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

• 批准号: 7495933
• 负责人: Stephen J. Lippard
• 金额: $ 2万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495933
• 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; 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; Range; Reaction; Research; Research Activity; Research Personnel; Rest; Roentgen Rays; Role; Route; Shunt Device; Site; Spectrum Analysis; 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 structure; small molecule; stoichiometry; toluene 2-xylene monooxygenase; vector

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, andprotons - 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 62 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. 62 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 Mb'ssbauer 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 02 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 62 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.

这项研究的长期目标是促进我们对多组分单组分的理解， 活化双氧以使烃类选择性羟基化的加氧酶。这些 值得注意的酶系统，通常由羟化酶、还原酶和调节酶组成， 蛋白质消耗四种底物--碳氢化合物、氧气、电子和质子--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）， 在其羟化酶组分的活性部位具有羧酸根桥接的非血红素二铁中心 其中实现甲烷选择性氧化成甲醇。在生物体中出现的类似单位 从植物到哺乳动物，激活62以执行各种相关功能。结构 来自三种细菌系统的单个组分蛋白质及其之间的复合物， sMMO、甲苯/邻二甲苯单加氧酶和苯酚羟化酶将通过X射线研究 晶体学和NMR光谱学。62在天然和突变体羟化酶中的活化将是 通过快速混合和冷冻骤冷方法学，包括新的亚质谱技术， 结合光学吸收、共振拉曼、IR、EPR、EXAFS和Mb'ssbauer 谱将研究动力学分离的中间体与底物的反应， 揭示了负责烷烃、烯烃、芳烃的立体定向羟基化的机制， 杂原子取代的底物。活化参数和动力学同位素效应将是 确定用于与理论推导值进行比较，以测试提出的机理途径。 还原酶或Rieske组分之间的电子转移和质子转移反应 将研究蛋白质和羟化酶。羟化酶的小分子类似物 将制备和表征二铁中心。中间体的结构和性质 通过使二铁（II）模型络合物与O2反应而产生， 将研究外源底物。一种新的制备方法将提供仿生 与羧酸根桥联的双金属中心的Fe-Fe载体syn的N-供体配合物。这 该项目与公共卫生有关，因为细菌单加氧酶可以防止温室效应 气体到达大气层后，会降解地下水中的氯化烃， 用于石油泄漏的生物修复。了解62活化的分子机制 该研究提供的底物羟基化将为生物降解提供新的策略， 环境净化和开发将甲烷转化为甲醇的催化剂。