FeMo-cofactor and in the surrounding protein during substrate reduction are necessary for substrate access and binding to the cofactor, and may further play a role in the reduction mechanism. The goal of this work is to determine what rearrangements occur in nitrogenase throughout turnover of substrate by harnessing the power of cryoEM and performing experiments outlined in two Aims. Aim 1: Structural characterization of resting states of nitrogenase: MoFe protein alone and the ADP-AlF4- stabilized complex with Fe-protein by single particle cryoEM. Aim 2: Structural characterization of nitrogenase turnover-related forms by single particle cryoEM. Through the pursual of Aims 1 and 2, I will obtain training in the field of single particle cryoEM, and each Aim will provide expertise in metalloenzyme chemistry. The research will be performed at the California Institute of Technology primarily in the well-known CryoEM Center with ample microscopes and expertise. These Aims will shed much needed light on transient intermediates within the nitrogenase turnover pathway thereby providing a better foundation for the rational design of efficient synthetic nitrogen fixation platforms. In addition, the insights and the methodology developed in this proposal can be applied to other poorly understood metalloenzymes related to human health.

项目摘要： 氮酶复合物是产生代谢有用形式的唯一生物学途径 大气二氮的氮。这两个组件蛋白系统由专性还原酶组成， Fe蛋白和催化蛋白MOFE蛋白仅存在于少数微生物中，但 产生近50％的生物利用氮。因此，生物氮固定机制极为 对全球作物生产和人类健康的重要影响。过去半个世纪的开创性作品还没有 仅揭示了氮酶的错综复杂的动力学途径，但同时也发现了最复杂的金属簇 MOFE蛋白活性位点内的性质。该群集被称为Femo-Cofactor，具有组成 [7FE：9S：1C：1MO] - R-HOMOCITRATE，仅由活跃部位中的两个残基进行协调。电子是 通过在Fe蛋白中的[4FE：4S]簇穿过[4fe：4s]的簇，到Mofe-蛋白质中的[8fe：7s]中心，称为P- 簇。然后将这些还原的等效物传递到底物还原的活性位点的FEMO-CACTOR 发生。需要四个连续的电子传递循环以结合二氮，需要另外四个 完全将一个二氮分子转换为两个氨分子。尚不清楚Femo-Cofactor是如何的 用于底物结合的启动，FEMO-CACTAROR如何结合或穿梭电子与底物或什么作用 周围的活动现场残留物可在底物减少中发挥作用。该提议的主要假设是 在Femo-cactor中的原子和周围蛋白质中原子的物理重排 底物减少对于底物访问和与辅因子的结合是必需的，并且可能会进一步播放 在还原机制中的作用。这项工作的目的是确定在 通过利用冷冻的能力和执行实验的氮化酶整个底物周转率 在两个目标中概述。目标1：氮酶静息状态的结构表征：单独使用MOFE蛋白和 通过单个颗粒冷冻剂，用Fe蛋白稳定ADP-ALF4的复合物。目标2：结构表征 单个颗粒冷冻的氮酶离职相关的形式。通过追求目标1和2，我将获得 在单个颗粒冷冻领域的训练，每个目标都将提供金属酶化学的专业知识。 该研究将在加利福尼亚技术学院进行，主要是在著名的低温中进行 具有丰富的显微镜和专业知识的中心。这些目标将为瞬态提供急需的灯光 氮化酶周转途径内的中间体，从而为理性提供了更好的基础 高效合成氮固定平台的设计。此外，洞察力和方法发展 在此提案中，可以应用于与人类健康有关的其他知识熟悉的金属酶。