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Chemical Approaches to Studying the Mechanisms and Biophysical Properties of Complex Metallocofactors

研究复杂金属辅因子的机制和生物物理性质的化学方法

基本信息

批准号：
10590756
负责人：
Daniel Leif Migdow Suess
金额：
$ 30.6万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10590756
关键词：
3-Dimensional Active Sites Address Air Ammonia Apoproteins Articulation Binding Biological Biological Availability Carrier Proteins Catalysis Chemicals Chemistry Complex Crystallization Crystallography Disease Electron Nuclear Double Resonance Electrons Elements Enzymes Fertilizers Generations Health Heart Human Incubated Iron Isotopes Kinetics Label Life Maps Metals Methodology Methods Modification Molecular Mononuclear Nature Nitrogen Nitrogen Fixation Nitrogenase Nuclear Oxidation-Reduction Planets Play Population Preparation Property Protocols documentation Reaction Reporting Research Rest Role Sampling Signal Transduction Site Spectrum Analysis Structure Sulfur Techniques Testing biophysical properties chemical bond cofactor electronic structure geometric structure insight iron deficiency metalloenzyme novel reconstitution spectroscopic survey three dimensional structure tool

项目摘要

Project Summary/Abstract Enzymes with complex metallocofactors in their active sites catalyze myriad transformations relevant to human health and disease. Understanding their reaction mechanisms requires molecular-level characterization of their resting states and intermediate states, and metal-specific spectroscopic techniques are especially useful in this endeavor. However, the high- nuclearity of many metallocofactors can limit the usefulness of such techniques; the signals arising from multiple metal sites can be challenging to resolve, especially in mixtures of reaction intermediates. Moreover, it is often impossible to map the rich spectroscopic information onto the geometric structure, and this severely limits our understanding of the chemical bonding—and therefore the reactivity—of complex metallocofactors. We propose to address these challenges by developing methods for modifying the isotopic and elemental compositions of complex metallocofactors, in particular the nitrogenase catalytic cofactors. Nitrogenases are responsible for supplying a significant portion of the fixed nitrogen on the planet, and they therefore play an important role in maintaining a healthy and growing human population. Their catalytic cofactors are among the most complex in Nature, and as a result their reaction mechanisms have been especially difficult to characterize. To overcome these challenges and gain new insights into the mechanism of biological nitrogen fixation, we will develop chemical methods for precisely altering the isotopic and elemental composition of nitrogenase cofactors. Our approach will be to discover mild protocols for removing specific Fe sites in nitrogenase cofactors and subsequently replacing them with 57Fe. The site-selectivity of the label will allow for the electronic structure (as elucidated spectroscopically) to be connected to the geometric structure (as defined crystallographically), and will thereby provide unprecedented insights into the chemical bonding and reactivity of nitrogenase cofactors. Studies of these cofactors in both their resting states and intermediate states comprise the heart of the proposal. We will also extend the site-selective 57Fe labeling protocol to incorporating different metals into specific sites of nitrogenase cofactors. This will yield artificial metalloenzymes that will serve as mechanistic probes with potentially unique properties and/or reactivity. Completion of this project will provide unprecedented mechanistic insights into biological nitrogen fixation and will articulate concepts and protocols for rendering complex metallocofactors as mechanistically tractable as mononuclear active sites.

项目总结/文摘