Nitrogenase Mechanism

固氮酶机制

• 批准号: 8054856
• 负责人: Lance C. Seefeldt
• 金额: $ 29.42万
• 依托单位: UTAH STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054856
• 关键词: Active Sites; Address; Amino Acid Substitution; Amino Acids; Binding; Biological; Catalysis; Complex; Computing Methodologies; Coupled; Development; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Enzymes; Foundations; Freezing; Funding; Goals; Health; Homologous Gene; Human; Hydrazine; Hydrolysis; Kinetics; Label; Lead; Light; Mediating; Membrane Proteins; Metals; Methods; MgATP; Modeling; Molecular; Molybdoferredoxin; Monitor; Movement; Nature; Nitrogen; Nitrogen Fixation; Nitrogenase; Nuclear; Nucleotides; Pathway interactions; Population; Process; Proteins; Reaction; Roentgen Rays; Role; Site; Spectrum Analysis; Structure; Techniques; Temperature; Testing; Time; Work; base; catalyst; diazene; electron nuclear double resonance spectroscopy; electronic structure; homocitrate; insight; interest; metalloenzyme; microorganism; novel; oxidation; protein function

DESCRIPTION (provided by applicant): Project Summary Nitrogen fixation (N2 reduction) represents the most significant input of nitrogen into the biogeochemical nitrogen cycle. The biological catalyst for nitrogen fixation is called nitrogenase, an enzyme that is only produced by certain microorganisms called diazotrophs. Nitrogenase contains two component proteins, one of these is a nucleotide-dependent agent of electron delivery (called the Fe protein) and the other (called MoFe protein) contains the site for substrate binding and reduction. The work proposed herein seeks to address some of the key unanswered questions about the nitrogenase catalytic mechanism. During the last funding cycle, methods were developed that allow the normal substrate N2, as well as proposed reduction intermediates, diazene (HN=NH and its homolog methydiazene HN=N-CH3), and hydrazine (H2N-NH2) to be trapped in high concentrations within the MoFe protein. Preliminary studies have revealed these trapped species are bound to a complex organo-metallocluster, called FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], which provides the nitrogenase active site. Each of the trapped states is paramagnetic thereby permitting the application of electron paramagnetic resonance (EPR) and enhanced nuclear double resonance (ENDOR) spectroscopies as powerful probes to deduce molecular details of the bound species. In the first aim, we seek to characterize each of these trapped states as an approach to explore intermediate states that occur during the catalytic reduction of N2. Studies proposed include optimization of the N2-trapped state, and a temperature step-annealing strategy to connect the intermediates to each other and to the mechanism. Collectively, these studies are expected to allow development of an experimentally driven model that describes the nature of the catalytic intermediates. A strategy is presented in the second aim that will probe a putative substrate channel within the MoFe protein component. This channel is proposed to provide a pathway for substrate movement from the protein surface to FeMo-cofactor. For these studies, amino acids that line the proposed channel within the MoFe protein will be substituted individually and in combinations and the impact of these substitutions on kinetic parameters for reduction of a range of substrates of varying size will be determined. X-ray crystal structures of the most interesting MoFe proteins will also be pursued. It is expected that these studies will provide the first experimental evidence that define a specific substrate channel within nitrogenase. The P-cluster is a second complex metallocluster ([8Fe-7S]) contained within the MoFe protein that is proposed to function in the intermolecular delivery of electrons to FeMo-cofactor. Studies presented in the third aim seek to define the role of the P-cluster in electron transfer during the catalytic cycle. We have developed a freeze-quench EPR spectroscopic approach that allows simultaneous observation of changes in the oxidation states of the P-cluster and FeMo-cofactor during the catalytic cycle. Preliminary results reveal that electron transfer from the P-cluster to FeMo-cofactor can be controlled and monitored by using a temperature step-annealing technique that avoids the confounding continuous addition of electrons from the Fe protein. These studies offer the first opportunity to monitor internal electron transfer within nitrogenase and are therefore expected to provide significant new insights into the role of the P-cluster. The fourth aim seeks to reveal the identity of X, the unknown atom that is located at the center of FeMo-cofactor. Preliminary ENDOR spectroscopic results reveal a strongly coupled N-atom in the wild-type MoFe protein trapped during turnover with N2 bound. The strongly coupled N is not coming from the bound N2. An experimental approach is presented to identify the origin of this strongly coupled N atom (possibly X) by using a MoFe protein assembled from an apo- MoFe protein labeled with either 14N or 15N and isolated FeMo-cofactor labeled with either 14N or 15N. Resolving the identity of X is important as it will provide mechanistic insight with respect to the electronic structure of the nitrogenase active site, it will impact the application of computational methods that seek to model the catalytic process, and it will provide information that is necessary to understand how FeMo-cofactor is formed biologically and how it might be produced synthetically. PUBLIC HEALTH RELEVANCE: Narrative The majority of N2 reduction that occurs today, ultimately supporting the nitrogen demand of an estimated 60% of the human population, is by biological nitrogen fixation within microorganisms, called diazotrophs. The catalyst for this N2 reduction is the enzyme nitrogenase. In this work, we seek to deduce key aspects of the mechanism of nitrogenase.

