Oxygen Activation by Mononuclear Copper(I) Active Sites

项目概要/摘要。(From原提案） 耦合双核铜中心和分子氧的相互作用已经被广泛研究， 生命这与单核铜位点和分子氧之间的类似相互作用形成对比， 尽管它们在临床、生物学和工业环境中很重要，但仍被很好地理解。因此，促进我们的 对这些酶的作用机制的理解将对一系列广泛的 学科本项目的重点是三种不同的单氧化合物的氧化行为的机理阐明， 具有单一铜活性位点的酶：酪胺单加氧酶（TM）、溶解性多糖单加氧酶 （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（DM），参与无脊椎动物神经递质的调节。作为一个非偶联双核 铜酶，其特征在于由11 A分开的两个铜中心，其中只有一个与O2接合。出去- 关于O2与还原酶结合的时间，氢原子提取（HAA）的长期问题，以及 金属间电子转移持续存在。第二种酶，LPMO，在可再生能源中具有很大的工业应用， 生物燃料，也被认为是几种病原体的毒力因子。它的活性部位由一个 铜离子与两个组氨酸残基和N -末端胺配位，形成罕见的“组氨酸支架”构型。 计算结果为计划的实验提供了信息，提出了几种机械的可能性;尽管，一些 最近的报道质疑O2的作用有利于H2 O2。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 具有耦合的双核铜活性位点，但最近被重新评估为具有单核铜位点 也参与组氨酸支架结构基序。这个新的建议意味着几乎没有机械的洞察力 虽然它在pMMO和LPMO之间画出了相似之处。该项目的最终目的是阐明 这些酶如何氧化其底物，并揭示有用的和可推广的结构-功能关系 以用于进一步的临床和工业应用。我们的具体目标包括调查每一个类别 酶使用一组光谱来发现信息中间体，利用广泛的 所罗门实验室的仪器和经验。由于这些转变中有许多涉及帕拉- 磁性物质、电子顺磁共振（EPR）和磁性圆二色性（MCD）实验 将允许直接询问铜中心，特别是与快速冷冻淬火（Rapid Freeze Quench，缩写为REQ）相结合， 技术.此外，停止低吸收、共振拉曼（rR）、X射线吸收（XAS）和X射线吸收（X-ray absorption，X-ray absorption，X-ray absorption）也被称为共振拉曼光谱。 发射（XES）将被大量采用，特别是在研究抗磁态时。所有这些研究都将 通过使用密度泛函理论（DFT）方法进行彻底的计算研究， 允许进一步深入了解电子结构和反应能量。培训计划包括沉浸式 在这些光谱技术和生物无机化学领域中，所有这些对于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.