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Heme Biosynthesis: Structure–Activity Studies of Murine Ferrochelatase

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Heme Biosynthesis: Structure–Activity Studies of Murine Ferrochelatase Heme Biosynthesis: Structure–Activity Studies of Murine Ferrochelatase by Zhen Shi A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biochemistry and Molecular Biology College of Medicine University of South Florida Major Professor: Gloria Ferreira, Ph.D. Michael Barber, Ph.D. Huntington Potter, Ph.D. George Blanck, Ph.D. Peter Medveczky, Ph.D. Kenton Rodgers, Ph.D. Date of Approval: February 10, 2006 Keywords: porphyrin, iron, resonance Raman, continuous assay, random mutagenesis © Copyright 2006, Zhen Shi ACKNOWLEDGEMENTS I wish to express my deep gratitude to the members of my committee, Dr. Michael Barber, Dr. Huntington Potter, Dr. George Blanck, Dr. Peter Medveczky, and most of all, to Dr. Gloria Ferreria, for their consistent guidance, understanding and support throughout the course of my graduate work. I am thankful to all the professors and colleagues in the Department of Biochemistry, the members of Dr. Ferreira lab, as well as the lab group of Dr. John Shelnutt at the University of New Mexico for their help and advice during the studies. I would like to acknowledge the financial support from the American Heart Association for a pre-doctoral fellowship from 2000 to 2002, and from the Institute of Biomolecular Sciences for a graduate fellowship from 1998 to 2000. I wish to express my appreciation to Ms. Kathy Zahn and Susan Chapman at the Office of Research and Graduate Affairs for their continuous administrative assistance. I am forever grateful to my family for their enduring understanding and encouragement. NOTE TO THE READER The original of this document contains color that is necessary for understanding the data. The original dissertation is on file with the USF library in Tampa, Florida. TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v LIST OF ABBREVIATIONS vii ABSTRACT ix CHAPTER ONE INTRODUCTION 1 The importance of heme in biological systems 1 Enzymes in the heme biosynthetic pathway 7 Aminolevulinic acid synthase 7 Porphobilinogen synthase 13 Porphobilinogen deaminase 18 Uroporphyrinogen III synthase 22 Uroporphyrinogen III decarboyxlase 27 Coproporphyrinogen III oxidase 32 Protoporphyrinogen IX oxidase 37 Ferrochelatase 42 CHAPTER TWO MATERIALS AND METHODS 55 Materials 55 Experimental Methods 57 Media preparation for bacterial cultures 57 Competent cell preparation and bacterial transformation 58 Glycerol stock preparation for bacterial cells 59 Plasmid DNA purification 60 Sodium dodecyl sulfate-polyacylamide gel electrophoresis and protein concentration determination 60 Construction of a random library and genetic selection of functional ferrochelatase loop variants 61 Large-scale purification of the wild-type ferrochelatase and loop variants 65 UV-visible absorbance spectra of purified ferrochelatase 67 Metal content analysis of purified ferrochelatase 68 Pyridine-hemochromogen assay 68 Continuous assay of ferrochelatase activity 69 Steady-state kinetic analysis of the loop variants 71 i Homology modeling of murine ferrochelatase 72 Resonance Raman spectroscopy of porphyrin binding to the wild-type ferrochelatase and loop variants 72 Profiling the active variants by high-throughput protein purification 74 Liposomal binding assays of ferrochelatase variants 75 Inhibition assay of ferrochelatase by N-methyl protoporphyrin on agar plates 76 Quantification of N-methyl protoporphyrin binding to ferrochelatase by fluorescence quenching measurements 77 Transient kinetic analysis of ferrochelatase activity 78 Ligand binding pocket size measurement 79 Enzymatic activity of ferrochelatase in the absence of FeS cluster synthesis 80 Molecular mass assessment of purified ferrochelatase 81 Electron paramagnetic resonance spectroscopy of purified ferrochelatase 82 CHAPTER THREE RESULTS 83 Purification of recombinant ferrochelatase 83 Large-scale purification of wild-type ferrochelatase and loop variants 83 Small-scale purification of ferrochelatase loop variants 84 Developing a continuous assay for steady-state kinetic analysis of ferrochelatase 87 Characterization of the functional ferrochelatase loop variants 92 Biological selection of the active loop variants 92 Distribution of the functional amino acid substitutions 96 Steady-state kinetic analysis of the active loop variants 96 Homology modeling of wild-type murine ferrochelatase and selected loop variants 98 Interaction of ferrochelatase with mitochondrial membrane lipids 103 Resonance Raman spectroscopic analysis of porphyrin binding in the loop variants 105 Binding of substrate protoporphyrin to the variants 105 Binding of hemin to the variants 108 Binding of nickel-protoporphyrin to the variants 