Methane Monoxygenase Structure and Function

描述（由申请人提供）：我们将研究可溶性甲烷单加氧酶（MMO）的三维结构、催化机制和调控。MMO启动甲烷营养细菌将CH4氧化为CO2。通过这种方式，厌氧细菌产生的大量CH4（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还会催化许多其他化学物质的氧化，促进在合成中的应用以及对大量具有人体毒性的污染物（如三氯乙烯）的生物降解。来自trichosporium Methylosinus OB3b的MMO由3种蛋白组成：羟化酶（MMOH）、还原酶（MMOR）和B （MMOB）。MMOH具有催化所需的双u-羟基桥接双核铁簇。光谱研究（光学、EPR、穆斯堡尔、EXAFS、ENDOR、拉曼、荧光、核磁共振、MCD和CD）、诊断底物的周转和瞬态动力学被用于研究结构和机制。瞬态动力学研究发现了2种稳定中间体和7种瞬态中间体。1中间体化合物Q含有一个双氧-铁(IV)-铁（IV）簇，它与CH4直接反应生成CH3OH。Q是第一个从加氧酶中分离出来的能够攻击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的形成速度和根据大小控制底物进入MMOH活性位点的速度来调节催化作用。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。我们最近的研究定义了MMO组件之间的交互界面。拟议的研究将利用荧光能量转移、交联、质谱和晶体学技术来确定组分的空间取向以及限制底物结合的构象变化。反应循环中间体将使用基于稳态稳定和表面冷冻淬火的新方法来捕获。这些将通过新开发的x射线吸收和低温还原技术进行光谱表征。这项工作将使我们深入了解：1)新的O2活化化学，2)Q的性质，3)新的调控策略，以及4)烃类氧化小分子催化剂的设计。最后，从MMO中吸取的经验教训应该适用于结构和机制类似的人类核糖核苷酸还原酶，它产生DNA的构建块。

DESCRIPTION (provided by applicant): We will investigate the 3D structure, catalytic mechanism, and regulation of soluble methane monooxygenase (MMO). MMO initiates the oxidation of CH4 to CO2 by methanotrophic bacteria. In this way, the atmospheric egress of nearly all of the enormous quantity of CH4 (greenhouse gas with 20 times the potency of CO2) generated by anaerobic bacteria is prevented. MMO also adventitiously catalyzes the oxidation of many other chemicals fostering applications in synthesis as well as biodegradation of abundant pollutants with human toxicity (e.g. trichloroethylene). MMO from Methylosinus trichosporium OB3b is composed of 3 proteins: hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB). MMOH has a bis-u-hydroxo-bridged dinuclear Fe cluster needed for catalysis. Spectroscopic studies (optical, EPR, Mossbauer, EXAFS, ENDOR, rRaman, fluorescence, NMR, MCD, and CD), turnover of diagnostic substrates, and transient kinetics are being used to study the structure and mechanism. Transient kinetic studies have revealed 2 stable and 7 transient intermediates in the reaction cycle. 1 intermediate, compound Q, contains a bis-u-oxo-Fe(IV)-Fe(IV) cluster which reacts directly with CH4 to give CH3OH. Q is the first intermediate isolated in an oxygenase that can attack unactivated hydrocarbons. Ongoing studies suggest that MMOR and MMOB regulate catalysis by increasing the rate of Q formation and by controlling the rate of substrate entry into the active site of MMOH based on size. MMOB mutants have been purified that allow the rate of each step in the catalytic cycle to be individually regulated. Our recent studies have defined the interaction surfaces between the MMO components. The proposed studies will utilize fluorescence energy transfer, cross-linking, mass spec, and crystallography techniques to define the spatial orientation of the components as well as conformational changes that gate substrate binding. Reaction cycle intermediates will be trapped using new approaches based on steady state stabilization and surface freeze quenching. These will be spectroscopically characterized by newly developed X-ray absorption and cryoreduction techniques. This work should give us insight into: 1) novel O2 activation chemistry, 2) the nature of Q, 3) a new regulation strategy, and 4) design of small molecule catalysts for hydrocarbon oxidation. Finally, lessons learned from MMO should apply to the structurally and mechanistically similar human ribonucleotide reductase, which generates the building blocks for DNA.