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研究生： 陳皇州
Kelvin, Huang Chou Chen
論文名稱： 嗜甲烷菌Methylococcus capsulatus (Bath)中微粒體甲烷單氧化酵素的結構與功能之研究
Studies Toward Understanding the Structure and Function of the Particulate Methane Monooxygenase in Methylococcus capsulatus (Bath)
指導教授： 陳長謙
Sunney I. Chan
口試委員:
學位類別： 博士
Doctor
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2003
畢業學年度： 91
語文別： 英文
論文頁數： 185
中文關鍵詞： 嗜甲烷菌 、微粒體甲烷單氧化酵素 、蛋白質純化 、電子自旋共振光譜 、X光吸收光譜
外文關鍵詞： Methylococcus capsulatus (Bath), Particulate Methane Monooxygenase, Protein purification, EPR, X-ray absorption
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    • 本論文中，利用多項不同之現代先進技術，針對嗜甲烷菌中微粒體型甲烷單氧化酵素的功能與結構，進行一系列系統性的研究。首先，藉由傳統發酵系統與平行微管束型過濾器的結合，在發酵技術上獲得突破性的進展，由於這項關鍵性的突破，使得我們獲得高產量、高活性之微粒體型甲烷單氧化酵素，以利各項實驗之進行，也正因為如此，後續蛋白質純化的程序亦得以簡化，縮短純化所需的時間，並提高純化後蛋白質的純度。微粒體型甲烷單氧化酵素活性位置的結構與其催化的反應機制是本研究的重心之一。為了深入探討酵素活性位置之相關特性，我們利用各種不同之還原劑，氧化劑及自殺性受質與氧氣，對酵素中大量的銅離子進行氧化還原滴定反應。藉由不同氧化狀態及自殺性受質修飾後的酵素樣品所獲取之電子自旋共振光譜及X光吸收光譜，確認每單位甲烷單氧化酵素中存在著約15個銅離子，區分為電子轉移功能之三核銅離子簇及基質催化中心之三核銅離子簇並深入探討其特性及其所扮演的角色。此外，我們利用膠體層析管柱將甲烷單氧化酵素加以純化後，以基質輔助雷射脫附離子化╱時間飛行式質譜儀技術所提供之序列片段對此一酵素加以鑑定，並確認酵素中各次單元之實際分子量。最後，在全反射─傅立葉轉換紅外線光譜的研究中，亦明確地指出甲烷單氧化酵素鑲入脂雙層之片段乃是以a-螺旋體的型式存在。

    The work described in this thesis is directed study toward understanding the structure and function of the pMMO from Methylococcus capsulatus (Bath). A variety of modern techniques have been brought to bear on this problem. An important development has been the development of a hollow-fiber bioreactor and fermentation technology toward scaling up the growth of the methanotropic bacteria. This new technology has allowed the purification of highly active pMMO in membranes, as well as in-depth biochemical/biophysical characterization of the membrane protein after the purification of the highly active pMMO.
    In order to explore the structure of the active site as well as the nature of the reaction intermediate(s) formed at the active site during turnover of the enzyme, we have subjected the pMMO to different levels of reductants, oxidants and suicide substrate acetylene under dioxygen tensions and looked for change at various stages of oxidation of the copper clusters. Both the catalytic and electron transfer clusters (C- and E-clusters, respectively) have been examined by EPR spectroscopy and X-ray K-edge absorption to distinguish between various multi-oxidation states of the copper clusters. To date, EPR and X-ray absorption measurements have confirmed the classification of the 15 copper ions into 3 trinuclear copper clusters for electron transfer (E-clusters) and 2 trinuclear copper clusters for dioxygen chemistry and alkane hydroxylation (C-clusters).

    Further purification by membrane solubilization in dodecyl b-D maltoside followed by fractionation of the protein-detergent complexes according to molecular size using gel filtration chromatography yielded the pMMO-detergent complex in good yield and high homogeneity. The purified pMMO-detergent complex has not only been identified by mass finger printing, but also by the determination of the actual molecular mass (99kDa) of the pMMO by MALDI-TOF mass analysis. Finally, ATR-FTIR spectroscopy combined with limited proteolysis has provided the direct evidence for the presence of a-helices in the membrane-embedded domains of pMMO.

