Electron Transport Chain Coupled Protoporphyrinogen IX Oxidase from Escherichia coli Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Kalle Möbius aus Wolfsburg 1. Referent: Prof. Dr. Dieter Jahn 2. Referent: Prof. Dr. Michael Steinert eingereicht am: 14.05.2008 mündliche Prüfung (Disputation) am: 23.06.2008 Druckjahr 2008 II VORVERÖFFENTLICHUNGEN DER DISSERTATION Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: EINGEREICHTE PUBLIKATION Kalle Möbius, Daniela Breckau, Rodrigo Arias, Anna-Lena Hännig, Claudia Schulz, Katrin Riedmann, Rebekka Biedendieck, Dörte Becher, Axel Magalon, Jürgen Moser and Dieter Jahn: Cofactor Biosynthesis is coupled to the Electron Transport mediated Energy Conservation. Under review. TAGUNGSBEITRÄGE Kalle Möbius, Corinna Lüer, Jürgen Moser, Dieter Jahn. The oxygen-independent magnesium protoporphyrin IX monomethyl ester oxidative cyclase of Chlorobaculum tepidum (Poster) ICTPPO, Luzern, Schweiz (2005) Kalle Möbius, Daniela Breckau, Anna-Lena Hännig, Jürgen Moser and Dieter Jahn. The oxygen-independent Protoporphyrinogen IX Oxidase HemG of E. coli couples to aerobic and anaerobic electron transfer chains (Poster) TPDG, Lund, Schweden (2007) III TABLE OF CONTENTS ABBREVIATIONS ______________________________________________________ VII 1 INTRODUCTION ______________________________________________________1 1.1 TETRAPYRROLES _____________________________________________________1 1.2 STRUCTURE AND FUNCTIONS OF TETRAPYRROLES ____________________________1 1.3 BIOSYNTHESIS OF HEMES AND CHLOROPHYLLS ______________________________4 1.4 FORMATION OF PROTOPORPHYRIN IX _____________________________________7 1.4.1 The Oxygen-Dependent Protoporphyrinogen IX Oxidase__________________7 1.4.2 The Oxygen-Independent Oxidation of Protoporphyrinogen IX ____________10 1.5 RESPIRATORY CHAINS OF ESCHERICHIA COLI _______________________________12 1.6 FORMATION OF PROTOCHLOROPHYLLIDE DURING CHLOROPHYLL BIOSYNTHESIS ___13 1.6.1 The Oxygen-Dependent oxidative Cyclisation of Mg-Protoporphyrin IX monomethyl ester ____________________________________________________14 1.6.2 The Oxygen-Independent Mg-Protoporphyrin IX monomethyl ester oxidative Cyclase ____________________________________________________________14 1.7 AIM OF THIS STUDY __________________________________________________17 2 MATERIALS AND METHODS _________________________________________18 2.1 INSTRUMENTS AND CHEMICALS _________________________________________18 2.1.2 Materials ______________________________________________________19 2.1.3 Chemicals, Enzymes and Kits ______________________________________19 2.2 BACTERIAL STRAINS , PLASMIDS AND PRIMERS _____________________________20 2.2.1 Bacterial Strains ________________________________________________20 2.2.2 Plasmids_______________________________________________________21 2.2.3 Primers________________________________________________________22 2.3 MEDIA AND ADDITIVES _______________________________________________22 2.3.1 Media _________________________________________________________22 2.4 MICROBIOLOGICAL TECHNIQUES ________________________________________24 2.4.1 Sterilisation ____________________________________________________24 2.4.2 Cultivation of Bacteria____________________________________________24 2.4.3 Determination of Cell Density ______________________________________25 2.4.4 Storage of Bacterial Strains________________________________________25 2.4.5 Harvesting of Bacterial Cells_______________________________________25 2.4.6 Disruption of Cells_______________________________________________25 2.5 MOLECULAR BIOLOGICAL TECHNIQUES __________________________________25 2.5.1 Preparation of Plasmid DNA (Miniprep) _____________________________25 2.5.3 Determination of DNA Concentration________________________________26 2.5.4 Amplification of DNA Fragments by Polymerase Chain Reaction __________27 2.5.5 Restriction of DNA_______________________________________________27 2.5.6 Ligation of DNA-Fragments _______________________________________28 2.5.7 Transformation of Escherichia coli by the RbCl Method__________________28 2.5.8 DNA Sequence Analysis of Plasmid DNA _____________________________29 2.5.9 Protoplast Transformation of Bacillus megaterium Cells _________________29 2.6 PROTEIN BIOCHEMICAL METHODS _______________________________________31 2.6.1 Determination of Protein-Concentration______________________________31 2.6.2 Concentrating Protein Solutions ____________________________________31 2.6.3 Dialysis _______________________________________________________31 2.6.4 Electrophoretic Separation of Proteins (SDS-PAGE) ____________________31 