Active-site models unravel mechanism of enzymatic alkane activation

项目摘要 人类肠道中的古生菌表达甲基辅酶M还原酶(MCR)来催化最后一步 产甲烷和甲烷厌氧氧化的第一步。这些反应发生在F430，a 镍辅因子，其第一配位球体--直接与之结合的配体--包括四个氮 一种独特的阴离子大循环的原子。该中心可逆地裂解硫醚甲基辅酶M(COM-1)。 SME)释放甲基自由基，与硫醇辅酶B(HS-CoB)二次配位结合 球体-活性中心附近但没有键合的残基-释放甲烷和二硫化物 S S-COB。镍在几个MCR状态中的第一和第二配位球是难以处理的， 这促使我们合成易于处理的小分子，这些小分子概括了提出的 这些有争议的州。比较模型和平均现金率的数据会告诉我们，这些建议有多可信。 我们的长期目标是合成高保真模型，帮助我们了解MCR的成败 甲烷中的C-H键。为了这个目标，这个项目的目标是准备和光谱 并用化学方法研究了具有与F430非常相似的配体环境的大环镍配合物 不同的MCR状态。中心假设是，为了模拟MCR光谱，镍络合物需要一个 高保真的第一配位球，而为了模拟MCR功能和裂解甲烷，它还需要一个 二次配位球根。在前期工作中，我们制备并晶化了四配位镍 易于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 这样的络合物应该与硫酸盐结合，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，在硫酸盐附近带有侧链硫基的相关络合物既有可能 热力学上有利于裂解甲烷。理由是，可调的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 镍在MCR中的配位球起到模拟作用。为实现目标1，我们将：(A)准备和说明 阴离子大环镍络合物，以及(B)将这些络合物与水、硫醚、硫醇、 甲基或氢化物配体。这些配体已被提出与MCR活性部位结合，但有证据表明， 特别是对于后三个配体，是稀有的，所以我们的模型将确定看似合理的第一配位范围。 为了实现目标2，我们将：(A)进一步发展镍配合物，使之具有硫基和近端硫基。 (B)研究这种硫酸盐-硫基物种对甲烷和其他烷烃的化学作用。 大环镍络合物及其加合物将是报道的最高保真度的合成模型和 对于这样的镍大循环来说，它们对甲烷的活化将是前所未有的。总的来说，这项工作将 补充生化研究，以填补我们对MCR的机理图景，MCR是一种中央代谢酶。

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.