Second-Sphere Influences on Oxygen Activation by Non-Canonical Heme Oxygenases

第二领域对非典型血红素加氧酶的氧活化的影响

• 批准号: 9750001
• 负责人: Matthew D Liptak
• 金额: $ 26.32万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750001
• 关键词: Active Sites; Air; Antibiotics; Azides; Biochemical; Biochemical Reaction; Biological Assay; Carbon; Chemistry; Circular Dichroism; Communities; Complex; Computer Simulation; Development; Dioxygen; Distal; Electron Spin Resonance Spectroscopy; Electrons; Enzymes; Equipment; Foundations; Funding; Genus staphylococcus; Goals; Heme; Heme Iron; Hemoglobin; Human; Hydroxylation; Investigation; Iron; Isomerism; Knowledge; Knowledge acquisition; Laboratories; Magnetism; Measures; Methods; Modeling; Molecular; Mycobacterium tuberculosis; NMR Spectroscopy; Nutrient; Optics; Oxygen; Oxygenases; Pathway interactions; Peroxidases; Pharmaceutical Preparations; Porphyrins; Procedures; Proteins; Public Health; Reaction; Reactive Oxygen Species; Reporting; Research; Research Project Grants; Spectrum Analysis; Staphylococcus aureus; Structural Protein; Structure; Temperature; Theoretical model; Variant; Water; absorption; analog; base; computer studies; computerized tools; density; electronic structure; experimental study; geometric structure; heme a; human disease; insight; magnetic field; member; mycobacterial; novel; novel strategies; pathogen; polypeptide; programs; protein structure; public health relevance; spectroscopic data; theories; tool

 DESCRIPTION (provided by applicant): This research program elucidates how two non-canonical heme oxygenases tune the electronic structure of heme in order to catalyze novel heme-dioxygen chemistry, and develops new research tools to achieve this objective. Second-sphere interactions within the MhuD and IsdG active sites tune the electronic structure and reactivity of a ferric-(hydro)peroxo intermediate to achieve regiospecific porphyrin oxygenation without the aid of the conserved water cluster found in canonical heme oxygenases. The observed reactivity cannot be attributed to purely steric control from the enzyme active sites, and the electronic structure of heme is far too complex for a purely theoretical approach, so the research team closely integrates spectroscopic characterization with computational modelling to understand the novel heme-dioxygen reactivity of MhuD and IsdG. In order to achieve the aims of this research project, the research team develops new spectroscopic experiments, including new approaches to determine the electron configuration of ferric heme and the orientation of heme-bound dioxygen. The research team also develops new computational models to aid analysis of the spectroscopic data, including a general theoretical model for the complex influence of porphyrin ruffling on the electronic absorption spectrum of heme. The results from this research program benefit both the fundamental heme community, by elucidating a novel reactive pathway and developing new research tools, and public health, by acquiring knowledge that lays the foundation for the development of new antibiotics. The objectives of this research program are achieved by characterizing the influence of four second- sphere interactions in the MhuD and IsdG active sites on the structure, electronic structure, and reactivity of two critical intermediates of non-canonical heme oxygenase-catalyzed heme degradation. The first aim of the research team is to identify variants of MhuD and IsdG with altered function due to active site changes. This aim is achieved by employing spectroscopic assays to identify variants with altered function, and additional spectroscopic characterization of these variants to determine whether these substitutions change the polypeptide secondary structure. The research team's second aim is to determine how these second sphere variants with altered function and unaltered secondary structure perturb the ferric-(hydro) peroxo intermediate. This aim is achieved by using optical spectroscopy to rapidly identify variants with perturbed substrate electronic structures, and employing magnetic spectroscopies and theoretical calculations to detail the electronic structure changes and their effect on heme-dioxygen reactivity. Finally, the third aim of the research team is to elucidate how the MhuD and IsdG active sites tune the reactivity of mesohydroxyheme. The research team employs air-sensitive equipment to prepare these reactive intermediates for spectroscopic and mechanistic characterization. Ultimately, this research program uses biochemical, spectroscopic, and computational tools to characterize two oxygenation reactions catalyzed by non-canonical heme oxygenases.

 描述（由申请人提供）：该研究计划阐明了两种非经典血红素加氧酶如何调节血红素的电子结构，以催化新型血红素-双氧化学，并开发新的研究工具来实现这一目标。在MhuD和IsdG活性位点内的第二球相互作用调节铁-（氢）过氧中间体的电子结构和反应性，以实现区域特异性卟啉氧合，而无需在典型血红素加氧酶中发现的保守水簇的帮助。观察到的反应性不能归因于酶活性位点的纯粹空间控制，血红素的电子结构对于纯理论方法来说太复杂了，因此研究小组将光谱表征与计算建模紧密结合，以了解MhuD和IsdG的新型血红素-双氧反应性。为了实现该研究项目的目标，研究小组开发了新的光谱实验，包括确定铁血红素的电子构型和血红素结合的双氧方向的新方法。研究小组还开发了新的计算模型来帮助分析光谱数据，包括卟啉褶皱对血红素电子吸收光谱的复杂影响的一般理论模型。这项研究计划的结果通过阐明新的反应途径和开发新的研究工具使基本血红素社区受益，通过获得为开发新抗生素奠定基础的知识使公共卫生受益。本研究计划的目标是通过表征MhuD和IsdG活性位点中的四个第二球相互作用对非典型血红素加氧酶催化血红素降解的两个关键中间体的结构、电子结构和反应性的影响来实现的。研究小组的第一个目标是鉴定由于活性位点变化而改变功能的MhuD和IsdG变体。该目的通过采用光谱测定来鉴定具有改变的功能的变体，以及这些变体的另外的光谱表征来确定这些取代是否改变多肽二级结构来实现。研究小组的第二个目标是确定这些功能改变和二级结构不变的第二个球体变体如何干扰铁-（氢）过氧中间体。这一目标是通过使用光谱学来快速识别具有扰动的基底电子结构的变体，并采用磁光谱学和理论计算来详细说明电子结构变化及其对血红素-二氧反应性的影响来实现的。最后，研究小组的第三个目标是阐明MhuD和IsdG活性位点如何调节mesohydroxyheme的反应性。研究团队采用空气敏感设备来制备这些活性中间体，以进行光谱和机械表征。最终，这项研究计划使用生物化学，光谱和计算工具来表征非典型血红素加氧酶催化的两个加氧反应。