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 甲烷氧化菌将 CH4 氧化为 CO2 的决定性第一步 细菌。 这样，几乎所有的大气排放 产生的大量 CH4（一种强效“温室”气体） 防止水生环境中的厌氧细菌。 大型多人在线游戏也 偶然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 其他加氧酶中没有发现；这意味着加氧酶的新策略 催化。 我们从 II 型甲烷氧化菌中纯化了 MMO， 毛孢甲基窦菌 OB3b；它由 3 种蛋白质组成，称为 羟化酶、还原酶和组分 B。该系统提供许多 与其他纯化 MMO 系统相比的优势包括更高的产量和 稳定性，羟化酶比活性增加 25 倍。 这些特性允许大量纯化，以便生物物理 技术（光学 EPR、穆斯堡尔、EXAFS、ENDOR、MCD 和 CD 光谱）可用于结构研究。 最近， 获得了令人满意的晶体结构研究。 小配体复合物、同位素标记底物的光谱学 和抑制剂，瞬态动力学被用来研究 分子机制。 协调光谱、化学和单一 周转研究表明，该反应是由 mu-(R- 或 H-) 氧桥双核 Fe 中心位于羟化酶中。 我们 假设 O2 添加到该簇的 [Fe(II)-Fe(II)] 状态 导致 O-O 键异裂，形成反应性 中间体，可能是[Fe(IV)-Fe(IV)=O] oxene。 这个物种是 被认为是通过中间形成的碳氢化合物来攻击碳氢化合物 底物自由基。 对该机制的大力支持正在 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，以及瞬态的检测 反应中间体。 MMO 的催化活性子系统 由羟化酶组成，不含其他两种或两种 组件被用来评估机械作用 还原酶和组分 B。初步结果表明，这些 成分在必要的还原当量的转移中发挥作用 用于催化，并​​确保能量消耗的有效耦合 随着甲烷周转。 这项工作应该产生一个基本的 了解新型生物氧活化化学， 铁在这种化学中的新作用，以及设计的指导 大量碳氢化合物氧化的催化剂。 也有可能 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。 此外，正如我们有 表明三氯乙烯等优先污染物正在迅速 通过MMO氧化和解毒，很可能获得的知识 从这项研究中将会发现环境应用。