METHANE MONOOXYGENASE STRUCTURE AND MECHANISM

甲烷单加氧酶的结构和机制

• 批准号: 2180353
• 负责人: JOHN D LIPSCOMB
• 金额: $ 20.81万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180353
• 关键词: Methanobacteriaceae; Mossbauer spectrometry; Raman spectrometry; chemical kinetics; chromophore; cofactor; crosslink; crystallization; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; free radical oxygen; halohydrocarbon; iron; metalloenzyme; methane; methane monooxygenase; microorganism metabolism; nuclear magnetic resonance spectroscopy; oxidation reduction reaction

We propose to investigate the 3 dimensional structure, active site architecture, catalytic mechanism, and mechanism of regulation of the soluble form of Methane Monooxygenase (MMO). This enzyme catalyzes the definitive first step in the oxidation of CH4 to CO2 by methanotrophic bacteria. In this way, the atmospheric egress of nearly all of the enormous quantity of CH4 (a potent "greenhouse" gas) generated by anaerobic bacteria in aquatic environments is prevented. MMO also adventitiously catalyzes the oxidation of many other saturated and unsaturated hydrocarbons. Although the detailed mechanism of MMO is unknown, our studies suggest that the reaction is catalyzed by a cofactor not found in other oxygenases; this implies a new strategy for oxygenase catalysis. We have purified MMO from the type II methanotroph, Methylosinus trichosporium OB3b; it is composed of 3 proteins termed hydroxylase, reductase, and component B. The system offers many advantages over other purified MMO systems including greater yield and stability, and a 25-fold increase in hydroxylase specific activity. These properties allow purification in quantity so that biophysical techniques (optical EPR, Mossbauer, EXAFS, ENDOR, MCD, and CD spectroscopies) can be applied for structural studies. Recently, satisfactory crystals for structural studies have been obtained. Spectroscopy of small ligand complexes, isotopically labeled substrates and inhibitors, and transient kinetics are being used to investigate the molecular mechanism. Coordinated spectroscopic, chemical, and single turnover studies, have shown that the reaction is catalyzed by a mu-(R- or H-)oxo-bridged dinuclear Fe center located in the hydroxylase. We hypothesize that O2 adds to the [Fe(II)-Fe(II)] state of this cluster resulting in heterolytic O-O bond cleavage to form a reactive intermediate, perhaps an [Fe(IV)-Fe(IV)=O] oxene. This species is thought to attack hydrocarbons with the intermediate formation of a substrate radical. Substantial support for this mechanism is being accumulated through the use of specially synthesized chiral substrates for the detection of substrate radicals, the elucidation of characteristic peroxide shunt chemistry, and the detection of transient reaction intermediates. Catalytically active subsystems of MMO consisting of the hydroxylase without one or both of the other two components are being used to evaluate the mechanistic roles of the reductase and component B. Preliminary results suggest that these components play roles in both transfer of reducing equivalents necessary for catalysis, and in assuring efficient coupling of energy expenditure with methane turnover. This work should yield a fundamental understanding of a new type of biological oxygen activation chemistry, a new role for iron in this chemistry, and guidance in the design of catalyst for oxidation of abundant hydrocarbons. It is also likely to contribute to our understanding of the role of protein-protein interactions in biological regulatory processes. Moreover, as we have shown that priority pollutants such as trichloroethylene are rapidly oxidized and detoxified by MMO, it is probable that the knowledge gained from this study will find environmental applications.

我们建议研究3维结构，活性位点 结构，催化机制和调节机制 甲烷单加氧酶（MMO）的可溶形式。 这种酶催化了 通过甲烷营养的CH4氧化至CO2的确定性第一步 细菌。 这样，几乎所有的大气出口 由CH4（一种有效的“温室”气体）产生的大量CH4 防止水生环境中的厌氧菌细菌。 也是MMO 异议地催化许多其他饱和和 不饱和烃。 虽然MMO的详细机制是 未知，我们的研究表明该反应是由辅因子催化的 在其他氧合酶中找不到；这意味着氧合酶的新策略 催化。 我们已经从II型甲烷营养中纯化MMO， Trichosporium ob3b甲基菌;它由称为3个蛋白质组成 羟化酶，还原酶和组件B。系统提供了许多 优于其他纯化的MMO系统，包括更高的收益和 稳定性，羟化酶特异性活性增加了25倍。 这些特性允许纯化数量，因此生物物理 技术（光学EPR，Mossbauer，Exafs，Endor，MCD和CD 光谱镜）可以应用于结构研究。 最近， 已经获得了令人满意的结构研究晶体。 小配体配合物的光谱，同位素标记的底物 和抑制剂和瞬时动力学被用于研究 分子机制。 协调的光谱，化学和单个 营业额研究表明，该反应是由mu-催化的（r- 或H-）位于羟化酶中的氧桥桥梁中心。 我们 假设O2添加了此群集的[Fe（II）-FE（II）]状态 导致杂化O-O键裂解形成反应性 中间，也许是[Fe（IV）-Fe（iv）= O]氧烯。 这个物种是 被认为是通过中间形成攻击碳氢化合物的 基材自由基。 对这种机制的大量支持正在 通过使用特殊合成的手性底物积累 为了检测底物自由基，阐明 特征过氧化物分流化学和瞬态检测 反应中间体。 MMO的催化活性子系统 由羟化酶组成，没有其他两个或两个 组件被用于评估 还原酶和成分B.初步结果表明这些 组件在减少等效物的两种转移中都起着作用 用于催化，并​​确保能量消耗的有效耦合 与甲烷更新。 这项工作应产生基本 了解一种新型的生物氧激活化学， 铁在这种化学中的新作用，以及在设计中的指导 氧化丰富的碳氢化合物。 它也可能 有助于我们理解蛋白质蛋白质的作用 生物调节过程中的相互作用。 而且，正如我们所拥有的 表明优先污染物（例如三氯乙烯）迅速 通过MMO氧化和排毒，很有可能获得的知识 从这项研究中将找到环境应用。