Mechanism of Energy Transduction and Substrate Activation in Biological Nitrogen

生物氮的能量转换和底物活化机制

• 批准号: 8516536
• 负责人: Faik Akif Tezcan
• 金额: $ 25.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516536
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Active Sites; Address; Agriculture; Ammonia; Binding; Bioavailable; Biochemical; Biological; Biological Assay; Biological Models; Biological Products; Carbon Dioxide; Catalysis; Cell physiology; Chemicals; Collaborations; Complex; Couples; Coupling; Crystallography; DNA Sequence Rearrangement; Data; Dependence; Docking; Electron Transport; Electronics; Electrons; Elements; Engineering; Enzymes; Event; Funding; Goals; Guanosine Triphosphate; Health; Human; Hydrogen Bonding; Kinetics; Laboratories; Mechanics; Metabolism; Metals; Molybdoferredoxin; Nature; Nitrogen; Nitrogen Fixation; Nitrogenase; Nucleotides; Nutritional; Outcome; Oxidation-Reduction; Pathway interactions; Population; Positioning Attribute; Process; Proteins; Protons; Reaction; Research; Rest; Role; Scheme; Site; Social Welfare; Spectrum Analysis; Structure; Surface; Techniques; Testing; Time; Variant; Work; base; catalyst; deprotonation; electron donor; enzyme activity; enzyme mechanism; enzyme model; experience; inhibitor/antagonist; insight; meetings; mutant; protonation; small molecule

DESCRIPTION (provided by applicant): This proposal aims to elucidate how the bacterial enzyme 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 four decades of extensive biochemical, biophysical and structural characterization, the two most important questions about nitrogenase mechanism are not answered: 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 on the nitrogenase active site cluster, FeMoco? To make any further progress toward answering these questions, new experimental approaches and testable hypotheses are needed. Toward this end, a new strategy was developed to photochemically activate nitrogenase catalysis in the absence of ATP hydrolysis, which opens up new avenues to populate discrete catalytic intermediates on FeMoco for structural characterization. At the same time, the capability was acquired to rapidly generate site-directed mutants of nitrogenase proteins. Motivated by these advances, recent crystallographic findings, extensive experience on nitrogenase and collaborations with world-class spectroscopy laboratories, the PI and his group are uniquely positioned to address outstanding mechanistic issues in biological nitrogen fixation. The objectives of this project are to: 1) Determine the mechanistic role of multiple ATP-dependent docking interactions between the two nitrogenase components, the MoFe-protein (catalytic component) and the Fe-protein (ATPase/electron donor). The complex between the Fe-protein and MoFe-protein was structurally characterized in five distinct nucleotide states, whereby the Fe-protein populates several docking zones on the MoFe-protein surface. These docking zones are hypothesized to enable rapid successive one-electron transfer (ET) reactions to FeMoco to promote the 8- electron catalytic turnover, and will be subjected to systematic structure-function studies. 2) Identify the structural/electronic features of the MoFe-protein that are critical for controlling electron flow between its two Fe-S clusters, the P-cluster and FeMoco. Several lines of research have indicated the necessity of a "conformational gate" to enable electron flow from P-cluster to FeMoco for catalysis, which is hypothesized to be a protonation/deprotonation event. The nature of this gate will be probed through enzyme activity assays, photo-initiated ET and various spectroscopic techniques, using MoFe-protein variants with perturbed electron and proton transfer pathways. 3) Characterize the FeMoco structure in an activated/substrate-bound state. FeMoco can only bind substrates/inhibitors upon reduction beyond its as-isolated state under constant ATP turnover conditions. The newly developed photocatalytic scheme will be exploited to populate FeMoco in a one-electron reduced state primed for substrate binding, and the structures of ensuing intermediates will be characterized by crystallography and an array of spectroscopic techniques (EPR, NRVS, IR, M"ssbauer). By meeting these project goals, the PI and his group will not only uncover the mechanistic details of this enzyme, but also provide general insights into biological multi-electron/proton redox catalytic processes and the transduction of ATP energy into chemical or mechanical work.

描述(申请人提供)：本提案旨在阐明细菌酶固氮酶如何催化化学上困难的大气氮素转化为生物可利用的形式氨，以及为什么/如何利用ATP水解来推动这一反应。固氮酶是唯一一种负责还原固氮的酶，维持着约40%人口的农业/营养需求。除了其全球重要性，固氮酶是一个独特的模型系统，与生物氧化还原催化以及依赖ATP/GTP的能量转导过程具有广泛的相关性，这两个过程都是细胞正常功能的核心，因此与人类健康直接相关。尽管四十年来对固氮酶进行了广泛的生化、生物物理和结构表征，但关于固氮酶机制的两个最重要的问题仍然没有得到回答：a)为什么以及如何最终利用ATP水解来还原氮气或替代底物？B)氮素对固氮酶活性部位簇FeMoco的密切作用机制是什么？为了在回答这些问题上取得任何进一步的进展，需要新的实验方法和可检验的假设。为此，开发了一种新的策略来在没有ATP水解的情况下光化学激活固氮酶催化，这为在FeMoco上填充离散的催化中间体以进行结构表征开辟了新的途径。同时，获得了快速产生固氮酶蛋白定点突变的能力。在这些进展、最新的结晶学发现、在固氮酶方面的丰富经验以及与世界级光谱实验室的合作的推动下，PI和他的团队在解决生物固氮中突出的机械问题方面具有独特的地位。本项目的目标是：1)确定固氮酶的两个组分，MoFe蛋白(催化组分)和Fe蛋白(ATPase/电子供体)之间依赖于ATP的多种对接相互作用的机制。铁蛋白和MoFe蛋白之间的复合体在结构上具有五种不同的核苷酸状态，从而使Fe蛋白聚集在MoFe蛋白表面的几个对接区。这些对接区域被认为能够使FeMoco发生快速连续的单电子转移(ET)反应，以促进8电子的催化周转，并将进行系统的结构-功能研究。2)确定MoFe-蛋白质的结构/电子特征，这些特征对控制其两个Fe-S团簇P-团簇和FeMoCo之间的电子流动至关重要。一些研究表明，为了催化，需要一个“构象门”使电子从P-团簇流向FeMoco，这被认为是质子化/去质子化事件。这一门的性质将通过酶活性分析、光引发的ET和各种光谱技术来探索，使用带有扰动的电子和质子转移路径的MoFe-蛋白质变体。3)表征激活/衬底结合态下的FeMoco结构。在恒定的ATP周转条件下，FeMoco只有在还原超过其隔离状态时才能结合底物/抑制剂。新开发的光催化方案将被用来在单电子还原状态下填充FeMoco，为底物结合做准备，随后的中间体的结构将通过结晶学和一系列光谱技术(EPR，NRVS，IR，M“SSbauer)来表征。通过满足这些项目目标，PI和他的团队将不仅揭示这种酶的机理细节，而且还将提供对生物多电子/质子氧化还原催化过程以及将ATP能量转化为化学或机械功的一般性见解。