Oxygen Activation by Mononuclear Copper(I) Active Sites

项目摘要/摘要。(摘自原建议) 偶联双核铜位和氧气的相互作用已被广泛地研究在关键的上下文中 为生活！干杯。这与单核铜位和氧气之间的类似相互作用形成对比，后者较少 尽管它们在临床、生物和工业环境中非常重要，但人们对它们的理解很好。因此，进一步推动我们的 了解这些酶的作用机制将对广泛的 纪律。本项目致力于从机理上阐明三种不同单氧基的氧化行为。 具有单一铜活性中心的酶：酪氨酸单加氧酶(T-M)、裂解多糖单加氧酶 (LPMO)和颗粒甲烷单加氧酶(PMMO)。第一个，T-M，与人类多巴胺密切相关 -单加氧酶(D-M)，参与无脊椎动物神经递质的调节。作为一个非偶联的双核 铜酶，它的特征是两个铜中心被11A隔开，其中只有一个与O2接合。出局- 关于O2与还原酶结合的时间、氢原子提取(HAA)以及 金属间电子转移持续存在。第二种酶，LPMO，在可再生能源方面有很大的工业应用 生物燃料，也被认为是几种病原体的致病因子。它的活动站点由单个 铜离子与两个组氨酸残基和N-端胺以稀有的组氨酸支撑构型配位。 计算结果为计划中的实验提供了依据，提出了几种机械上的可能性；然而，一些 最近的报道质疑O2对过氧化氢的作用。最后一种酶，pMMO，在CON中起着重要作用。 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 具有偶联的双核铜活性中心，但最近被重新评估为具有单核铜中心 还从事组氨酸支撑的结构主题。这一新建议意味着几乎没有机械论的见解。 可用，尽管它将pMMO和LPMO相提并论。该项目的最终目的是阐明 这些酶是如何氧化它们的底物的，并揭示了有用和普遍的结构-功能关系 将在进一步的临床和工业应用中加以开发。我们的具体目标是对每个班级进行调查。 使用一组光谱来发现信息丰富的中间体，利用广泛的 所罗门实验室提供的仪器和经验。由于这些转变中的许多都涉及到对- 磁性物种、电子顺磁共振(EPR)和磁性圆二色(MCD)实验 将允许直接询问铜中心，特别是与快速冷冻淬火(RFQ)结合使用时 技巧。此外，阻止了低吸收、共振拉曼(RR)、X射线吸收(XAS)和X射线 发射(XES)将被大量使用，特别是在研究抗磁状态时。所有这些研究都将是 由使用密度泛函理论(DFT)方法的彻底计算研究支持，这将 允许进一步了解电子结构和反应能量。训练计划包括沉浸其中 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。

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.