Kinetic Simulation of Methane and Ethane Hydroxylation by Methane Monooxygenase

According to the mechanism of alkane hydroxylation, whose main postulate is the formation of an intermediate complex containing pentacoordinated carbon, the hydroxylation of methane and ethane by methane monooxygenase was kinetically simulated by the numerical method. The published data on the kinetic isotope effects of oxidation of deuterium-substituted methane molecules (CHD₃, CH₂D₂, and CH₃D) and the distribution of products of chiral ethane (R- and S-MeCHDT) oxidation by methane monooxygenase were examined. The kinetic models proposed for the oxidation of isotopically substituted methane and ethane are in good agreement with experimental data.     

Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins A list of abbreviations can be found in Section 7

Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O(2). The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.     

甲烷单加氧酶催化机理的研究进展

甲烷单加氧酶是甲烷营养细菌代谢过程中的重要酶系,同时也是一类能够很好地催化底物分子单加氧反应的生物催化剂,在工业应用、医药和环境治理方面有着广泛的应用前景。文章综述了近年来在甲烷单加氧酶催化机理方面的研究,着重阐述了含非血红素双铁核的结构及活化氧分子和底物的机理。     

甲烷单加氧酶的催化反应机理研究*

本文就甲烷单加氧酶近年来在催化反应机理方面的最新研究成果进行了详细阐述。甲烷C—H 键的活化机理主要包括自由基回弹机理和协调的氧插入机理。运用自由基探针底物和量化计算等方法对烷烃羟基化反应机理的直接研究表明, 目前没有一个统一的机理来解释甲烷单加氧酶的反应过程。反应机理的类型可能取决于MMO 的来源或者其他因素。对甲烷单加氧酶的几种中间化合物的各种光谱学研究有力地推动了机理研究的发展。     

甲烷单加氧酶化学模拟的初步研究

制备了两种新的甲烷单加氧酶模拟酶：（1）固载于分子筛上的单氧桥联双核铁配合物Fe2(O)(H2O)2-(phen)4(ClO4)；（2）在表面修饰的介孔分子筛上原位合成的氧桥、羧基桥联双核铁配合物Fe2(O)（μ－CH3COO）2－(H2O)2-(phen)2Cl2。利用紫外漫反射、红外漫反射、拉曼光谱、N2吸脱附分析及元素分析等手段，对模拟酶进行结构分析。结果表明，Fe2(O)(H2O)2-(phen)4(ClO）4主要是以配合物中的桥氧与分子筛表面硅羧基成氢键固载；原位合成的Fe2(O)（μ－CH3COO）2(H2O)2-(phen)2Cl2中，一个羟基来自表面修饰的分子筛，催化反应结果表明在，温和条件下、以叔丁基过氧化氢为氧化剂，这两种模拟酶均催化环已烷氧化。     

The Methane Monooxygenase Intrinsic Activity of Kinds of Methanotrophs

Methanotrophs have promising applications in the epoxidation of some alkenes and some chlorinated hydrocarbons and in the production of a biopolymer, poly-β-hydroxybutyrate (poly-3-hydroxybutyrate; PHB). In contrast with methane monooxygenase (MMO) activity and ability of PHB synthesis of four kinds of methanotrophic bacteria Methylosinus trichosporium OB3b, M. trichosporium IMV3011, Methylococcus capsulatus HD6T, Methylomonas sp. GYJ3, and the mixture of the four kinds of strains, M. trichosporium OB3b is the highest of the four in the activity of propene epoxidation (10.72 nmol/min mg dry weight of cell [dwc]), the activity of naphthalene oxidation (22.7 mmol/mg dwc), and ability in synthesis of PHB(11% PHB content in per gram dry weight of cell in 84 h). It could be feasible to improve the MMO activity by mixing four kinds of methanotrophs. The MMO activity dramatically decreased when the cellular PHB accumulated in the second stage. The reason for this may be the dilution of the MMO system in the cells with increasing PHB contents. It has been found that the PHB contents at the level of 1–5% are beneficial to the cells for maintenance of MMO epoxidation activity when enough PHB have been accumulated. Moreover, it was also found that high particulate methane monooxygenase activity may contribute to the synthesis of PHB in the cell, which could be used to improve the yield of PHB in methanotrophs.     

戊烷与颗粒型甲烷单加氧酶的相互作用研究

颗粒型甲烷单加氧酶(Particulate methane monooxygenase, PMMO)是一个与细胞膜结合的金属酶, 能将烷烃生物催化为醇. 研究PMMO与烷烃的结合模式及催化机制将有利于设计合成一个新的模拟酶, 进而有效地利用烷烃作为新能源. 用分子对接方法获得了PMMO单体与一系列烷烃的结合模式, 并对PMMO单体和PMMO-戊烷复合物进行了6 ns的分子动力学模拟, 最后对复合物进行了构象成簇及结合能分析. 结果表明, 戊烷结合到靠近Zn^2＋的疏水口袋中, 该口袋由pmoA亚基的M45～W60和R190～T193以及pmoC亚基的Q161三个片段组成. 动力学结果表明, 与PMMO单体比, PMMO-戊烷复合物保持着相近的运动模式, 但幅度更明显, 另外, 戊烷在疏水口袋中的大幅度运动对于PMMO发挥催化作用是必须的. 结合能计算揭示疏水相互作用是戊烷与PMMO稳定识别的主要驱动力, 所有模拟结果与实验数据吻合较好.     

颗粒性甲烷单加氧酶分离纯化方法的研究进展

颗粒性甲烷单加氧酶(pMMO)是甲烷氧化菌的特征酶之一,在生物催化方面具有广泛的应用前景,但由于其内膜蛋白的性质以及纯化过程中的不稳定性,使其生物化学性质、金属活性位点等方面仍存在许多未知和争议.着重总结了颗粒性甲烷单加氧酶的分离纯化方法,并对其活性以及与甲烷氧化菌素-Cu(methanobactin-Cu)和其他物质之间的作用关系进行了概述,以促进颗粒性甲烷单加氧酶的深入研究和应用.     

热力学抑制剂作用下甲烷水合物分解过程的分子动力学模拟

采用正则系综(NVT)分子动力学方法模拟研究277.0 K、11.45 mol·L-1的热力学抑制剂乙二醇(EG)溶液作用下甲烷水合物分解微观过程. 模拟显示甲烷水合物的分解从甲烷水合物固体表面开始, 逐渐向内部推移, 固态水合物在分解过程中逐渐缩小, 直至消失. 固态水合物的分解从晶格扭曲变形开始, 之后笼形框架结构破裂, 最后形成笼形结构碎片. 同时已经分解的甲烷水合物在外层形成水膜, 包裹里层正在分解的甲烷水合物, 增大里层甲烷水合物分解传质阻力.     

Regio‐ and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C?H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity.     

Reactivity of Compound II: Electronic Structure Analysis of Methane Hydroxylation by Oxoiron(IV) Porphyrin Complexes

The methane hydroxylation reaction by a Compound II (Cpd II) mimic PorFe(IV)=O and its hydrosulfide-ligated derivative [Por(SH)Fe(IV)=O](-) is investigated by density functional theory (DFT) calculations on the ground triplet and excited quintet spin-state surfaces. On each spin surface both the σ- and π-channels are explored. H-abstraction is invariably the rate-determining step. In the case of PorFe(IV)=O the H-abstraction reaction can proceed either through the classic π-channel or through the nonclassical σ-channel on the triplet surface, but only through the classic σ-mechanism on the quintet surface. The barrier on the quintet σ-pathway is much lower than on the triplet channels so the quintet surface cuts through the triplet surfaces and a two state reactivity (TSR) mechanism with crossover from the triplet to the quintet surface becomes a plausible scenario for C-H bond activation by PorFe(IV)=O. In the case of the hydrosulfide-ligated complex the H-abstraction follows a π-mechanism on the triplet surface: the σ* is too high in energy to make a σ-attack of the substrate favorable. The σ- and π-channels are both feasible on the quintet surface. As the quintet surface lies above the triplet surface in the entrance channel of the oxidative process and is highly destabilized on both the σ- and π-pathways, the reaction can only proceed on the triplet surface. Insights into the electron transfer process accompanying the H-abstraction reaction are achieved through a detailed electronic structure analysis of the transition state species and the reactant complexes en route to the transition state. It is found that the electron transfer from the substrate σ(CH) into the acceptor orbital of the catalyst, the Fe-O σ* or π*, occurs through a rather complex mechanism that is initiated by a two-orbital four-electron interaction between the σ(CH) and the low-lying, oxygen-rich Fe-O σ-bonding and/or Fe-O π-bonding orbitals of the catalyst.     

Investigation of the Hydroxylation Mechanism of Noncoupled Copper Oxygenases by Ab Initio Molecular Dynamics Simulations

In Nature, the family of copper monooxygenases comprised of peptidylglycine α‐hydroxylating monooxygenase (PHM), dopamine β‐monooxygenase (DβM), and tyramine β‐monooxygenase (TβM) is known to perform dioxygen‐dependent hydroxylation of aliphatic C? H bonds by using two uncoupled metal sites. In spite of many investigations, including biochemical, chemical, and computational, details of the C? H bond oxygenation mechanism remain elusive. Herein we report an investigation of the mechanism of hydroxylation by PHM by using hybrid quantum/classical potentials (i.e., QM/MM). Although previous investigations using hybrid QM/MM techniques were restricted to geometry optimizations, we have carried out ab initio molecular dynamics simulations in order to include the intrinsic flexibility of the active sites in the modeling protocol. The major finding of this study is an extremely fast rebound step after the initial hydrogen‐abstraction step promoted by the cupric–superoxide adduct. The hydrogen‐abstraction/rebound sequence leads to the formation of an alkyl hydroperoxide intermediate. Long‐range electron transfer from the remote copper site subsequently triggers its reduction to the hydroxylated substrate. We finally show two reactivity consequences inherent in the new mechanistic proposal, the investigation of which would provide a means to check its validity by experimental means.     

Regioselectivity and Deuterium Isotope Effects in Geraniol Hydroxylation by the Cytochrome P-450 Monooxygenase from Catharanthus roseus (L.) G. DON

The hydroxylation of geraniol ( 8 ) by cytochrome P-450 (P-450_Cath.) from the subtropical plant Catharanthus roseus (L.) G. DON was optimised to give 8-hydroxygeraniol ( 9 ) as the single product in 35% yield. Incubations of different ¹³C- and ²H-labelled geraniols revealed that H-abstraction is completely regioselective in favour of the CH₃ group trans to the chain at C(6) of 8 . An intramolecular isotope effect k_H/k_D = 8.0 was determined, suggesting that H-abstraction is one of the major rate-contributing steps; however, the intermolecular isotope effect was surprisingly inverse at low conversion k_H/k_D = 0.50, indicating the existence of rate-contributing steps preceding the first irreversible, isotope-sensitive reaction in the sequence.     

Enantioselective Allylic Hydroxylation of ω‐Alkenoic Acids and Esters by P450 BM3 Monooxygenase

Chiral allylic alcohols of ω‐alkenoic acids and derivatives thereof are highly important building blocks for the synthesis of biologically active compounds. The direct enantioselective C? H oxidation of linear terminal olefins offers the shortest route toward these compounds, but known synthetic methods are limited and suffer from low selectivities. Described herein is an enzymatic approach using the P450 BM3 monooxygenase mutant A74G/L188Q, which catalyzes allylic hydroxylation with high to excellent chemo‐ and enantioselectivities providing the desirable secondary alcohols.     

Production of Hydrogen from Methane by a CO2 Emission-Suppressed Process: Methane Decomposition and Gasification of Carbon Nanofibers

Catalytic methane decomposition into hydrogen and carbon nanofibers and the oxidations of carbon nanofibers with CO₂, H₂O and O₂ were overviewed. Supported Ni catalysts (Ni/SiO₂, Ni/TiO₂ and Ni/carbon nanofiber) were effective for the methane decomposition. The activity and life of the supported Ni catalysts for methane decomposition strongly depended on the particle size of Ni metal on the catalysts. The modification of the catalysts with Pd enhanced the catalytic activity and life for methane decomposition. In particular, the supported Ni catalysts modified with Pd showed high turnover number of hydrogen formation at temperatures higher than 973 K with a high one-pass methane conversion (>70%). However, sooner or later, every catalyst completely lost their catalytic activities due to the carbon layer formation on active metal surfaces. In order to utilize a large quantity of the carbon nanofibers formed during methane decomposition as a chemical feedstock or a powdered fuel for heat generation, they were oxidized with CO₂, H₂O and O₂ into CO, synthesis gas and CO₂, respectively. In every case, the conversion of carbon was greater than 95%. These oxidations of carbon nanofibers recovered or enhanced the initial activities of the supported Ni catalysts for methane decomposition.     

