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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：
9979903
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 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979903
关键词：
Active Sites Air Antibiotics Azides Biochemical Biochemical Reaction Biological Assay Carbon Chemistry Circular Dichroism Communities Complex Computer Models 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 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.