2.6.5 Western-Blot ___________________________________________________33 IV 2.6.6 Immunodetection of Immobilised Proteins ____________________________33 2.6.7 Compliance of Anaerobic Conditions ________________________________34 2.6.8 UV-Vis Spectroscopy _____________________________________________34 2.7 DETERMINATION OF ESCHERICHIA COLI PROTOPORPHYRINOGEN IX OXIDASE ACTIVITY _____________________________________________________________________35 2.7.1 Principle of the Activity-Assay______________________________________35 2.7.2 Preparation of the Substrate Protoporphyrinogen IX ____________________35 2.7.2.1 Reduction of Protoporphyrin IX with Sodium Amalgam______________35 2.7.2.2 Palladium-catalysed Reduction of Protoporphyrin IX with Molecular Hydrogen_________________________________________________________35 2.7.3 Conditions for the Activity-Assay____________________________________35 2.8 PURIFICATION OF THE OXYGEN -INDEPENDENT PROTOPORPHYRINOGEN IX OXIDASE 37 2.8.1 Cultivation of Escherichia coli BL21 ( λDE3) __________________________37 2.8.2 Isolation of Membrane Fractions of Escherichia coli ____________________37 2.8.3 Purification of Membrane Fractions performing Sucrose Density Gradient Centrifugation_______________________________________________________37 2.8.4 Solubilisation of Membrane Proteins ________________________________37 2.8.5 Anion Exchange Chromatography___________________________________38 2.9 RECOMBINANT PRODUCTION , PURIFICATION AND CHARACTERISATION OF ESCHERICHIA COLI HEM G ___________________________________________________________38 2.9.1 Cultivation of Escherischia coli Cells for the Production of HemG _________38 2.9.2 Cultivation of Bacillus megaterium Cells for the Production of HemG ______38 2.9.3 Affinity-Purification of Recombinant HemG under Anaerobic Conditions____39 2.9.4 Mass-Spectrometry ______________________________________________39 2.9.5 Gel Permeation Chromatography of HemG ___________________________39 2.9.6 Cofactor Determination by High Performance Liquid Chromatography (HPLC) ___________________________________________________________________40 2.10 PRODUCTION AND PURIFICATION OF RECOMBINANT ESCHERICHIA COLI FUMARATE REDUCTASE ___________________________________________________________40 2.10.1 Cultivation of Cells _____________________________________________40 2.10.2 Preparation of Membrane Fractions and Solubilisation of Fumarate Reductase ___________________________________________________________________41 2.10.3 Purification of Fumarate Reductase by "Fast Performance Liquid Chromatography" (FPLC) _____________________________________________41 2.10.4 Determination of Fumarate Reductase-Activity _______________________42 2.11 PRODUCTION AND PURIFICATION OF RECOMBINANT ESCHERICHIA COLI CYTOCHROME BO OXIDASE ___________________________________________________________42 2.11.1 Recombinant Production of Cyo ___________________________________42 2.11.2 Preparation of Membrane Fractions and Solubilisation of Cyo ___________43 2.11.3 Purification of Cyo______________________________________________43 2.12 PRODUCTION AND PURIFICATION OF RECOMBINANT ESCHERICHIA COLI CYTOCHROME BD OXIDASE ___________________________________________________________44 2.12.1 Recombinant Production of Cyd ___________________________________44 2.12.2 Preparation of Membrane Fractions and Solubilisation of Cyd ___________44 2.12.3 Purification of Cyd______________________________________________44 2.13 PRODUCTION , ISOLATION AND CHARACTERISATION OF CHLOROBACULUM TEPIDUM OXYGEN -INDEPENDENT MG-PROTOPORPHYRIN IX MONOMETHYL ESTER OXIDATIVE CYCLASE BCH E ________________________________________________________45 2.13.1 Recombinant Production of BchE __________________________________45 2.13.2 Isolation of tagged BchE Cyclase __________________________________45 2.13.3 PreScission™ Protease Cleavage and second Chromatography __________46 V 2.13.4 Determination of Iron Content_____________________________________46 2.13.5 Chemical Reconstitution of Iron-Sulphur Clusters _____________________47 2.13.6 Cyclase Activity assays __________________________________________47 2.13.7 HPLC-Analysis of Cyclase Activity Test Products _____________________48 3 RESULTS AND DISCUSSION __________________________________________49 3.1 CHROMATOGRAPHIC PURIFICATION OF OXYGEN -INDEPENDENT PPO FROM ESCHERICHIA COLI ______________________________________________________49 3.2 PRODUCTION AND PURIFICATION OF RECOMBINANT ESCHERICHIA COLI HEM G _____54 3.2.1 Overproduction of recombinant HemG in Escherichia coli _______________54 3.2.2 Purification of HemG from