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: 8645652
• 负责人: Faik Akif Tezcan
• 金额: $ 26.39万
• 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/8645652
• 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（由申请人提供）：该提案旨在阐明细菌固氮酶如何催化大气中的二氮化学上困难的转化为生物可利用的形式氨，以及为什么/如何利用 ATP 水解来驱动该反应。作为唯一负责还原固氮的酶，固氮酶可以满足约 40% 人口的农业/营养需求。除了其全球重要性之外，固氮酶还是一种独特的模型系统，与生物氧化还原催化以及 ATP/GTP 依赖性能量转导过程具有广泛的相关性，这些过程对于正常的细胞功能至关重要，因此与人类健康直接相关。尽管四十年来进行了广泛的生物化学、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未得到解答：a) 为何以及如何最终利用 ATP 水解来还原 N2 或替代底物？ b) 二氮在固氮酶活性位点簇 FeMoco 上的内在机制是什么？为了在回答这些问题方面取得进一步进展，需要新的实验方法和可检验的假设。为此，开发了一种在没有 ATP 水解的情况下光化学激活固氮酶催化的新策略，这为在 FeMoco 上填充离散催化中间体以进行结构表征开辟了新途径。同时，获得了快速生成固氮酶蛋白定点突变体的能力。在这些进展、最新的晶体学发现、固氮酶方面的丰富经验以及与世界一流的光谱实验室的合作的推动下，首席研究员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优势。该项目的目标是： 1) 确定两种固氮酶成分、MoFe 蛋白（催化成分）和 Fe 蛋白（ATP 酶/电子供体）之间多种 ATP 依赖性对接相互作用的机制作用。 Fe-蛋白质和MoFe-蛋白质之间的复合物在结构上以五种不同的核苷酸状态为特征，其中Fe-蛋白质占据MoFe-蛋白质表面上的几个对接区。假设这些对接区能够实现与 FeMoco 的快速连续单电子转移 (ET) 反应，以促进 8 电子催化转换，并将进行系统的结构功能研究。 2) 确定 MoFe 蛋白的结构/电子特征，这些特征对于控制两个 Fe-S 簇（P 簇和 FeMoco）之间的电子流至关重要。多项研究表明，需要“构象门”来使电子从 P 团簇流向 FeMoco 进行催化，这被假设为质子化/去质子化事件。该门的性质将通过酶活性测定、光引发 ET 和各种光谱技术，使用具有扰动电子和质子转移途径的 MoFe 蛋白变体来探测。 3) 表征处于激活/基质结合状态的 FeMoco 结构。 FeMoco 仅在恒定 ATP 周转条件下还原至超出其分离状态时才能结合底物/抑制剂。新开发的光催化方案将用于以单电子还原态填充 FeMoco，为底物结合做好准备，随后的中间体的结构将通过晶体学和一系列光谱技术（EPR、NRVS、IR、M"ssbauer）进行表征通过实现这些项目目标，PI 和他的团队不仅将揭示这种酶的机制细节，还将提供对生物多电子/质子氧化还原的一般见解。催化过程以及将 ATP 能量转化为化学或机械功。