Active-site models unravel mechanism of enzymatic alkane activation

项目摘要 人肠道中的大肠杆菌表达甲基辅酶M还原酶（MCR），催化 产甲烷和甲烷厌氧氧化的第一步。这些反应发生在F430， 镍辅因子，其第一配位层-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。该中心可逆地裂解硫醚甲基-辅酶M（CoM-1）。 SMe）释放出甲基自由基，甲基自由基与巯基辅酶B（HS-Co B）以第二配位结合 球体-接近但不与活性位点结合的残基-以释放甲烷和二硫化物 CoM-S-S镍的第一和第二配位球在几个MCR状态下是难以处理的， 这促使我们合成易于处理的小分子，这些小分子重现了活性位点的特征， 这些有争议的国家。比较模型和MCR的数据可以告诉我们这些建议的合理性。 我们的长期目标是合成高保真模型，帮助我们了解MCR如何制造和破坏 甲烷中的碳氢键为了实现这一目标，本项目的目标是准备，光谱 和化学询问大环镍配合物与F430中非常相似的配体环境， 不同的MCR状态。中心假设是，为了模拟MCR光谱，镍络合物需要 高保真第一配位球，而为了模拟MCR功能和裂解甲烷，它还需要 第二配位圈基。在前期工作中，我们制备并晶化了四配位镍 容易调节的阴离子大环的络合物。我们的密度泛函理论计算预测， 这种络合物应该结合硫醇盐以模拟甲烷裂解步骤中F430的第一配位球。 进一步的计算预测，在硫醇盐附近有一个悬挂的硫基自由基的相关络合物是合理的， 并且在热力学上有利于裂解甲烷。基本原理是，可调模型将让我们梳理出 C-H激活所必需的基序。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中镍的第一配位球以模拟光谱，目标2：模拟第二配位球 MCR中镍的配位球以模拟功能。为实现目标1，我们将：（a）编写和描述 阴离子大环的镍络合物，和（B）将这些络合物与水、硫醚、硫醇盐、硫醇， 甲基或氢化物配体。已经提出这些配体结合MCR活性位点，但证据表明， 特别是对于最后三个配体，是稀缺的，所以我们的模型将确定合理的第一协调领域。 为了实现目标2，我们将：（a）进一步开发镍配合物，以具有硫醇盐和近端硫基 自由基，和（B）研究这种硫醇盐-硫基物质对甲烷和其它烷烃的化学性质。 大环镍配合物及其加合物将是报道的最高保真的合成模型， 它们对甲烷的活化对于这种镍大环化合物来说是前所未有的。总的来说，这项工作将 补充生化研究，以填补我们的机制图片MCR，一个中央代谢酶。

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.