108 Inhibition of ferrochelatase by N-methyl protoporphyrin 112 Equilibrium binding of inhibitor to ferrochelatase 114 Kinetic pathway of inhibition 114 Size measurement of the active site pocket 119 FeS cluster and oligomeric assembly in ferrochelatase variants 121 UV-visible absorbance spectra of the variants 121 Metal content analysis 123 Dependence of enzymatic activity on FeS cluster synthesis 123 Subunit assembly of purified ferrochelatase 125 Electron paramagnetic resonance spectra of ferrochelatase 128 CHAPTER FOUR DISCUSSION 131 Continuous assay for ferrochelatase activity 131 Characterization of the active site loop variants 135 Resonance Raman spectroscopy analysis of ferrochelatase-induced porphyrin distortion 142 ii Inhibition of ferrochelatase by N-methyl protoporphyrin 151 FeS cluster assembly and oligomeric organization in ferrochelatase 155 REFERENCES 161 APPENDICES 205 ABOUT THE AUTHOR END PAGE iii LIST OF TABLES Table 1. Steady-state kinetic parameters of wild-type ferrochelatase and selected loop variants 99 Table 2. Comparison of the steady-state kinetic parameters of ferrochelatase determined by various assay methods 134 Table 3. Results of simulation for the low-frequency resonance Raman spectra of ferrochelatase-bound protoporphyrin 145 iv LIST OF FIGURES Figure 1. The heme biosynthetic pathway in animal cells 3 Figure 2. The heme degradation pathway in mammalian cells 6 Figure 3. The reaction catalyzed by ferrochelatase 43 Figure 4. Random mutagenesis of the ferrochelatase active site loop motif and biological selection of the functional variants 62 Figure 5. The UV-visible absorbance spectra of purified wild-type murine ferrochelatase and selected loop variants 85 Figure 6. SDS-polyacrylamide gel electrophoresis of purified ferrochelatase 86 Figure 7. The fluorescence spectra of protoporphyrin 88 Figure 8. Time course for the disappearance of protoporphyrin in the ferrochelatase-catalyzed reaction 89 Figure 9. Dependence of the initial rate of protoporphyrin consumption on ferrochelatase concentration 90 Figure 10. Determination of the steady-state kinetic parameters of wild-type murine ferrochelatase 91 Figure 11. Sequence alignment of the loop motif in ferrochelatase 93 Figure 12. Activity assessment and distribution of the number of the functional loop variants 95 Figure 13. Spectrum and frequency of amino acid substitutions in the functional loop variants 97 Figure 14. Molecular modeling of wild-type murine ferrochelatase and selected loop variants 101 v Figure 15. Interaction between ferrochelatase and the mitochondrial membrane lipids 104 Figure 16. The resonance Raman spectra of protoporphyrin incubated with ferrochelatase at a porphyrin-to-protein molar ratio of 0.1 106 Figure 17. The resonance Raman spectra of hemin incubated with ferrochelatase at a hemin-to-protein molar ratio of 0.1 109 Figure 18. The resonance Raman spectra of nickel-protoporphyrin incubated with ferrochelatase at a porphyrin-to-protein molar ratio of 0.1 111 Figure 19. The structural diagram of N-methyl protoporphyrin 113 Figure 20. The intrinsic fluorescence of ferrochelatase 115 Figure 21. Binding curves generated from protein fluorescence quenching measurements following titration with N-methyl protoporphyrin 116 Figure 22. Transient kinetic analysis of the ferrochelatase-catalyzed reaction 118 Figure 23. Dependence of the rate constants for ferrochelatase binding on N- methyl protoporphyrin concentration 120 Figure 24. The UV-visible absorbance spectra of purified wild-type ferrochelatase and variants 122 Figure 25. Dependence of ferrochelatase activity on FeS cluster synthesis 124 Figure 26. Molecular weight assessment of purified wild-type ferrochelatase and variants by gel filtration chromatography 126 Figure 27. Molecular size determination of purified wild-type ferrochelatase and variants by dynamic light scattering 127 Figure 28. EPR spectra of purified wild-type ferrochelatase and variants 129 Figure 29. Temperature-dependence of the EPR signal intensity for a purified ferrochelatase variant 130 Figure 30. Structural features of the murine ferrochelatase models 141 Figure 31. Three-dimensional views of the porphyrin-binding cleft in ferrochelatase 149 Figure 32. Position of the FeS cluster relative to the active site in ferrochelatase 156 vi LIST OF ABBREVIATIONS AIP, acute intermittent porphyria ALA, δ-aminolevulinic acid ALAS, δ-aminolevulinic acid synthase AONS, 8-amino-7-oxononanoate synthase Biotin-DHPE, N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt CEP, congenital erythropoietic porphyria CPO, coproporphyrinogen III oxidase ECL, enhanced chemiluminescence EPP, erythropoietic protoporphyria EPR, electron paramagnetic resonance FAD, flavin adenine dinucleotide HCP, hereditary coproporphyria Hemin, iron(III) protoporphyrin IX
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