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Methanobactin and the Membrane-Associated Methane Monooxygenase in Methanotrophy: a Tale of Two Proteins Dongwon Choi Iowa State University

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Methanobactin and the Membrane-Associated Methane Monooxygenase in Methanotrophy: a Tale of Two Proteins Dongwon Choi Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 Methanobactin and the membrane-associated methane monooxygenase in methanotrophy: a tale of two proteins DongWon Choi Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, and the Microbiology Commons Recommended Citation Choi, DongWon, "Methanobactin and the membrane-associated methane monooxygenase in methanotrophy: a tale of two proteins" (2007). Retrospective Theses and Dissertations. 15600. https://lib.dr.iastate.edu/rtd/15600 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Methanobactin and the membrane-associated methane monooxygenase in methanotrophy: A tale of two proteins by DongWon Choi A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Microbiology Program of Study Committee: Alan A. DiSpirito, Major Professor Gregory J. Phillips Aubrey F. Mendonca Thomas A. Bobik Mark S. Hargrove Iowa State University Ames, Iowa 2007 Copyright © DongWon Choi, 2007. All rights reserved. UMI Number: 3289402 UMI Microform 3289402 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii TABLE OF CONTENTS CHAPTER 1: GENERAL INTRODUCTION 1 Thesis organization 3 References 7 CHAPTER 2: THE MEMBRANE-ASSOCIATED METHANE MONOOXYGENASE (pMMO) AND pMMO-NADH: QUINONE OXIDOREDUCTASE COMPLEX FROM Methylococcus capsulatus Bath 12 Abstract 12 Introduction 13 Materials and Methods 15 Organism and cultivation 15 Enzyme Activity 16 Isolation of membranes and soluble fractions 17 Solubilization of pMMO 18 Purification of pMMO, NDH, and NDH-pMMO complex 18 Dodecyl β-D maltoside treatment of pMMO 19 Electrophoresis and immunoblot analysis 19 Preparation of antibodies against the pMMO 20 Protein, cell count, and metal determinations 20 Fatty Acid analysis 21 Amino acid analysis and sequence 22 Spectroscopy 22 Total RNA extraction from pure cultures 22 Results 23 Effect of copper and iron in the culture medium on pMMO activity 23 Effect of copper in the culture medium on pMMO expression and membrane development 24 Detergent to protein ratio 27 Effect of detergent concentration on purified pMMO 28 Purification of pMMO and NDH-pMMO complex 29 Physiological reductant 30 Discussion 31 Acknowledgement 33 References 33 CHAPTER 3: EFFECT OF METHANOBACTIN ON THE ACTIVITY AND ELECTRON PARAMAGNETIC RESONANCE SPECTRA OF THE MEMBRANE-ASSOCIATED METHANE MONOOXYGENASE IN Methylococcus capsulatus Bath 49 Abstract 49 iii Introduction 50 Materials and methods 52 Organisms, culture conditions, and isolation of membrane fractions 52 Isolation of mb 53 Molecular mass determinations 54 Enzyme activity, isolation of cell fraction, and protein determinations 55 Cu-mb and substrate effects on the EPR spectra of washed membranes 56 UV-visible absorption spectroscopy 57 EPR spectrospcopy 57 Metal, and protein determinations 57 Results 57 Isolation of mb in the absence of copper 57 Effects of Cu-mb on pMMO activity 59 EPR spectra of mb 61 Cu(II), Cu-mb, and substrate effects on the EPR spectra in membrane samples 63 Effects of mb on the cupric signal in the g⊥ region 65 Discussion 67 Acknowledgements 70 References 70 Supporting Online Material 82 Supporting Materials and Methods 82 Supporting Results and Discussion 82 Supporting References 86 CHAPTER 4: SPECTRAL, KINETIC, AND THERMODYNAMIC PROPERTIES OF Cu(I)- AND Cu(II)-BINDING BY METHANOBACTIN FROM Methylosinus trichosporium OB3b 92 Abstract 92 Introduction 93 Materials and methods 96 Organism, culture conditions, and isolation of methanobactin 96 Metal titration of mb and EDTA-mb 96 Spectroscopic measurements 97 X-ray photoelectron spectroscopy (XPS) 98 Isothermal Titration Calorimetry (ITC) 99 Kinetics of copper binding 100 Metal, thiol, and protein determinations 101 Statistical analysis 101 Results 101 UV-visible absorption spectra of mb 101 Kinetics of copper binding 104 Fluorescence spectroscopy 105 Circular dichroism spectra 107 X-ray photoelectron spectroscopy 107 iv Thermodynamic properties of Cu(II) binding by mb 109 Solublization and binding of Cu(I) by mb and EDTA-mb 110 Discussion 112 Acknowledgements 116 References 116 CHAPTER 5: SPECTRAL AND THERMODYNAMIC PROPERTIES OF Ag(I), Au(III), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), AND Zn(II) BINDING BY METHANOBACTIN FROM Methylosinus trichosporium OB3b 137 