描述（由申请人提供）： 项目摘要 固氮（N2 还原）是生物地球化学氮循环中最重要的氮输入。固氮的生物催化剂称为固氮酶，这种酶仅由某些称为固氮微生物的微生物产生。固氮酶含有两种蛋白质，其中一种是核苷酸依赖性电子传递剂（称为 Fe 蛋白），另一种（称为 MoFe 蛋白）包含底物结合和还原位点。本文提出的工作旨在解决有关固氮酶催化机制的一些尚未解答的关键问题。在上一个资助周期中，开发了一些方法，允许将正常底物 N2 以及拟议的还原中间体、二氮烯（HN=NH 及其同系物甲基二氮烯 HN=N-CH3）和肼（H2N-NH2）捕获在MoFe 蛋白内浓度高。初步研究表明，这些被捕获的物质与一种称为 FeMo 辅因子 [7Fe-9S-Mo-X-homocitrate] 的复杂有机金属簇结合，它提供固氮酶活性位点。每个俘获态都是顺磁性的，因此允许应用电子顺磁共振（EPR）和增强核双共振（ENDOR）光谱作为强大的探针来推断束缚物质的分子细节。第一个目标是，我们试图描述每一种俘获态的特征，作为探索 N2 催化还原过程中出现的中间态的方法。提出的研究包括 N2 捕获态的优化，以及将中间体彼此连接并与机制连接的温度步进退火策略。总的来说，这些研究有望开发出描述催化中间体性质的实验驱动模型。第二个目标提出了一种策略，该策略将探测 MoFe 蛋白质成分内的假定底物通道。该通道被提议为底物从蛋白质表面移动到 FeMo 辅因子提供途径。对于这些研究，MoFe 蛋白内拟议通道内的氨基酸将被单独或组合取代，并且将确定这些取代对还原一系列不同尺寸底物的动力学参数的影响。最有趣的 MoFe 蛋白的 X 射线晶体结构也将被研究。预计这些研究将提供定义固氮酶内特定底物通道的第一个实验证据。 P 簇是 MoFe 蛋白中包含的第二种复杂金属簇 ([8Fe-7S])，其功能是在电子向 FeMo 辅因子的分子间传递中发挥作用。第三个目标中提出的研究试图确定 P 团簇在催化循环期间电子转移中的作用。我们开发了一种冷冻淬灭 EPR 光谱方法，可以同时观察催化循环期间 P 团簇和 FeMo 辅因子氧化态的变化。初步结果表明，可以通过使用温度步进退火技术来控制和监测从 P 团簇到 FeMo 辅因子的电子转移，该技术避免了来自 Fe 蛋白的电子连续添加的混乱。这些研究首次提供了监测固氮酶内部电子转移的机会，因此有望为 P 簇的作用提供重要的新见解。第四个目标旨在揭示位于 FeMo 辅因子中心的未知原子 X 的身份。初步的 ENDOR 光谱结果显示，野生型 MoFe 蛋白中存在一个强耦合的 N 原子，在 N2 结合的周转过程中被捕获。强耦合 N 不是来自绑定 N2。提出了一种实验方法，通过使用由标记有 14N 或 15N 的 apo-MoFe 蛋白质和标记有 14N 或 15N 的分离 FeMo 辅因子组装而成的 MoFe 蛋白质来鉴定这种强耦合 N 原子（可能是 X）的起源。解决 X 的身份非常重要，因为它将提供有关固氮酶活性位点电子结构的机制见解，它将影响寻求模拟催化过程的计算方法的应用，并将提供必要的信息。了解 FeMo 辅因子是如何生物形成的以及如何合成生产。公共卫生相关性：叙述 如今发生的大部分氮减少，最终满足了估计 60% 人口的氮需求，是通过微生物（称为固氮菌）内的生物固氮作用实现的。这种 N2 还原的催化剂是固氮酶。在这项工作中，我们试图推断固氮酶机制的关键方面。