Methane Monooxygenase Structure and Function

我们打算研究可溶性甲烷单加氧酶(MMO)的三维结构、催化机制和调控方法。MMO催化甲烷氧化细菌将甲烷氧化为二氧化碳的第一步。通过这种方式，几乎所有由厌氧细菌产生的大量甲烷(一种温室气体，其效力是二氧化碳的20倍)的大气排放都被阻止了。MMO还意外地催化许多其他碳氢化合物的氧化，导致在合成和生物降解方面的应用。我们从第二类嗜甲烷菌--三孢甲氧甲藻OB3b中纯化了MMO，它由羟基酶(MMOH)、还原酶(MMOR)和“B”(MMOB)三种蛋白质组成。我们的MMOH的晶体结构表明，它含有一个新的活性中心-双羟基桥联双核铁簇，这对于催化光谱研究(包括光学、EPR、穆斯堡尔谱、EXAFS、Endor、共振拉曼、荧光、核磁共振、MCD和CD)、诊断底物的周转和瞬时动力学也被用来研究其结构和分子机理。我们假设O2导致团簇的[Fe(II)-Fe(II)]状态，导致异解O-O键断裂形成反应中间体，形式为[oxo-Fe(IV)-Fe(IV)]物种，通过底物自由基或阳离子的中间形成来攻击碳氢化合物。该项目的瞬时动力学研究表明，在反应周期中有2个稳定中间体和7个瞬时中间体。光谱研究表明，一个中间体化合物Q含有一个双钼氧铁(IV)簇合物，该簇合物直接与CH4反应生成CH3OH，支持机理提议。Q是第一个从加氧酶中分离出来的中间体，可以附着未活化的碳氢化合物。正在进行的研究表明，MMOR和MMOB通过增加Q的形成速度来调节催化。MMOB突变体已被提纯。已经提纯了MMOB突变体，使催化循环中每一步的速度都可以单独调节。建议的研究集中在定位MMO三元蛋白质复合体和使用MMOB突变体来捕获和表征反应周期中间产物的化学。这项工作将对一种新型的O2活化化学有一个基本的了解，对新型化合物Q的结构和反应活性有深入的了解，并对丰富的碳氢化合物的氧化催化剂的设计具有指导意义。这也可能有助于我们理解蛋白质-蛋白质相互作用在生物调控过程中的新角色。

We propose to investigate the 3D structure, catalytic mechanism, and method of regulation of soluble methane monooxygenase (MMO). MMO catalyzes the first step in the oxidation of CH4 to CO2 by methanotrophic bacteria. In this way, the atmospheric egress of nearly all of the enormous quantify of CH4 (a greenhouse gas with 20 times the potency of C02) generated by anaerobic bacteria is prevented. MMO also adventitiously catalyzes the oxidation of many other hydrocarbons leading to applications in synthesis and in biodegradation. We have purified MMO from the type II methanotroph, Methylosinus trichosporius OB3b; it is composed of 3 proteins termed hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB). Our crystal structure of MMOH shows it to contain a novel active site bis-mu-hydroxo bridged dinuclear iron cluster which is essential for catalysis Spectroscopic studies (including optical, EPR, Mossbauer, EXAFS, ENDOR, resonance Raman, fluorescence, NMR, MCD, and CD), turnover of diagnostic substrates, and transient kinetics are also being used to investigate the structure and molecular mechanism. We hypothesize that O2 leads to the [Fe(II)-Fe(II)]state of the cluster resulting in heterolytic O-O bond cleavage to form a reactive intermediate, formally an [oxo-Fe(iv)-Fe(IV)]species, which attack hydrocarbons with the intermediate formation of a substrate radical or cation. Transient kinetic studies from the project have revealed 2 stable and 7 transient intermediates in the reaction cycle. Spectroscopic studies show that one intermediate, compound Q, contains a bis-mu-oxo-Fe(IV) cluster which reacts directly with CH4 to give CH3OH in support of the mechanistic proposal. Q is the first intermediate to be isolated in an oxygenase that can attach unactivated hydrocarbons. Ongoing studies suggest that MMOR and MMOB regulate catalysis by increasing the rate of Q formation. MMOB mutants have been purified. MMOB mutants have been purified that allow the rate of each step in the catalytic cycle to be individually regulated. Proposed studies focus on mapping the MMO ternary protein complex and using the MMOB mutants to trap and characterize the chemistry of reaction cycle intermediates. This work should yield a fundamental understanding of a new type of O2 activation chemistry, insight into the structure and reactivity of the novel compound Q, and guidance in the design of catalysts for oxidation of abundant hydrocarbons. It is also likely to contribute to our understanding of a new role for protein-protein interactions in biological regulatory processes.