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Biogenesis of Iron-Sulfur Clusters and Intracellular Iron Metabolism

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Biogenesis of Iron-Sulfur Clusters and Intracellular Iron Metabolism Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2009 Biogenesis of iron-sulfur clusters and intracellular iron metabolism Jacob Philip Bitoun Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Bitoun, Jacob Philip, "Biogenesis of iron-sulfur clusters and intracellular iron metabolism" (2009). LSU Doctoral Dissertations. 670. https://digitalcommons.lsu.edu/gradschool_dissertations/670 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. BIOGENESIS OF IRON-SULFUR CLUSTERS AND INTRACELLULAR IRON METABOLISM A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences By Jacob Philip Bitoun B.S., Louisiana State University, 2004 August, 2009 ACKNOWLEDGEMENTS In retrospect, it has become evident that I would not have been able to complete this journey through graduate school without the help of many people. I would like to thank my major professor, Dr. Huangen Ding, for his patience and mentorship. Only now, as a Ph.D candidate, do I realize how much time and commitment he has invested into me and my research. I would like to thank my committee members: Dr. John R. Battista, Dr. Joomyeong Kim, Dr. Yong-Hwan Lee, and Dr. Donghui Zhang for their insightful discussions. I would like to thank other professors who impacted my time at LSU: Dr. Jacqueline Stephens, Dr. Simon Chang, Dr. Greg Pettis, Dr. Grover Waldrop, Dr. Jayne Garno and Dr. Megan Macnaughtan. I also would like to thank Dr. Erin Hawkins. I would like to thank my colleagues in the Ding Lab: Binbin Ren, Juanjuan Yang, Dr. Xuewu Duan, Guoqiang Tan, and Huang Hao for their insights into certain experiments, discussions in the lab, and friendship. Also I would like to thank the numerous graduate and undergraduate students who have helped over the years. I would like to thank my family for their encouragement and support. Of course, I would not be in this position without the love from my mother, Ann Woods. I would like to thank my brother Joshua Bitoun for supplying me with endless white noise while conducting my experiments. I would like to thank Philip Fricano Jr., Inez Fricano, Roy Woods, and Jacques Bitoun for all listening to me when I needed support. I would like especially thank Abby Gandolfi for her support and encouragement. Without her it would have been a more challenging road. Thank you for always believing in me, and challenging me. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ......................................................................................................... ii LIST OF TABLES .........................................................................................................................v LIST OF FIGURES ..................................................................................................................... vi LIST OF SCHEMES ................................................................................................................. viii LIST OF ABBREVIATIONS ..................................................................................................... ix ABSTRACT .................................................................................................................................. xi CHAPTER 1: LITERATURE REVIEW ...................................................................................1 IRON IN BIOLOGY....................................................................................................................1 HISTORICAL PERSPECTIVE ...................................................................................................2 IRON-SULFUR CLUSTER VERSATILITY ..............................................................................6 IRON-SULFUR CLUSTER BIOGENESIS ..............................................................................10 IRON ACQUISTION AND STORAGE ....................................................................................13 OXIDATIVE AND NITROSATIVE STRESS ..........................................................................17 STATEMENT OF RESEARCH PROBLEMS ..........................................................................22 STATEMENT OF RESEARCH OBJECTIVES ........................................................................24 REFERENCES ...........................................................................................................................25 CHAPTER 2: CHARACTERIZATION OF A HUMAN IRON-SULFUR CLUSTER ASSEMBLY PROTEIN, HISCA ................................................................................................48 INTRODUCTION ......................................................................................................................48 MATERIALS AND METHODS ...............................................................................................50 RESULTS ...................................................................................................................................53 DISCUSSION ............................................................................................................................59 REFERENCES ...........................................................................................................................61 CHAPTER 3: FERRITIN A ACTS AS AN IRON RESERVOIR FOR IRON-SULFUR CLUSTER RE-ASSEMBLY AFTER OXIDATIVE STRESS ................................................66 INTRODUCTION .....................................................................................................................66 MATERIALS AND METHODS ...............................................................................................68 RESULTS ...................................................................................................................................71 DISCUSSION ............................................................................................................................83 REFERENCES ...........................................................................................................................85 CHAPTER 4: NSRR, A TRANSCRIPTIONAL REPRESSOR OF THE NITROSATIVE STRESS RESPONSE, RESPONDS TO NITRIC OXIDE AND CELLULAR REDOX STATUS ........................................................................................................................................91 INTRODUCTION .....................................................................................................................91 MATERIALS AND METHODS ...............................................................................................93 iii RESULTS ..................................................................................................................................99 DISCUSSION ..........................................................................................................................116 REFERENCES .........................................................................................................................118 CHAPTER 5: CONCLUSIONS ..............................................................................................125 SUMMARY .............................................................................................................................125 FUTURE RESEARCH GOALS ..............................................................................................129 REFERENCES ........................................................................................................................133 APPENDIX: PERMISSION TO INCLUDE PUBLISHED WORK ....................................139 VITA............................................................................................................................................142 iv LIST OF TABLES 1.1 E. coli K-12 [Fe-S] Proteins or [Fe-S] Cluster Assembly Proteins ....................................3 2.1 Oligonucleotide Sequences of the hIscA Primers ..............................................................50 3.1 Oligonucleotide Sequences of the ftnA Primers.................................................................69 4.1 Oligonucleotide Sequences of the nsrR Primers ................................................................94 4.2 Oligonucleotide Sequences of the Primers for Site Directed Mutagenesis of NsrR ..........95 4.3 Oligonucleotide Sequences of the hmpA Primers ..............................................................98 v LIST OF FIGURES 1.1 Iron-Sulfur Clusters .............................................................................................................7 1.2 The isc Operon of E. coli ..................................................................................................12 1.3 Reduction of Molecular Oxygen ........................................................................................18 1.4 Reaction Catalyzed by Nitric Oxide Synthase ..................................................................20 2.1 hIscA is the Human Homolog of E. coli IscA ..................................................................53
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