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研究生:陳皇州
研究生(外文):Kelvin, Huang Chou Chen
論文名稱:嗜甲烷菌Methylococcuscapsulatus(Bath)中微粒體甲烷單氧化酵素的結構與功能之研究
論文名稱(外文):Studies Toward Understanding the Structure and Function of the Particulate Methane Monooxygenase in Methylococcus capsulatus (Bath)
指導教授:陳長謙陳長謙引用關係
指導教授(外文):Sunney I. Chan
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
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 MonooxygenaseProtein purificationEPRX-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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