    ABSTRACT ABSTRACT (IN CHINESE) ACKONWLEDGEMENTS TABLE OF CONTENTS ABBREVIATIONS AND NOMENCLATURE CHAPTER 1: INTRODUCTION METHANOTROPHS METHANE MONOOXYGENASE BASIC THEORY OF X-RAY ABSORPTION SPECTROSCOPY The physical basis of X-ray absorption Extended X-ray Absorption Fine Structure (EXAFS Advantages of XAS BASIC THEORY OF EPR SPECTROSCOPY Energy of Magnetic Dipoles in a Magnetic Field The Zeeman Effect Thermal Equilibrium and Spin-Lattice Relaxation g-Value Hyperfine Interactions INTRODUCTION OF MALDI-TOF MASS OVERVIEWS OF THIS THESIS REFERENCES CHAPTER 2: PRODUCTION OF HIGH QUALITY PMMO IN HIGH YIELDS FROM METHYLOCOCCUS CAPSULATUS (BATH) WITH A HOLLOW-FIBER MEMBRANE BIOREACTOR ABSTRACT INTRODUCTION MATERIALS AND METHODS Culturing of Bacteria Regulation of the Copper Concentration Preparation of pMMO-Enriched Membranes Metal contents in the pMMO-Enriched Membranes Derived from Cells Cultured at Various Copper Concentrations in the Growth Media X-ray Absorption Spectroscopy pMMO Activity Assay Instrumentation RESULTS Copper Regulation and Hollow Fiber Bioreactor Quantitation of the pMMO Specific Activity of the pMMO Copper Uptake Ttermination Other Metals: Fe and Zn NADH versus Duroquinol Activity DISCUSSION Quality of the pMMO Copper Content in the pMMO CONCLUSIONS REFERENCES CHAPTER 3: HIGHLY EXPRESSED PMMO MEMBRANE PROTEIN PURIFICATION AND CHARACTERIZATION. ABSTRACT INTRODUCTION MATERIALS AND METHODS… Membrane Isolation and Solubilization Protein Purification by Size-Exclusion Chromatograph Purified pMMO-Detergent Complex Activity Assays SDS-PAGE ElectrophoresisNative Gel Electrophoresis In-gel Protein Digestion and Analysis of the Peptide Fragments of the pMMO Subunits Molecular Weight Identification of the pMMO Subunits Instrumentation RESULTS AND DISCUSSION Purification of the pMMO from pMMO-Enriched Membranes Peptide Mass Fingerprinting of the pMMO Subunits Properties of the Purified pMMO K- edge X-ray Absorption Spectroscopy of the Purified pMMO-Detergent Complex EPR Spectroscopy of the Purified pMMO-Detergent Complex Molecular Weight Identification of the Three Subunits in pMMO The Specific Activity of Purified pMMO-Detergent Complex SUMMARY AND CONCLUSIONS REFERENCES CHAPTER 4: THE COPPER CLUSTERS IN PARTICULATE METHANE MONOOXYGENASE ABSTRACT INTRODUCTION MATERIALS AND METHODS The Culturing of Methylococcus capsulatus (Bath) Cell Breakage and Isolation of pMMO-Enriched Membranes 15N pMMO-Enriched Membranes Preparation Modification of the Protein by the Suicide Substrate Acetylene Purging of the Acetylene-Modified pMMO with Pure Dioxygen Reductive Titration with Sodium Dithionite Oxidative Titration with Potassium Ferricyanide Oxidative Titration with Hydrogen Peroxide EPR Spectroscopy X-ray Absorption Spectroscopy FTIR Spectroscopy RESULTS 4K EPR of the As-Isolated pMMO-Enriched Membranes 77 K EPR of the As-Isolated pMMO-Enriched Membranes and the Purified pMMO-Detergent Complex 15N-Labeling of the pMMO EPR and XAS Spectroscopy of pMMO-Enriched Membranes. The Effect of the Suicide Substrate Reductive Titration of the As-Isolated pMMO. Oxidative Titration of the Fully Reduced pMMO by HydrogenPeroxide Oxidative Titration of the Fully Reduced pMMO by Ferricyanide DISCUSSION SUMMARY REFERENCES CHAPTER 5: POLARIZED ATR-FTIR SPECTROSCOPY THE MEMBRANE-EMBEDDED DOMAINS OF THE PMMO ABSTRACT INTRODUCTION MATERIALS AND METHODS Sample Preparation ATR–FTIR Measurements 1H / 2H-Exchange Secondary Structure Determination Secondary Structure Orientation from ATR–FTIR Dichroism RESULTS DISSCUSSION AND SUMMARY REFERENCES

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