Active-site models unravel mechanism of enzymatic alkane activation

活性位点模型揭示了酶促烷烃活化的机制

• 批准号: 10711929
• 负责人: David Schilter
• 金额: $ 18.05万
• 依托单位: TEXAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10711929
• 关键词: Active Sites; Alkanes; Apical; Archaea; Binding; Biochemical; Charge; Chemicals; Chemistry; Coenzyme M; Complement; Complex; Crystallization; Data; Disulfides; Educational process of instructing; Energy Metabolism; Environment; Enzymes; Equilibrium; Gases; Gastrointestinal Diseases; Goals; Health; Human; Hydrocarbons; Hydrogen; Hydrogen Bonding; In Situ; Learning; Ligands; Measurement; Measures; Mediating; Medicine; Metabolic; Metabolic Diseases; Metabolism; Methane; Methods; Modeling; Nickel; Nitrogen; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Periodicity; Pharmacologic Substance; Phase; Planet Earth; Process; Reaction; Regulation; Reporting; Research; Spectrum Analysis; Structural Models; Structure; Structure-Activity Relationship; Sulfhydryl Compounds; Testing; Thermodynamics; Water; Work; adduct; analog; coenzyme B; cofactor; density; expectation; functional mimics; gastrointestinal; gut microbiota; methyl radical; oxidation; small molecule; theories; thioether

Project Summary Archaea in the human gut express methyl-coenzyme M reductase (MCR) to catalyze the last step of methanogenesis and the first step of the anaerobic oxidation of methane. These reactions occur at F430, a nickel cofactor whose first coordination sphere — the ligands directly bound to it — includes the four nitrogen atoms of a unique anionic macrocycle. This center reversibly cleaves the thioether methyl-coenzyme M (CoM– SMe) to release methyl radical, which combines with thiol coenzyme B (HS–CoB) in the second coordination sphere — the residues proximal to but not bonded to the active site — to liberate methane and the disulfide CoM–S–S–CoB. The first and second coordination spheres of nickel in several MCR states are intractable, which motivates us to synthesize tractable small molecules that recapitulate active-site features proposed for these contentious states. Comparing data for models and MCR tells us how plausible these proposals are. Our long-term goal is to synthesize high-fidelity models that help us understand how MCR makes and breaks the C–H bonds in methane. Towards this goal, the objective of this project is to prepare, and spectroscopically and chemically interrogate macrocyclic nickel complexes with very similar ligand environments to F430 in different MCR states. The central hypothesis is that to mimic MCR spectroscopy a nickel complex needs a high-fidelity first coordination sphere, while to mimic MCR function and cleave methane it further requires a second-coordination-sphere radical. In preliminary work, we prepared and crystallized four-coordinate nickel complexes of a readily tunable anionic macrocycle. Our density functional theory calculations predict that one such complex should bind thiolate to mimic the first coordination sphere of F430 in the methane-cleaving step. Further calculations predict that a related complex with a pendant thiyl radical near the thiolate is both plausible and thermodynamically favored to cleave methane. The rationale is that tunable models will let us tease out the motifs necessary for C–H activation. We will test our hypothesis by focusing on two specific aims. Aim 1: Model the first coordination sphere of nickel in MCR to mimic spectroscopy and Aim 2: Model the second coordination sphere of nickel in MCR to mimic function. Towards Aim 1 we will: (a) prepare and characterize nickel complexes of anionic macrocycles, and (b) bind these complexes to water, thioether, thiolate, thiol, methyl or hydride ligands. These ligands have been proposed to bind the MCR active site but evidence, particularly for the last three ligands, is scarce, so our models will identify plausible first coordination spheres. Towards Aim 2 we will: (a) further develop the nickel complexes to feature a thiolate and a proximal thiyl radical, and (b) investigate the chemistry of this thiolate–thiyl species towards methane and other alkanes. The macrocylic nickel complexes and their adducts will be the highest-fidelity synthetic models reported and their activation of methane would be unprecedented for such nickel macrocycles. Overall, this work will complement biochemical studies to fill in our mechanistic picture for MCR, a central metabolic enzyme.

项目摘要 人类肠道中的古细菌表达甲基辅酶M还原（MCR），以催化最后一步 甲烷的甲烷发生和甲烷厌氧氧化的第一步。这些反应发生在F430，A 镍辅因子的第一个配位球 - 配体直接与之结合的配体 - 包括四个氮 独特的阴离子大环的原子。该中心可逆地切割硫醚甲基辅酶M（com– SME）释放甲基自由基，并在第二个配位中与硫醇辅酶B（HS -COB）结合 球 - 残差靠近但不粘结到活跃部位 - 以释放甲烷和二硫化物 com – s – s -cob。在几个MCR​​状态中镍的第一和第二配位球很棘手， 这激励我们合成可牵引的小分子，该分子概括了提议的活动位置特征 这些有争议的状态。比较模型和MCR的数据告诉我们这些建议的合理性。 我们的长期目标是综合高保真模型，以帮助我们了解MCR的生产方式和破坏方式 甲烷中的C – H键。为了实现这一目标，该项目的目的是准备和光谱 并在化学询问的大环镍复合物具有与F430非常相似的配体环境的大型镍配合物中 不同的MCR状态。中心假设是要模仿MCR光谱，镍复合物需要 高保真性第一协调领域，而要模仿MCR功能并清除甲烷，它需要进一步 第二配合 - 球体自由基。在初步工作中，我们准备并结晶了四坐标镍 易于调谐的阴离子大环的复合物。我们的密度功能理论计算预测一个 这种复合物应在甲烷切割步骤中结合硫醇与模仿F430的第一个配位球体。 进一步的计算预测，与甲基酸酯靠近硫醇的硫基自由基相关的复合物都是合理的 在热力学上更喜欢清除甲烷。理由是可调型会让我们取笑 C – H激活所需的基序。我们将通过关注两个具体目标来检验我们的假设。目标1： 模拟MCR中镍至模拟光谱法的第一个配位球，AIM 2：模型第二 镍在MCR中的配位领域至模拟功能。朝目标1我们将：（a）准备和表征 阴离子大环的镍复合物，（b）将这些复合物与水，硫乙醚，硫醇，硫代，，， 甲基或氢化物配体。已经提出了这些配体来结合MCR活性位点，但证据，但 特别是对于最后三个配体，很少，因此我们的模型将确定合理的第一协调领域。 朝目标2我们将：（a）进一步开发镍络合物以具有硫醇酸盐和近端硫醇 自由基，（b）研究这种硫醇硫酸盐 - 甲烷和其他烷烃的化学。 大环镍复合物及其加合物将是报告的最高保真性合成模型，并且 对于这种镍大环，它们的激活将是前所未有的。总的来说，这项工作将 补体生化研究以填补我们的机械图片MCR，一种中央代谢酶。