Structure and Function of Cytochrome c Nitrite Reductase Dissertation submitted to Fakultät für Biologie, Universität Konstanz for the degree of Doctor of Natural Sciences by Dipl.-Biol. Oliver F. Einsle Max-Planck-Institut für Biochemie Abteilung Strukturforschung D-82152 Martinsried Konstanz, December 1999 Examiner: Prof. Dr. Peter M.H. Kroneck Coexaminer: Prof. Dr. Robert Huber Dissertation der Universität Konstanz Datum der mündlichen Prüfung: 25. 2. 2000 Referenten: Prof. Dr. P. M. H. Kroneck Prof. Dr. R. Huber Dedicated to my father, Dr. Ulrich K. Einsle † 24. 12. 1996 TABLE OF CONTENTS 5 Table of Contents 1 ZUSAMMENFASSUNG....................................................................................................... 7 2 SUMMARY ............................................................................................................................ 9 3 INTRODUCTION ...............................................................................................................11 3.1 THE BIOGEOCHEMICAL NITROGEN CYCLE .......................................................................... 11 3.2 DISSIMILATORY NITRATE REDUCTION ................................................................................ 12 3.2.1 Physiology ............................................................................................................... 13 3.2.2 Occurence ................................................................................................................ 16 3.2.3 Regulation............................................................................................................... 17 3.2.4 Dissimilatory Nitrate Reductase............................................................................ 17 3.3 NITRITE REDUCTASES ........................................................................................................ 19 3.3.1 Copper-containing Nitrite Reductase..................................................................... 22 3.3.2 Cytochrome cd1 Nitrite Reductase.......................................................................... 22 3.3.3 Assimilatory Siroheme Nitrite Reductase.............................................................. 24 3.3.4 Dissimilatory Cytochrome c Nitrite Reductase...................................................... 24 3.4 PROTEIN CRYSTALLOGRAPHY ............................................................................................. 25 3.5 SCOPE OF THIS WORK......................................................................................................... 26 4 MATERIALS AND METHODS ........................................................................................27 4.1 MICROBIOLOGY .................................................................................................................. 27 4.1.1 Growth of S. deleyianum and W. succinogenes ..................................................... 27 4.1.2 Growth of Escherichia coli ..................................................................................... 28 4.2 MOLECULAR BIOLOGY........................................................................................................ 28 4.2.1 Preparation of Bacterial Genomic DNA ................................................................ 28 4.2.2 Preparation of Plasmid DNA ................................................................................. 29 4.2.3 Sequencing Strategy ............................................................................................... 30 4.2.3.1 Polymerase Chain Reaction .............................................................................. 30 4.2.3.2 Design of PCR Primers...................................................................................... 30 4.2.3.3 Inverse PCR....................................................................................................... 30 4.2.3.4 Anchor-ligated Genome Walking...................................................................... 31 4.3 PROTEIN BIOCHEMISTRY .................................................................................................... 31 4.3.1 Purification of Nitrite Reductase ........................................................................... 31 4.3.2 Sequencing of cytochrome c3................................................................................... 32 4.4 CRYSTALLOGRAPHY ............................................................................................................ 33 4.4.1 Theoretical Background ......................................................................................... 33 4.4.1.1 Crystal Growth .................................................................................................. 33 4.4.1.2 Crystals .............................................................................................................. 34 4.4.1.3 X-ray Diffraction by Crystals ............................................................................ 35 4.4.1.4 The Electron Density Function......................................................................... 37 4.4.1.5 The Phase Problem............................................................................................ 39 4.4.1.6 MAD and Metalloproteins................................................................................. 40 4.4.2 Crystal Growth and Data Collection ..................................................................... 44 4.4.2.1 Crystallization Experiments ............................................................................. 44 4.4.2.2 Crystal Seeding.................................................................................................. 45 4.4.2.3 Cryocrystallography .......................................................................................... 46 4.4.2.4 Substrate Complexes......................................................................................... 47 4.4.2.5 Data Collection .................................................................................................. 47 4.4.3 Graphical Representation ...................................................................................... 48 4.4.4 Database Searches.................................................................................................. 48 5 RESULTS ............................................................................................................................50 5.1 A PROBE FOR NRFA............................................................................................................ 50 5.2 FURTHER NRF OPERON SEQUENCES ................................................................................... 51 5.3 ORGANIZATION OF THE NRF OPERON .................................................................................. 52 5.3.1 The nrf Promoter .................................................................................................... 53 5.3.2 NrfH; Electron Carrier and Membrane Anchor .................................................... 55 6 TABLE OF CONTENTS 5.3.3 NrfA; Cytochrome c Nitrite Reductase................................................................... 57 5.3.4 NrfI; Heme Transport............................................................................................. 59 5.3.5 NrfJ; A Crippled Thioredoxin? .............................................................................. 60 5.3.6 The nrf Operon of Porphyromonas gingivalis ....................................................... 62 5.4 THE CRYSTAL STRUCTURE OF NRFA................................................................................... 63 5.4.1 Purification of Nitrite Reductase for Crystallization ............................................ 63 5.4.2 Crystallization ........................................................................................................ 65 5.4.3 Nitrite Reductase from S. deleyianum................................................................... 68 5.4.3.1 MAD Data Collection......................................................................................... 68 5.4.3.2 Self-rotation Function ....................................................................................... 69 5.4.3.3 Location of the Iron Positions ........................................................................... 69 5.4.3.4 Phase Calculations ............................................................................................ 70 5.4.3.5 Electron Density Modifications......................................................................... 71 5.4.3.6 Model Building and Refinement ....................................................................... 71 5.4.3.7 A Second Crystal Form...................................................................................... 73 5.4.4 Nitrite Reductase from W. succinogenes ................................................................ 73 5.4.4.1 Data Collection and Molecular Replacement ................................................... 73 5.4.4.2 Model Building and Refinement ....................................................................... 74 5.4.5 Description of the Structure ................................................................................... 75 5.4.5.1 Overall Structure............................................................................................... 75 5.4.5.2 Differences between S. deleyianum and W. succinogenes................................ 77 5.4.5.3 Heme Cofactor Arrangement ...........................................................................