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
基本信息
- 批准号:8839784
- 负责人:
- 金额:$ 26.22万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2012
- 资助国家:美国
- 起止时间:2012-08-01 至 2016-04-30
- 项目状态:已结题
- 来源:
- 关键词:ATP HydrolysisATP phosphohydrolaseActive SitesAddressAgricultureAmmoniaBindingBioavailableBiochemicalBiologicalBiological AssayBiological ModelsBiological ProductsCarbon DioxideCatalysisCell physiologyChemicalsCollaborationsComplexCouplesCouplingCrystallographyDataDependenceDockingElectron TransportElectronicsElectronsElementsEngineeringEnzymesEventFundingGoalsGuanosine TriphosphateHealthHumanHydrogen BondingKineticsLaboratoriesMechanicsMetabolismMetalsMolybdoferredoxinNatureNitrogenNitrogen FixationNitrogenaseNucleotidesNutritionalOutcomeOxidation-ReductionPathway interactionsPopulationPositioning AttributeProcessProteinsProtonsReactionResearchRestRoleSchemeSiteSocial WelfareSpectrum AnalysisStructureSurfaceTechniquesTestingTimeVariantWorkbasecatalystdeprotonationelectron donorenzyme activityenzyme mechanismenzyme modelexperienceinhibitor/antagonistinsightmeetingsmutantprotonationsmall molecule
项目摘要
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):本提案旨在阐明细菌固氮酶如何催化大气二氮转化为生物可利用形式氨的化学困难转化,以及为什么/如何利用ATP水解来驱动该反应。作为唯一负责还原性固氮的酶,固氮酶维持了约40%人口的农业/营养需求。除了其全球重要性之外,固氮酶是一种独特的模型系统,与生物氧化还原催化以及ATP/GTP依赖的能量转导过程具有广泛的相关性,这两者都是正常细胞功能的核心,因此与人类健康直接相关。尽管40年来广泛的生物化学,生物物理和结构表征,固氮酶机制的两个最重要的问题没有得到回答:a)为什么和如何ATP水解最终用于减少N2或替代底物?B)双氮对固氮酶活性中心簇FeMoco的作用机制是什么?为了进一步回答这些问题,需要新的实验方法和可验证的假设。为此,开发了一种新的策略,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体的能力。受这些进展、最近的晶体学发现、固氮酶方面的丰富经验以及与世界一流光谱实验室的合作的推动,PI和他的团队在解决生物固氮中突出的机制问题方面具有独特的优势。1)确定固氮酶的两个组分MoFe蛋白(催化组分)和Fe蛋白(ATP酶/电子供体)之间的多个ATP依赖的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的结构特征在于在五个不同的核苷酸状态,其中Fe-蛋白质填充几个对接区的MoFe-蛋白质表面上。假设这些对接区能够使FeMoco发生快速连续的单电子转移(ET)反应,以促进8电子催化转换,并将进行系统的结构-功能研究。2)确定的结构/电子功能的钼铁蛋白质是至关重要的控制电子流之间的两个Fe-S集群,P-集群和FeMoco。几条研究线已经表明了“构象门”的必要性,以使电子流从P-簇到FeMoco用于催化,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,光引发的ET和各种光谱技术,使用具有扰动的电子和质子转移途径的MoFe蛋白变体来探测。3)表征处于活化/基底结合状态的FeMoco结构。FeMoco仅在恒定ATP周转条件下还原超过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被用来填充FeMoco在一个单电子还原状态为基板结合,并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,穆斯堡尔谱)的特点。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,而且还将提供对生物多电子/质子氧化还原催化过程以及ATP能量转化为化学或机械功的一般见解。
项目成果
期刊论文数量(0)
专著数量(0)
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Faik Akif Tezcan其他文献
Faik Akif Tezcan的其他文献
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{{ truncateString('Faik Akif Tezcan', 18)}}的其他基金
Mechanism of Energy Transduction and Substrate Activation in Biological Nitrogen Fixation
生物固氮中的能量转换和底物激活机制
- 批准号:
10566582 - 财政年份:2023
- 资助金额:
$ 26.22万 - 项目类别:
Mechanism of Energy Transduction and Substrate Activation in Biological Nitrogen Fixation
生物固氮中的能量转换和底物激活机制
- 批准号:
10795182 - 财政年份:2023
- 资助金额:
$ 26.22万 - 项目类别:
Design and Evolution of Metal-Based Functions in Supramolecular Protein Scaffolds
超分子蛋白支架中金属基功能的设计与演化
- 批准号:
10221740 - 财政年份:2020
- 资助金额:
$ 26.22万 - 项目类别:
Design and Evolution of Metal-Based Functions in Supramolecular Protein Scaffolds
超分子蛋白支架中金属基功能的设计与演化
- 批准号:
10033233 - 财政年份:2020
- 资助金额:
$ 26.22万 - 项目类别:
Design and Evolution of Metal-Based Functions in Supramolecular Protein Scaffolds
超分子蛋白支架中金属基功能的设计与演化
- 批准号:
10387560 - 财政年份:2020
- 资助金额:
$ 26.22万 - 项目类别:
Design and Evolution of Metal-Based Functions in Supramolecular Protein Scaffolds
超分子蛋白支架中金属基功能的设计与演化
- 批准号:
10413065 - 财政年份:2020
- 资助金额:
$ 26.22万 - 项目类别:
Design and Evolution of Metal-Based Functions in Supramolecular Protein Scaffolds
超分子蛋白支架中金属基功能的设计与演化
- 批准号:
10645201 - 财政年份:2020
- 资助金额:
$ 26.22万 - 项目类别:
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 - 财政年份:2012
- 资助金额:
$ 26.22万 - 项目类别:
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:
8217963 - 财政年份:2012
- 资助金额:
$ 26.22万 - 项目类别:
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 - 财政年份:2012
- 资助金额:
$ 26.22万 - 项目类别: