Nonheme Diiron Centers and the Biological Oxidation of Hydrocarbons

这项研究的长期目标是促进我们对多组分单核磁共振的理解。 为碳氢化合物的选择性羟化激活氧气的加氧酶。这些 显著的酶系统，通常由羟基酶、还原酶和调节性酶组成 蛋白质需要消耗四种底物--碳氢化合物、氧气、电子和质子--来生产酒精 还有水。该家族的旗舰成员是可溶性甲烷单加氧酶(SMMO)，它 在其羟基酶成分的活性部位有一个羧酸桥联的非血红素二铁中心 在那里实现了甲烷选择性氧化为甲醇。出现在生物体中的类似单位 从植物到哺乳动物，激活62来执行各种相关的功能。的结构 来自三个细菌系统的单个组成蛋白质，以及它们之间的复合体， SMMO，甲苯/邻二甲苯单加氧酶和苯酚羟基酶，将通过X射线进行研究 结晶学和核磁共振波谱。62天然和突变羟基酶的激活将是 通过快速混合和冷冻淬火方法进行研究，包括一种新的亚毫秒技术， 结合光吸收、共振拉曼、IR、EPR、EXAFS和穆斯堡尔谱 光谱学。动力学分离的中间体与底物的反应将被研究 揭示了烷烃、烯烃、芳烃和 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的值进行比较，以测试提出的机械路径。 还原酶或Rieske组分之间的电子转移和质子转移反应 将对蛋白质和羟基酶进行研究。羟基酶的小分子类似物 将准备双铁中心并进行表征。中间体的结构和性质 二铁(11)模型络合物与02反应生成的化合物及其氧化束缚的能力 将研究外源底物。一种新开发的制备路线将提供仿生 与N-给体的配合物与羧酸桥联的双金属中心的Fe-Fe载体合成。这 该项目与公共健康相关，因为细菌单加氧酶可以防止温室气体 气体，从进入大气，降解地下水中的氯代烃，并且是 被激活用于石油泄漏的生物修复。对62激活分子机制的认识 而本研究提供的底物羟化将推进新的策略 环境净化和甲烷转化为甲醇催化剂的开发。

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.