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在环境条件下将CH4、O2、H+和NADH选择性地转化为CH3OH、H2O和NAD+的蛋白质的可溶性甲烷单加氧酶(SMMO)系统。从其中分离出sMMO的甲烷氧化菌都可以防止温室气体CH4进入大气，并促进生物修复。CH4和其他底物的氧化是在羧酸桥联的非血红素二铁中心的羟基酶(MMOH)中实现的，这也存在于相关的氧激活蛋白中。SMMO蛋白通过在MMOH、还原酶(MMOR)和辅助蛋白(MMOB)之间形成复合体来协同工作，MMOB将02的消耗与CH4的氧化结合在一起。为了了解蛋白质的相互作用，将用X射线结晶学和先进的EPR方法对复合物的结构进行表征，这些方法使用带有自旋标记的蛋白质突变体。MMOR的结构将通过核磁共振波谱确定。这些信息将被用来解释从末端还原剂NADH通过MMOR到MMOH中双铁中心的电子转移(ET)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和激光闪光光解结合ET中间体的光谱监测来研究。MMOH的催化循环将通过溶液中的单混合和双混合停流和快速冷冻急冷法以及一种新的冷冻还原方法进行研究，在这种方法中，在冻结的基质中生成的还原酶通过退火法形成含氧中间体。EPR、Endor、穆斯堡尔和EXAFS谱将有助于中间体的表征。羟化中间体Q与各种底物的反应，结合先进量子力学技术的分析，将提供对C-H键活化步骤的本质的洞察。MmoH将被表达并产生突变体，以测试特定的蛋白质残基如何帮助催化机制。第四种蛋白质成分MMOD的功能将通过在天然生物体中删除它，并研究它帮助MMOH中金属簇组装的能力来研究。将制备MMOH中双铁中心的合成模型。他们羟化系留碳氢化合物底物的能力将被机械地研究。将寻求一种新的用于氧转移化学的羧酸桥联二铁催化剂。提供更接近MMOH模拟的双核配体将被用来提供更好的碳氢化合物氧化催化剂。

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.