Nitrogenase Mechanism

固氮酶机制

• 批准号: 7646819
• 负责人: Lance C. Seefeldt
• 金额: $ 30.99万
• 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/7646819
• 关键词: Active Sites; Address; Amino Acid Substitution; Amino Acids; Binding; Biological; Catalysis; Complex; Computing Methodologies; Coupled; Development; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Enzymes; Foundations; Freezing; Funding; Goals; Homologous Gene; Human; Hydrazine; Hydrazines; 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; electronic structure; homocitrate; insight; interest; metalloenzyme; microorganism; novel; oxidation; protein function; public health relevance; Active Sites; Address; Amino Acid Substitution; Amino Acids; Binding; Biological; Catalysis; Complex; Computing Methodologies; Coupled; Development; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Enzymes; Foundations; Freezing; Funding; Goals; Homologous Gene; Human; Hydrazine; Hydrazines; 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; electronic structure; homocitrate; insight; interest; metalloenzyme; microorganism; novel; oxidation; protein function; public health relevance

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）： 项目摘要氮固定（N2还原）代表了氮对生物地球化学氮循环的最显着输入。固定氮的生物催化剂称为氮酶，这种酶是一种仅由某些称为重生营养的微生物产生的酶。氮酶含有两种成分蛋白，其中一种是电子递送的核苷酸依赖性药物（称为Fe蛋白），另一个是核苷酸依赖性蛋白（称为Fe蛋白）（称为MOFE蛋白），其中包含用于底物结合和还原的位点。本文提出的工作旨在解决有关氮酶催化机制的一些关键未解决问题。在最后一个融资周期中，开发了允许正常基板N2的方法，以及提议的还原中间体，重点（HN = NH及其同源甲基二苯HN = N-CH3）和hildrazine（H2N-NH2），在MOFE蛋白内被捕获高浓度。初步研究表明，这些被困的物种与复杂的有机甲簇结合，称为FEMO-COFACTOR [7FE-9S-MO-X-HOMOCITRATE]，该物种提供氮酶的活性位点。每个被困的状态都是顺磁性的，因此允许电子顺磁共振（EPR）和增强的核双共振（Endor）光谱镜作为强大的探针来推断结合物种的分子细节。在第一个目标中，我们试图将这些被困状态中的每一个都表征为探索N2催化减少过程中发生的中间状态的一种方法。提出的研究包括优化N2陷阱状态，以及将中间体连接到彼此和机制的温度逐步退缩策略。总的来说，这些研究有望允许开发一个实验驱动的模型，该模型描述了催化中间体的性质。在第二个目标中提出了一种策略，该策略将探测MOFE蛋白质成分内推定的底物通道。提出该通道为从蛋白质表面到FEAMO-CACTOR的底物运动提供了途径。在这些研究中，将单独和组合替换为MOFE蛋白内提出的通道的氨基酸，这些取代对动力学参数的影响将确定降低大小不同的底物的动力学参数。还将追求最有趣的MOFE蛋白质的X射线晶体结构。预计这些研究将提供第一个实验证据，以定义氮气中特定的底物通道。 P簇是MOFE蛋白中包含的第二个复合物金属簇（[8FE-7S]），该蛋白质中提议在电子向FEMO-CACTAR的分子递送中发挥作用。在第三个目的中提出的研究旨在定义催化周期中p簇在电子转移中的作用。我们已经开发了一种冻结EPR光谱方法，该方法允许同时观察催化循环期间P簇和FEAMO-CACTOR的氧化状态的变化。初步结果表明，可以通过使用温度逐步退缩技术来控制和监测电子从p群集到人含量的转移，从而避免了Fe蛋白中的电子连续添加的混杂添加。这些研究为监测氮气中的内部电子转移提供了第一个机会，因此有望为P群的作用提供重要的新见解。第四个目标旨在揭示X的身份，X是位于Femo-Cofactor中心的未知原子。初步的内虫光谱结果揭示了与N2结合的失误期间，野生型MOFE蛋白质中有强耦合的N原子。强耦合n不是来自界的n2。提出了一种实验方法，以通过使用由14N或15N标记的apo-MOFE蛋白组装的MOFE蛋白来识别这种强耦合N原子（可能是X）的起源，并使用14N或14N或15N标记的孤立的Femo-cactor。解决X的身份很重要，因为它将提供有关氮酶活性位点的电子结构的机械洞察力，它将影响试图建模催化过程的计算方法的应用，并且它将提供为了解如何形成生物学和如何形成的femo-Cactactor所必需的信息。公共卫生相关性：叙述今天发生的大多数N2减少，最终支持估计有60％人口的氮需求，是通过微生物中的生物氮固定，称为重物营养性。 N2还原的催化剂是氮氮。在这项工作中，我们试图推断氮酶机制的关键方面。