甲烷单加氧酶的模型化合物:以树脂为载体的双铁(Ⅲ)催化剂的合成、表征及催化性能的研究

使用树脂为载体,合成了6种新的双铁(Ⅲ)催化剂,成功的模拟了可溶性甲烷单加氧酶(MMO)的双核铁中心.这是首次使用树脂作为载体合成模拟MMO的非均相催化剂.在室温下以乙腈为溶剂,30%的过氧化氢或特丁基过氧化氢做氧化剂,环己烷或乙苯作为底物的反应中,催化剂具有MMO相似的催化活性.其中,使用大孔氨甲基树脂为载体合成的催化剂能有效地催化乙苯氧化得到较好的转化率(总收率达69.9%,TON值达55.3).同时研究了树脂的颗粒大小及阴离子供体对催化性能的影响.使用粒径0.675～0.280 mm的树脂和ClO-4的阴离子供体所合成的催化剂具有很好的催化活性.最后探讨了催化剂的回收和再利用.     

Benchmarking the Polyatomic Reaction Dynamics of X+Methane

With recent developments of sophisticated experimental techniques and advanced theoretical methods/computations, the field of chemical dynamics has reached the point that theoryexperiment comparisons can be made at a quantitative level in very fine details for a prototypical A+BC system. As the system becomes larger, more degrees of freedom are involved and the complexity increases exponentially. At the same time, the multifaceted nature of polyatomic systems also opens up the possibilities for observing many new chemistry and novel phenomena|a land of opportunities. For the past 15 years or so my laboratory has delved into the reaction dynamics of methane+X (X: F, Cl, O(³P), and OH). This effort shifts the paradigm in the field of reaction dynamics by making the title reaction a benchmark polyatomic system. In this account, I shall disclose my thinking behind some of the key concepts and methods we introduced and how the unexpectedly discovered phenomena led to other uncharted territories. Those ndings not only enrich our understanding of the specific reactions we studied at the most fundamental level and inspire the theoretical developments, but also shape our thinking and lay the foundation for future explorations of different aspects of the multifaceted nature of polyatomic reactivity.     

Quantum Refinement Does Not Support Dinuclear Copper Sites in Crystal Structures of Particulate Methane Monooxygenase

Particulate methane monooxygenase (pMMO) is one of the few enzymes that can activate methane. The metal content of this enzyme has been highly controversial, with suggestions of a dinuclear Fe site or mono‐, di‐, or trinuclear Cu sites. Crystal structures have shown a mono‐ or dinuclear Cu site, but the resolution was low and the geometry of the dinuclear site unusual. We have employed quantum refinement (crystallographic refinement enhanced with quantum‐mechanical calculations) to improve the structure of the active site. We compared a number of different mono‐ and dinuclear geometries, in some cases enhanced with more protein ligands or one or two water molecules, to determine which structure fits two sets of crystallographic raw data best. In all cases, the best results were obtained with mononuclear Cu sites, occasionally with an extra water molecule. Thus, we conclude that there is no crystallographic support for a dinuclear Cu site in pMMO.     

Analysis of Decomposition for Structure I Methane Hydrate by Molecular Dynamics Simulation

Under multi-nodes of temperatures and pressures, microscopic decomposition mechanisms of structure I methane hydrate in contact with bulk water molecules have been studied through LAMMPS software by molecular dynamics simulation. Simulation system consists of 482 methane molecules in hydrate and 3027 randomly distributed bulk water molecules. Through analyses of simulation results, decomposition number of hydrate cages, density of methane molecules, radial distribution function for oxygen atoms, mean square displacement and coefficient of diffusion of methane molecules have been studied. A significant result shows that structure I methane hydrate decomposes from hydrate-bulk water interface to hydrate interior. As temperature rises and pressure drops, the stabilization of hydrate will weaken, decomposition extent will go deep, and mean square displacement and coefficient of diffusion of methane molecules will increase. The studies can provide important meanings for the microscopic decomposition mechanisms analyses of methane hydrate.