Nonheme Diiron Centers and the Biological Oxidation of Hydrocarbons

非血红素二铁中心和碳氢化合物的生物氧化

基本信息

批准号：
7923548
负责人：
Stephen J. Lippard
金额：
$ 5.74万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7923548
关键词：
Active Sites Address Alcohols Alkanes Alkenes Amines Amino Acids Area Binding Biological Biomimetics Bioremediations Chemistry Chlorinated Hydrocarbons Complex Coupling Decontamination Development Dioxygen Electron Transport Electrons Enzymes Family member Freezing Future Genetic Structures Goals Heme Homologous Protein Human Resources Hydrocarbons Hydrogen Peroxide Hydroxylation Individual Investigation Isotopes Kinetics Knowledge Learning Mammals Measures Methane Methane hydroxylase Methanol Methodology Methods Mixed Function Oxygenases Modeling Molecular Mono-S Mossbauer Spectroscopy Movement NADH NMR Spectroscopy Nature Oils Optics Organism Oxidation-Reduction Oxidoreductase Oxygenases Pathway interactions Peroxides Phenol 2-monooxygenase Plants Preparation Process Property Proteins Protons Public Health Quantum Mechanics Reaction Research Research Activity Research Personnel Rest Roentgen Rays Role Route Shunt Device Site Structure Sulfides System Techniques Testing Water Work X ray diffraction analysis X-Ray Crystallography X-Ray Diffraction absorption analog carboxylate catalyst chemical property design genetic regulatory protein greenhouse gases ground water insight macromolecule molecular mechanics mutant novel novel strategies oxidation planetary Atmosphere prevent programs protein complex protein structure small molecule stoichiometry toluene 2-xylene monooxygenase vector

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this research is to advance our understanding of multicomponent mono-oxygenases that activate dioxygen for the selective hydroxylation of hydrocarbons. These remarkable enzyme systems, which typically consist of hydroxylase, reductase, and regulatory proteins, consume four substrates - a hydrocarbon, O2, electrons, and protons - to produce alcohol and water. The flagship member of the family is soluble methane monooxygenase (sMMO), which has a carboxylate-bridged non-heme diiron center at the active site of its hydroxylase component where selective oxidation of methane to methanol is achieved. Similar units that occur in organisms ranging from plants to mammals activate O2 to perform a variety of related functions. Structures of the individual component proteins, and of complexes between them, from three bacterial systems, sMMO, toluene/o-xylene monooxygenase, and phenol hydroxylase, will be investigated by X-ray crystallography and NMR spectroscopy. O2 activation in native and mutant hydroxylases will be studied by rapid-mixing and freeze-quench methodologies, including a novel sub-ms technique, combined with optical absorption, resonance Raman, IR, EPR, EXAFS, and Mossbauer spectroscopy. Reactions of kinetically isolated intermediates with substrates will be investigated to reveal mechanisms responsible for stereospecific hydroxylation of alkanes, alkenes, arenes, and heteroatom-substituted substrates. Activation parameters and kinetic isotope effects will be determined for comparison with theoretically derived values to test proposed mechanistic pathways. Electron-transfer and proton-translocation reactions between the reductase or Rieske component proteins and the hydroxylases will be investigated. Small molecule analogs of the hydroxylase diiron centers will be prepared and characterized. The structures and properties of intermediates generated by reacting diiron(ll) model complexes with O2 and their ability to oxidize tethered or exogenous substrates will be studied. A newly developed preparative route will afford biomimetic complexes with N-donors syn to the Fe-Fe vector of the carboxylate-bridged dimetallic center. This project is relevant to public health because bacterial monooxygenases prevent ChU, a greenhouse gas, from reaching the atmosphere, degrade chlorinated hydrocarbons in ground water, and are activated for bioremediation of oil spills. Knowledge of the molecular mechanisms of O2 activation and substrate hydroxylation provided by this research will advance novel strategies for environmental decontamination and the development of catalysts to convert methane to methanol.

描述（由申请人提供）：这项研究的长期目标是增进我们对多组分单加氧酶的理解，这些单加氧酶可激活双氧以实现碳氢化合物的选择性羟基化。这些非凡的酶系统通常由羟化酶、还原酶和调节蛋白组成，消耗四种底物——碳氢化合物、氧气、电子和质子——来产生酒精和水。该家族的旗舰成员是可溶性甲烷单加氧酶（sMMO），其羟化酶组分的活性位点上有一个羧酸桥联的非血红素二铁中心，可实现甲烷选择性氧化为甲醇。从植物到哺乳动物的生物体中都存在类似的单位，它们可以激活 O2 以执行各种相关功能。来自三个细菌系统（sMMO、甲苯/邻二甲苯单加氧酶和苯酚羟化酶）的各个成分蛋白质及其之间的复合物的结构将通过 X 射线晶体学和 NMR 光谱进行研究。天然和突变羟化酶中的 O2 活化将通过快速混合和冷冻猝灭方法进行研究，包括一种新颖的亚毫秒技术，结合光学吸收、共振拉曼、红外、EPR、EXAFS 和穆斯堡尔光谱。将研究动力学分离的中间体与底物的反应，以揭示烷烃、烯烃、芳烃和杂原子取代的底物立体特异性羟基化的机制。将确定激活参数和动力学同位素效应，以便与理论推导值进行比较，以测试所提出的机制途径。将研究还原酶或 Rieske 成分蛋白与羟化酶之间的电子转移和质子易位反应。将制备并表征羟化酶二铁中心的小分子类似物。将研究二铁(II)模型配合物与O2反应生成的中间体的结构和性质及其氧化束缚或外源底物的能力。新开发的制备路线将提供仿生复合物，其 N 供体与羧酸桥双金属中心的 Fe-Fe 载体顺式。该项目与公共卫生相关，因为细菌单加氧酶可防止温室气体 ChU 进入大气，降解地下水中的氯化碳氢化合物，并被激活用于石油泄漏的生物修复。这项研究提供的关于氧气活化和底物羟基化分子机制的知识将推动环境净化的新策略和开发将甲烷转化为甲醇的催化剂。