Nonheme Iron and the Biological Oxidation of Methane

描述（由申请人提供）：本提案的长期目标是了解蛋白质的可溶性甲烷单加氧酶（sMMO）系统，该系统在环境条件下将CH 4、O2 H+和NADH选择性转化为CH 3OH、H2O和NAD+。从sMMO中分离出的甲烷氧化菌既能防止温室气体CH 4到达大气，又能促进生物修复。在羟化酶（MMOH）中，CH 4和其他底物的氧化在羧酸根桥接的非血红素二铁中心实现，这些中心也出现在相关的双氧活化蛋白中。sMMO蛋白通过在MMOH、还原酶（MMOR）和辅助蛋白（MMOB）之间形成复合物协同工作，所述辅助蛋白将O2消耗与CH 4氧化偶联。为了了解蛋白质的相互作用，复合物的结构特征将通过X射线晶体学和先进的EPR方法，采用蛋白质突变体轴承自旋标记。MMOR的结构将通过NMR光谱测定。这一信息将被用来解释的电子转移（ET）从终端还原剂，NADH，通过MMOR的二铁中心在MMOH的溶液动力学研究的结果。MMOR还原MMOH的各个步骤将通过停流光谱法和激光闪光光解结合ET中间体的光谱监测进行研究。MMOH的催化循环将研究通过单和双混合停流和快速冷冻淬火方法在溶液中，并通过一种新的冷冻还原方法，其中在冷冻基质中产生的还原酶被允许通过退火形成含氧中间体。EPR、ENDOR、Mossbauer和EXAFS谱将有助于表征中间体。反应的Q，羟基化中间体，与各种基板，结合先进的量子力学技术的分析，将提供洞察的性质的C-H键活化步骤。将表达MMOH并产生突变体以测试特定蛋白质残基如何辅助催化机制。MMOD是第四种蛋白质成分，将通过在天然生物体中删除它并研究其帮助组装MMOH中金属簇的能力来研究其功能。将制备MMOH中二铁中心的合成模型。将从机理上研究它们羟基化系留烃底物的能力。一种新的羧酸根桥联的二铁催化剂的氧转移化学将追求。提供更接近MMOH模拟物的双核配体将用于提供更好的烃氧化催化剂。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the soluble methane monooxygenase (sMMO) system of proteins that converts CH4, O2 H+, and NADH selectively to CH3OH, H2O, and NAD+ under ambient conditions. Methanotrophs from which sMMO is isolated both prevent CH4, a greenhouse gas, from reaching the atmosphere and facilitate bioremediation. Oxidation of CH4 and other substrates is achieved in the hydroxylase enzyme (MMOH) at carboxylate-bridged non-heme diiron centers that also occur in related dioxygen-activating proteins. The sMMO proteins work in concert through the formation of complexes between MMOH, a reductase (MMOR), and an auxiliary protein (MMOB) that couples 02 consumption with CH4 oxidation. In order to understand the protein interactions, the complexes will be structurally characterized by Xray crystallography and advanced EPR methods that employ protein mutants bearing spin labels. The structure of MMOR will be determined by NMR spectroscopy. This information will be used to interpret the results of solution kinetics studies of electron-transfer (ET) from the terminal reductant, NADH, through MMOR to the diiron centers in MMOH. Individual steps of MMOR reduction of MMOH will be investigated by stopped-flow spectroscopy and by laser flash photolysis combined with spectroscopic monitoring of ET intermediates. The catalytic cycle of MMOH will be studied by single- and double-mixing stopped-flow and rapid freeze-quench methods in solution and by a novel cryoreduction approach in which the reduced enzyme generated in a frozen matrix is allowed to form oxygenated intermediates by annealing. EPR, ENDOR, Mossbauer, and EXAFS spectra will help characterize intermediates. Reactions of Q, the hydroxylating intermediate, with various substrates, in conjunction with analysis by advanced quantum mechanical techniques, will provide insight into the nature of the C-H bond activation step. MMOH will be expressed and mutants generated to test how specific protein residues assist the catalytic mechanism. The function of MMOD, a fourth protein component, will be studied by deleting it in the native organism and investigating its ability to help assemble the metal clusters in MMOH. Synthetic models of the diiron center in MMOH will be prepared. Their ability to hydroxylate tethered hydrocarbon substrates will be mechanistically studied. A new carboxylate-bridged diiron catalyst for oxo-transfer chemistry will be pursued. Dinucleating ligands that afford closer MMOH mimics will be employed to afford better hydrocarbon oxidation catalysts.