Methane Monooxygenase Structure and Function

本文旨在研究可溶性甲烷单加氧酶（MMO）的三维结构、催化机理和调控方法。MMO催化甲烷氧化细菌将CH 4氧化为CO2的第一步。以这种方式，防止了几乎所有由厌氧细菌产生的大量CH 4（一种温室气体，其效力是CO2的20倍）的大气排放。MMO还间接催化许多其他烃类的氧化，导致在合成和生物降解中的应用。我们已经从II型甲烷氧化菌，发孢甲基弯菌OB 3 B中纯化了MMO;它由3种蛋白质组成，称为羟化酶（MMOH）、还原酶（MMOR）和“B”（MMO B）。我们的晶体结构的MMOH表明，它包含一个新的活性位点双μ-羟基桥双核铁簇，这是必不可少的催化光谱研究（包括光学，EPR，穆斯堡尔，EXAFS，ENDOR，共振拉曼，荧光，NMR，MCD，和CD），周转的诊断底物，和瞬态动力学也被用来研究结构和分子机制。我们假设O2导致簇的[Fe（II）-Fe（II）]状态，导致异裂O-O键裂解以形成反应性中间体，形式上为[oxo-Fe（IV）-Fe（IV）]物种，其攻击烃类，中间体形成底物自由基或阳离子。该项目的瞬态动力学研究揭示了反应循环中的2种稳定中间体和7种瞬态中间体。光谱研究表明，一个中间体，化合物Q，包含一个bis-mu-oxo-Fe（IV）簇，直接与CH 4反应，得到CH 3OH，支持的机制建议。Q是在加氧酶中分离的第一个中间体，该加氧酶可以连接未活化的烃。正在进行的研究表明，MMOR和MMOB通过增加Q形成的速率来调节催化。MMOB突变体已被纯化。MMOB突变体已被纯化，允许催化循环中的每个步骤的速率被单独调节。拟议的研究集中在映射MMO三元蛋白质复合物，并使用MMOB突变体来捕获和表征反应循环中间体的化学。这项工作应该会对新型O2活化化学有一个基本的了解，深入了解新型化合物Q的结构和反应性，并为丰富碳氢化合物氧化催化剂的设计提供指导。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控过程中的新作用。

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.