Methane Monooxygenase Structure and Function

本文拟对可溶性甲烷单加氧酶（MMO）的三维结构、催化机理和调控方法进行研究。MMO催化甲烷营养菌将CH4氧化为CO2的第一步。通过这种方式，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）几乎全部被阻止向大气排放。MMO还可以催化许多其他碳氢化合物的氧化，从而在合成和生物降解中得到应用。我们从II型甲烷化菌Methylosinus trichosporius OB3b中纯化了MMO；它由3种蛋白质组成，分别是羟化酶（MMOH）、还原酶（MMOR）和B蛋白（MMOB）。我们的MMOH晶体结构表明，它含有一个新的活性位点双羟基桥接双核铁簇，这对于催化光谱研究（包括光学、EPR、穆斯堡尔、EXAFS、ENDOR、共振拉曼、荧光、核磁共振、MCD和CD）、诊断底物的转换和瞬态动力学也被用于研究结构和分子机制至关重要。我们假设O2导致簇的[Fe(II)-Fe(II)]状态，导致O-O键的异裂，形成反应中间体，正式形式为[oxo-Fe(iv)-Fe(iv)]物种，它们通过中间形成底物自由基或阳离子来攻击碳氢化合物。该项目的瞬态动力学研究揭示了反应周期中2种稳定中间体和7种瞬态中间体。光谱研究表明，其中一个中间体化合物Q含有一个双mu-oxo- fe （IV）簇，该簇与CH4直接反应生成CH3OH，支持了机理建议。Q是第一个从加氧酶中分离出来的能附着非活性碳氢化合物的中间体。正在进行的研究表明，MMOR和MMOB通过增加Q的形成速率来调节催化作用。MMOB突变体已被纯化。已经纯化的MMOB突变体允许催化循环中每个步骤的速率单独调节。建议的研究重点是绘制MMOB三元蛋白复合物，并利用MMOB突变体捕获和表征反应循环中间体的化学性质。这项工作将有助于对一种新型O2活化化学的基本理解，对新化合物Q的结构和反应性的深入了解，并指导丰富碳氢化合物氧化催化剂的设计。它也可能有助于我们理解蛋白质-蛋白质相互作用在生物调节过程中的新作用。

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.