NONHEME IRON AND THE BIOLOGICAL OXIDATION OF METHANE

这项研究的主要目的是阐明化学和 可溶性甲烷单加氧酶（sMMO）的物理性质， 甲烷氧化细菌用来转化甲烷的三种蛋白质， 氧气选择性地转化为甲醇和水。甲烷氧化菌消耗 大量的甲烷，一种温室气体，也是他们唯一的 碳和能源。这些生物体用于生物修复的 环境，例如，去除饮用水中的氯化烃 供水。了解酶系统的原理 羟基甲烷可以提供关键的见解， 用于实现这一重要工业目标的合成催化剂。一 sMMO的主要成分是羟化酶（MMOH），它含有 两个非血红素，羧酸根桥接的二铁中心，其中还原 分子氧的活化发生，进化出最终 氧化甲烷。相关的化学反应发生在位于 核糖核苷酸还原酶（RNR）的一个小亚基， 是DNA生物合成的第一步，是抗肿瘤的靶点， 抗病毒剂。该项目的具体目标之一是 了解这些非血红素铁中心如何实现这种功能的细节 在生理条件下的显著变化。先进 方法将被应用于捕获和确定的结构， MMOH反应循环中的中间体，包括快速冷冻淬灭 EPR和ENDOR光谱。和双混合停流实验， 并检查时间分辨中间体与 印刷受体.在研究这种酶的同时， 将制备MMOH和RNR的羧酸根桥接的二铁核心，以帮助 了解它们的活性位点，并复制它们的催化步骤， 自行车.动力学和机械学实验将在学习过程中进行。 控制烷烃羟基化，烯烃环氧化， 酪氨酰自由基的产生，氧化酶和过氧化物酶的活性， 桥连二铁中心。sMMO系统的另外两个组件， 还原酶（MMOR）和小偶联蛋白（MMOB）形成复合物， MMOH和显着改变其催化活性和氧化还原性能。 另外的目标是确定这两种蛋白质的结构， NMR和X-射线衍射方法，并研究形成的 通过热力学和动力学分析所有三种组分之间的络合物 测量.黄素和[2Fe-2S]发色团的光谱 在MMOR将用于跟踪电子转移反应，通过 系统所有三种蛋白质的定点诱变研究将在 进行鉴定的关键氨基酸残基假定参与 在络合物形成、电子转移、质子转移、底物进入 到二铁中心和羟基化化学。

The major objective of this research is to elucidate the chemical and physical properties of soluble methane monooxygenase (sMMO), a system of three proteins used by methanotrophic bacteria to convert methane and oxygen selectively to methanol and water. Methanotrophs consume significant amounts of methane, a greenhouse gas and their sole source of carbon and energy. These organisms are used in bioremediation of the environment, for example, to remove chlorinated hydrocarbons from drinking water supplies. Understanding the principles by which the enzyme system hydroxylates methane can provide key insights into the development of synthetic catalysts for achieving this important industrial goal. A principle component of sMMO is the hydroxylase enzyme (MMOH), which house two non-heme, carboxylate-bridged diiron centers where reductive activation of dioxygen takes places, evolving species that ultimately oxidize methane. Related chemistry occurs at similar cores located in the small subunit of ribonucleotide reductase (RNR), an enzyme which catalyzes the first step in DNA biosynthesis and which is a target of anti-tumor and anti-viral agents. Among the specific aims of this project is to understand the details of how these non-heme iron centers achieve such remarkable transformations under physiological conditions. Advanced methodologies will be applied to trap and determine the structures of intermediates in the MMOH reaction cycle, including rapid freeze-quench EPR and ENDOR spectroscopic. and double-mixing stopped-flow experiments, and to examine the reactivity of time-resolved intermediates with substrates. In parallel with work on the enzyme, synthetic analogs of the carboxylate-bridged diiron cores of MMOH and RNR will be prepared to help understand their active sites and to reproduce steps in their catalytic cycles. Kinetic and mechanistic experiments will be performed the learn the factors which control alkane hydroxylation, alkene epoxidation, tyrosyl radical generation, oxidase and peroxidase activities of the bridged diiron centers. The other two components of the sMMO system, a reductase (MMOR) and a small coupling protein (MMOB), form complexes with MMOH and significantly alter its catalytic activity and redox properties. Additional goals are to determine the structures of both these proteins by NMR and X-ray diffraction methods and to investigate the formation of complexes between all three components by thermodynamic and kinetic measurements. The optical spectra of the flavin and [2Fe-2S] chromophores in MMOR will be used to track electron-transfer reactions through the system. Site-directed mutagenesis studies of all three proteins will be carried out to identify key amino acid residues postulated to be involved in complex formation, electron transfer, proton transfer, substrate access to the diiron center, and the hydroxylation chemistry.