Nonheme Diiron Centers and the Biological Oxidation of Hydrocarbons

描述(申请人提供)：这项研究的长期目标是促进我们对多组分单加氧酶的理解，这些加氧酶激活氧气用于碳氢化合物的选择性羟化。这些非凡的酶系统通常由羟基酶、还原酶和调节蛋白组成，需要四种底物--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)，它在其羟基酶成分的活性位置具有一个羧酸桥联的非血红素二铁中心，在那里实现甲烷选择性氧化为甲醇。在从植物到哺乳动物的各种生物体中都存在类似的单位，激活O2来执行各种相关的功能。将用X射线结晶学和核磁共振波谱研究来自三个细菌系统--sMMO、甲苯/邻二甲苯单加氧酶和苯酚羟基酶--的单个组分蛋白质及其之间的复合体的结构。我们将通过快速混合和冷冻猝灭方法来研究天然和突变羟基酶中的O2激活，包括一种新的亚毫秒技术，结合光学吸收、共振拉曼、IR、EPR、EXAFS和穆斯堡尔光谱。动力学分离的中间体与底物的反应将被研究，以揭示烷烃、烯烃、芳烃和杂原子取代的底物的立体特异性羟化反应的机理。将确定活化参数和动力学同位素效应，以与理论推导的值进行比较，以检验所提出的机理途径。我们将研究还原酶或Rieske组分蛋白质与羟基酶之间的电子转移和质子转移反应。羟基酶双铁中心的小分子类似物将被制备和表征。将研究双铁(11)模型络合物与O2反应生成的中间体的结构和性质，以及它们氧化束缚或外源底物的能力。一条新开发的制备路线将提供N-供体与羧酸桥联双金属中心的Fe-Fe载体的仿生配合物。该项目与公众健康相关，因为细菌单加氧酶阻止温室气体Chu进入大气，降解地下水中的氯代烃，并被激活以用于石油泄漏的生物修复。本研究提供的氧气活化和底物羟化的分子机制将为环境净化和甲烷转化为甲醇的催化剂的开发提供新的策略。

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.