METHANE MONOOXYGENASE STRUCTURE AND MECHANISM

我们建议研究三维结构，活性中心 的结构、催化机制和调节机制 甲烷单加氧酶(MMO)的可溶性形式。这种酶催化 甲烷营养氧化制二氧化碳的第一步反应 细菌。通过这种方式，几乎所有的大气出口 大量的甲烷(一种强有力的温室气体)由 防止了水环境中的厌氧细菌。MMO还 外来催化氧化许多其他饱和的和 不饱和碳氢化合物。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。 在其他加氧酶中没有发现；这意味着加氧酶的一种新策略 催化作用。我们已经从第二类嗜甲烷菌中提纯了MMO， Tichosporium OB3b；它由3种蛋白质组成，命名为 羟基酶、还原酶和B组分。该系统提供许多 与其他纯化的MMO系统相比，具有更高的产率和 稳定性，羟基酶比活力提高25倍。 这些性质允许在数量上进行纯化，从而使生物物理 技术(光学EPR、穆斯堡尔、EXAFS、Endor、MCD和CD 光谱分析)可用于结构研究。最近， 得到了用于结构研究的满意的晶体。 小分子配体络合物、同位素标记底物的光谱 和抑制剂，以及瞬时动力学正在被用来研究 分子机制。协调光谱、化学和单一光谱 周转研究表明，该反应是由Mu-(R-)催化的。 或H-)氧桥联双核铁中心位于羟基酶中。我们 假设O2添加到该团簇的[Fe(II)-Fe(II)]状态 导致异解O-O键断裂，形成反应性 中间体，可能是[Fe(IV)-Fe(IV)=O]氧。这个物种是 认为以一种中间体的形式来攻击碳氢化合物 底物自由基。对这一机制的实质性支持正在 通过使用特殊合成的手性底物积累起来的 对于底物自由基的检测，阐明了 过氧化氢分流化学特性及瞬变的检测 反应中间体。MMO的催化活性亚系统 由羟基酶组成的，没有另外两个中的一个或两个 组件正被用来评估 还原酶和B组分初步结果表明这些 组件在两种必要的减排当量转移中都发挥了作用 用于催化，并确保能源消耗的有效耦合 甲烷周转率高。这项工作应该产生一个基本的 对一种新型生物氧活化化学的认识 铁在这种化学中的新作用，以及对设计的指导 富含碳氢化合物的氧化催化剂。它也有可能 有助于我们理解蛋白质-蛋白质的作用 生物调控过程中的相互作用。此外，正如我们所做的那样 表明优先污染物，如三氯乙烯，很快 用MMO氧化解毒，很可能所获得的知识 从这项研究中将发现环境方面的应用。

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.