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

基本信息

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
项目类别：
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

项目摘要

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）：该研究项目阐明了两种非经典血红素加氧酶如何调整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以实现这一目标。 MhuD 和 IsdG 活性位点调节铁-（氢）过氧中间体的电子结构和反应性，无需辅助即可实现区域特异性卟啉氧化观察到的反应性不能归因于酶活性位点的纯粹空间控制，而且血红素的电子结构对于纯粹的理论方法来说过于复杂，因此研究小组将光谱学紧密结合起来。通过计算模型进行表征，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，研究团队开发了新的光谱实验，包括确定的新方法。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通用理论模型。该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，使基础血红素界受益，并通过获取为新抗生素的开发奠定基础的知识，使公众健康受益。该研究计划是通过表征 MhuD 和 IsdG 活性位点中的四个第二球相互作用对非经典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。研究小组的目标是识别由于活性位点变化而导致功能改变的 MhuD 和 IsdG 变体，这一目标是通过采用光谱分析来识别功能改变的变体以及这些变体的额外光谱表征来实现的。研究小组的第二个目标是确定这些具有改变的功能和未改变的二级结构的第二球体变体如何扰乱铁-（氢）过氧中间体。该目标是通过使用光谱来实现的。快速识别基底电子结构受到扰动的变体，并利用磁谱和理论计算来详细说明电子结构的变化及其对血红素-双氧反应性的影响最后，研究小组的第三个目标。目的是阐明 MhuD 和 IsdG 活性位点如何调节内消旋羟基血红素的反应性。该研究团队采用空气敏感设备来制备这些反应中间体，以进行光谱和机械表征，最终，该研究项目使用生物化学、光谱和计算工具来表征。由非经典血红素加氧酶催化的两个氧合反应。