Nonheme Diiron Centers and the Biological Oxidation of Hydrocarbons

描述（由申请人提供）：本研究的长期目标是促进我们对多组分单加氧酶的理解，该酶可激活双氧以选择性地羟基化碳氢化合物。这些非凡的酶系统通常由羟化酶、还原酶和调节蛋白组成，它们消耗四种底物——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），它在其羟化酶组分的活性位点有一个羧酸桥接的非血红素二铁中心，在那里甲烷被选择性氧化成甲醇。类似的单位存在于从植物到哺乳动物的生物体中，激活O2来执行各种相关功能。本文将利用x射线晶体学和核磁共振波谱技术研究sMMO、甲苯/邻二甲苯单加氧酶和苯酚羟化酶这三种细菌体系的单个组成蛋白及其复合物的结构。O2在原生和突变羟化酶中的活化将通过快速混合和冻灭方法进行研究，包括一种新的亚质谱技术，结合光学吸收、共振拉曼、IR、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、芳烃和杂原子取代底物立体特异性羟基化的机制。将确定活化参数和动力学同位素效应，与理论推导值进行比较，以测试提出的机制途径。还原酶或Rieske组分蛋白与羟化酶之间的电子转移和质子易位反应将被研究。制备并表征了羟化酶二铁中心的小分子类似物。二铁（ll）模型配合物与O2反应生成的中间体的结构和性质以及它们氧化系缚或外源底物的能力将被研究。一种新开发的制备路线将提供与羧酸桥接双金属中心的Fe-Fe载体同步的n给体仿生配合物。该项目与公共卫生有关，因为细菌单加氧酶可以防止温室气体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。本研究提供的O2活化和底物羟基化的分子机制知识将促进环境净化的新策略和甲烷转化为甲醇的催化剂的开发。

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.