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氮酶如何催化大气二氮的化学困难转化为可生物利用形式，氨，以及为什么/如何利用ATP水解来驱动这一反应。作为唯一负责还原氮固定的酶，氮酶可以维持约40％人口的农业/营养需求。除全球重要性外，氮酶是一个独特的模型系统，与生物氧化还原催化以及ATP/GTP依赖性能量转导过程相关，这既是适当的细胞功能，因此与人类健康直接相关。尽管有四十年的广泛的生化，生物物理和结构表征，但未回答有关氮酶机制的两个最重要的问题：a）为什么最终以及如何最终将ATP水解用于减少N2或替代性底物？ b）氮酶活性位点簇（Femoco）在二氮中的亲密机制是什么？为了在回答这些问题方面取得进一步的进展，需要采用新的实验方法和可检验的假设。为此，在没有ATP水解的情况下，制定了一种新的策略来激活氮酶催化，这为FEAMOCO上的离散催化中间体开辟了新的途径，以实现结构表征。同时，获得了能力以快速生成氮酶蛋白的位置定向突变体。在这些进步的推动下，最近的晶体学发现，对氮酶的丰富经验以及与世界一流的光谱实验室的合作，PI及其小组在生物氮固定方面的出色机械问题是独特的。该项目的目的是：1）确定两个氮酶成分之间的多个ATP对接相互作用的机械作用，即MOFE蛋白（催化成分）和Fe蛋白（ATPase/Electron供体）。 Fe蛋白和MOFE-蛋白质之间的复合物在五个不同的核苷酸态中进行了结构表征，因此，Fe蛋白在MOFE蛋白表面上填充了几个对接区。假设这些对接区域可以使FEMOCO的快速连续的单电子转移（ET）反应促进8-电子催化转移，并将进行系统的结构功能研究。 2）确定MOFE-蛋白质的结构/电子特征对于控制两个Fe-S簇之间的电子流量至关重要。几条研究线表明需要“构象门”才能使电子流从p群集到femoco进行催化，这被认为是质子化/去质子化事件。该门的性质将通过酶活性测定，光发出的ET和各种光谱技术进行探测，使用具有扰动的电子和质子转移途径的MOFE蛋白质变体。 3）在激活/底物结合的状态中表征Femoco结构。在恒定的ATP周转条件下，Femoco只能在降低其AS溶解状态后减少底物/抑制剂。新开发的光催化方案将被利用，以在单电子降低的状态下填充Femoco进行底物结合的状态，随后的中间体的结构将以晶体学和一系列光谱技术（EPR，NRV，IRV，IR，IR，M'SSSBAUER）的方式来表征。酶，但还提供了对生物多电子/质子氧化还原催化过程以及将ATP能量转导向化学或机械工作的一般见解。