Abstract 137 Introduction 138 Experimental 140 Organisms culture conditions and isolation of mb 140 Metal titrations 140 Spectroscopy, isothermal titration calorimetry (ITC), and metal determinations 141 X-ray photoelectron spectroscopy 142 Transmission electron microscroscopy 142 Results and discussion 143 Metals bound by mb and metals binding groups 143 UV-visible absorption spectra 144 Fluorescence Spectroscopy 146 Circular Dichroism Spectroscopy 147 Electron paramagnetic resonance and X-ray photoelectron spectroscopy: oxidation state of metals bound to mb 149 Isothermal titration calorimetry 150 Summary and concluding remarks 151 Abbreviations 153 Acknowledgements 154 References 155 CHAPTER 6: OXIDASE, SUPEROXIDE DISMUTASE, AND HYDROGEN PEROXIDE REDUCTASE ACTIVITIES OF METHANOBACTIN FROM TYPE I AND TYPE II METHANOTROPHS 170 Abstract 170 Introduction 171 Experimental 173 Organisms, culture conditions and isolation of membrane fraction 173 Isolation of mb 173 Superoxide dismutase (SOD) activity assay 173 Anaerobic NBT reduction 174 Oxidase and hydrogen peroxide reductase (HPR) activities 175 v Methane oxidation activity, protein determination, electron paramagnetic resonance (EPR) spectroscopy, UV-visible absorption spectroscopy, and metal analysis 176 Molecular-mass determinations 176 X-ray photoelectron spectroscopy (XPS) 176 Results and discussion 177 Effect of mb on pMMO-H activity 177 Superoxide dismutase-like activity 178 Oxidase activity by mb 178 Reduction of nitroblue tetrazolium by mb 180 Hydrogen peroxide reductase activity 181 Cu2+ binding and reduction 183 EPR spectroscopy 184 Summary and concluding remarks 185 Abbreviations 187 References 188 CHAPTER 7: MÖSSBAUER STUDIES OF THE MEMBRANE- ASSOCIATED METHANE MONOOXYGENASE FROM Methylococcus capsulatus Bath: EVIDENCE FOR A DIIRON CENTER 200 Abstract 200 Introduction 201 Results and discussion 202 Acknowledgement 207 References 207 Supporting information 215 Materials and methods 215 Results 217 References 223 CHAPTER 8: GENERAL CONCLUSIONS 229 Metal centers of membrane-associated methane monooxygenase 229 Methanobactin: A moonlighting protein 231 Methanobactin: Other properties 232 References 233 1 CHAPTER 1: GENERAL INTRODUCTION Modified from a book chapter “Respiration in Methanotrophs” published in Respiration in Archaea and Bacteria1 Alan A. DiSpirito, Ryan C. Kunz, DongWon Choi, and James A. Zahn Methanotrophs are Gram-negative bacteria characterized by the utilization of methane or methanol as a sole carbon and energy source. Two general categories of methanotrophs have been identified, Type I and Type II, based on several characteristics including the pattern of internal membranes, carbon assimilation pathway, and predominant fatty acid chain length (1, 6, 25). Methanotrophs play a key role in the global carbon cycle, and may be a significant sink for atmospheric methane (7, 18, 23). In addition to their ecological significance, the potential use of these microorganisms in bioremediation and biotransformations processes has provided incentive for the biochemical characterization of methanotrophs (4, 6, 10, 12). The oxidation of methane to carbon dioxide by methanotrophs involves a series of two electron steps with methanol, formaldehyde, and formate as intermediates (Fig. 1) (1, 6). The first enzyme in this pathway, the methane monooxygenase (MMO) catalyzes the energy-dependent oxidation of methane to methanol. In some methanotrophs, methane is oxidized to methanol by two different methane monooxygenases (MMOs), a membrane- associated MMO or particulate MMO (pMMO) and a soluble cytoplasmic MMO (sMMO) depending on the copper concentration during growth (19, 20). In cells cultured under a 1Respiration in Archaea and Bacteria, Zannoni, D. ed (2004) 149-168. Copyright 2004 Kluwer Scientific 2 low copper-to-biomass ratio, the sMMO is predominately expressed, with low, but detectable levels of pMMO expression (12, 16, 17, 20, 26). Cells cultured under higher copper-to-biomass ratios express the pMMO exclusively, with no detectable expression of sMMO (12, 16, 21). While sMMO is a well-characterized enzyme that consists of a hydroxylase component composed of three polypeptides and a hydroxo-bridged binuclear iron cluster, an NADH-dependent reductase component composed of one polypeptide containing both FAD and [Fe2S2] cofactors, and a regulatory polypeptide (4, 6, 8, 19), information on the molecular properties of the pMMO is limited due to the instability of the pMMO in cell free fractions. Purification of the pMMO has been reported
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