Oxygen Activation by Mononuclear Copper(I) Active Sites

单核铜 (I) 活性位点的氧活化

• 批准号: 10556191
• 负责人: Wesley John Transue
• 金额: $ 2.91万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10556191
• 关键词: Active Sites; Address; Amines; Behavior; Binding; Bioinorganic Chemistry; Biological; Biological Process; Catalysis; Cations; Chemistry; Chronic Disease; Clinical; Communities; Copper; Coupled; Dangerousness; Data; Detection; Dioxygen; Discipline; Distant; Dopamine; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Environment; Enzymes; Equilibrium; Freezing; Geometry; Goals; Health; Histidine; Human; Hydrogen; Hydrogen Peroxide; Immersion; Industrialization; Invertebrates; Investigation; Ions; Lead; Life; Ligands; Light; Lytic; Measures; Mediating; Methane; Methane hydroxylase; Methanol; Methionine; Methods; Mixed Function Oxygenases; Monitor; Mononuclear; N-terminal; Neurotransmitters; Outcome; Oxidants; Oxidases; Oxidative Stress; Oxides; Oxygen; Particulate; Pattern; Peroxidases; Peroxides; Polysaccharides; Process; Publishing; Raman Spectrum Analysis; Reaction; Regulation; Reporting; Roentgen Rays; Role; Site; Site-Directed Mutagenesis; Spectrum Analysis; Starch; Structure; Suggestion; Sulfur; Techniques; Testing; Thermodynamics; Training; Triplet Multiple Birth; Tyramine; Virulence; Virulence Factors; absorption; circular magnetic dichroism; cold temperature; computer studies; density; electronic structure; enzyme activity; enzyme mechanism; experience; experimental study; greenhouse gases; insight; instrumentation; man; neurochemistry; oxidation; oxidative damage; pathogen; spectroscopic data; theories

Project Summary/Abstract. (From Original Proposal) The interactions of coupled binuclear copper sites and dioxygen have been extensively studied in contexts crucial to life. This contrasts with analogous interactions between mononuclear copper sites and dioxygen, which are less well understood despite their importance in clinical, biological, and industrial settings. As such, furthering our understanding of these enzymes' mechanisms of action would have far reaching implications across a wide set of disciplines. This project focuses on mechanistic elucidation of the oxidative behaviors of three di erent monooxyge- nases featuring single copper active sites: tyramine -monooxygenase (T M), lytic polysaccharide monooxygenase (LPMO), and particulate methane monooxygenase (pMMO). The rst, T M, is closely related to human dopamine -monooxygenase (D M) and participates in invertebrate neurotransmitter regulation. As a noncoupled binuclear copper enzyme, it features two copper centers separated by 11 A, only one of which engages with O2. Out- standing questions on the timing of O2 binding to the reduced enzyme, hydrogen atom abstraction (HAA), and intermetallic electron transfer persist. The second enzyme, LPMO, has large industrial applications in renewable biofuels and has also been implicated as a virulence factor in several pathogens. Its active site comprises a single copper ion coordinated to two histidine residues and the N -terminal amine in a rare \histidine brace" geometry. Computational results inform the planned experiments, suggesting several mechanistic possibilities; though, some recent reports have questioned the role of O2 in favor of H2O2. The last enzyme, pMMO, is important in con- version of methane, a dangerous greenhouse gas, into methanol, a renewable fuel. It has long been thought to possess a coupled binuclear copper active site, but has recently been reappraised to have a mononuclear copper site also engaged in a histidine brace structural motif. This new suggestion means there is little mechanistic insight available, though it draws parallels between pMMO and LPMO. The project ultimately aims to shed light on how these enzymes oxidize their substrates, and to uncover useful and generalizable structure-function relations to be exploited in further clinical and industrial applications. Our speci c aims involve investigation of each class of enzyme using a battery of spectroscopies to uncover informative intermediates, capitalizing on the extensive instrumentation and experience available in the Solomon lab. As many of these transformations involve para- magnetic species, electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) experiments will allow direct interrogation of the copper center, particularly in combination with rapid freeze quench (RFQ) techniques. Additionally, stopped ow absorption, resonance Raman (rR), X-ray absorption (XAS), and X-ray emission (XES) will be heavily employed, especially when studying diamagnetic states. All of these studies will be supported by thorough computational investigations using density functional theory (DFT) methods, which will allow for further insight into the electronic structures and reaction energies. The training plan involves immersion in these spectroscopic techniques and in the eld of bioinorganic chemistry, all of which are new to the applicant.

项目摘要/摘要。 （来自原始提案） 耦合双核铜位点和双氧的相互作用已在关键环境中得到广泛研究 生活。这与单核铜位点和双氧之间的类似相互作用形成鲜明对比，后者较少 尽管它们在临床、生物和工业环境中很重要，但人们已经很好地理解了它们。因此，进一步推进我们的 了解这些酶的作用机制将对广泛的领域产生深远的影响 学科。该项目的重点是三种不同单氧化合物的氧化行为的机制阐明 具有单一铜活性位点的鼻：酪胺单加氧酶 (TM)、裂解多糖单加氧酶 （LPMO）和颗粒甲烷单加氧酶（pMMO）。第一个，TM，与人类多巴胺密切相关 -单加氧酶（DM）并参与无脊椎动物神经递质调节。作为非耦合双核 铜酶，它具有两个由 11 A 隔开的铜中心，其中只有一个与 O2 结合。出去- 关于 O2 与还原酶结合的时间、氢原子提取 (HAA) 和 金属间电子转移持续存在。第二种酶 LPMO 在可再生能源领域具有广泛的工业应用 生物燃料，也被认为是多种病原体的毒力因子。其活性位点包括一个 铜离子以罕见的“组氨酸大括号”几何结构与两个组氨酸残基和 N 末端胺配位。 计算结果为计划的实验提供了信息，提出了几种机械可能性；不过，有些 最近的报告质疑 O2 对 H2O2 的作用。最后一种酶，pMMO，在 con- 将危险的温室气体甲烷转化为可再生燃料甲醇。长期以来人们一直认为 具有耦合双核铜活性位点，但最近被重新评估为具有单核铜活性位点 还参与组氨酸支撑结构基序。这个新建议意味着几乎没有机械洞察力 可用，尽管它在 pMMO 和 LPMO 之间进行了相似之处。该项目的最终目标是揭示 这些酶如何氧化其底物，并揭示有用且可推广的结构功能关系 进一步用于临床和工业应用。我们的具体目标包括对每个班级进行调查 利用一系列光谱学来发现信息丰富的中间体，利用广泛的酶 所罗门实验室提供仪器和经验。由于许多这些转变涉及对 磁性物种、电子顺磁共振 (EPR) 和磁圆二色性 (MCD) 实验 将允许直接询问铜中心，特别是与快速冷冻淬火 (RFQ) 结合使用 技术。此外，停止流吸收、共振拉曼 (rR)、X 射线吸收 (XAS) 和 X 射线 发射（XES）将被大量使用，特别是在研究抗磁态时。所有这些研究都将 使用密度泛函理论（DFT）方法进行彻底的计算研究支持，这将 允许进一步了解电子结构和反应能量。培训计划涉及沉浸式 在这些光谱技术和生物无机化学领域，所有这些对申请人来说都是新的。