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Use of Mathematical Modeling and Other Biophysical Methods For USE OF MATHEMATICAL MODELING AND OTHER BIOPHYSICAL METHODS FOR INSIGHTS INTO IRON-RELATED PHENOMENA OF BIOLOGICAL SYSTEMS A Dissertation by JOSHUA D. WOFFORD Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Paul A. Lindahl Committee Members, David P. Barondeau Simon W. North Vishal M. Gohil Head of Department, Simon W. North December 2018 Major Subject: Chemistry Copyright 2018 Joshua D. Wofford ABSTRACT Iron is a crucial nutrient in most living systems. It forms the active centers of many proteins that are critical for many cellular functions, either by themselves or as Fe-S clusters and hemes. However, Fe is potentially toxic to the cell in high concentrations and must be tightly regulated. There has been much work into understanding various pieces of Fe trafficking and regulation, but integrating all of this information into a coherent model has proven difficult. Past research has focused on different Fe species, including cytosolic labile Fe or mitochondrial Fe-S clusters, as being the main regulator of Fe trafficking in yeast. Our initial modeling efforts demonstrate that both cytosolic Fe and mitochondrial ISC assembly are required for proper regulation. More recent modeling efforts involved a more rigorous multi- tiered approach. Model simulations were optimized against experimental results involving respiring wild-type and Mrs3/4-deleted yeast. Simulations from both modeling studies suggest that mitochondria possess a “respiratory shield” that prevents a vicious cycle of nanoparticle formation, ISC loss, and subsequent loading of mitochondria with iron. Work has also been done in understanding an accumulation of Fe in stationary grown yeast cells. This accumulated Fe was found to be localized to the cell wall, and can be used as cells are metabolically reactivating by being placed into fresh media. A maethematical model has been developed to describe the metabolism of oxygen and nutrients in the autocatalytic production of active cells, with subsequent deactivation of cells as nutrients became limiting. E. coli have similar Fe contents relative to mitochondria, and they also appear to also employ a “respiratory shield”. This hypothesis was tested by either inhibiting respiratory complexes with CN, or by growing cells into a metabolically inactive stationary growth state. ii The generated nanoparticles were not associated with ferritins, which is surprising given that much of the literature claims that ferritin Fe makes up a large portion of cellular Fe. The iron content of murine hearts was also studied. Previous work from the Lindahl lab focused on murine brains and livers, which contain ferritin at young and old ages, while losing it in middle age. Hearts differ from these two organs, in that they mainly contain respiratory iron- sulfur clusters, and only gain ferritin as the mice approach old age. iii ACKNOWLEDGEMENTS I dedicate this work, and the time it went into it, to so many people in my life that have supported me in multiple ways that have made my doctorate possible. Firstly, I want to thank Almighty God, for creating the universe we live in, and giving humanity the minds capable of studying it and thinking His thoughts after him. I want to thank my beautiful wife, Elizabeth, who provides daily support and is my partner in life. I want to thank my parents, John and Jennifer, for bringing me up, and encouraging my academic pursuits, even when I didn’t want to. I want to thank my siblings, Jacob, Kaitlyn, and Joseph, for being there and giving me healthy competition. I want to thank the members of the Lindahl lab, past and present, for allowing me to learn from them and their assistance in completing this research. I want to thank my collaborators from groups within and without Texas A&M, for allowing me to assist and learn about other research going on around me. I also want to thank the many staff members of the Texas A&M Chemistry department that have assisted with running instruments, navigating paperwork, and fabricating parts that I need, with special thanks to Will Seward, Greg Wylie, Bill Merka, and Sandy Horton. Lastly, I want to thank my advisor, Paul Lindahl, for taking me into his lab and letting me pursue this crazy combination of chemistry, biology, and mathematics. iv CONTRIBUTORS AND FUNDING SOURCES This work was supported by a dissertation committee consisting of Professors Paul A. Lindahl, David P. Barondeau, and Simon W. North of the Department of Chemistry, and Professor Vishal M. Gohil of the Department of Biochemistry and Biophysics. This research was funded with grants from the National Institutes of Health and the Welch Foundation v NOMENCLATURE MB Mössbauer TB Blocking temperature BPS Bathophenanthroline disulfonate DFO Desferrioxamine ISC Iron-Sulfur Cluster CD Central Doublet CIA Cytosolic Iron-sulfur assembly complex Isomer shift EQ Quadrupole splitting Linewidth EPR Electron paramagnetic resonance LMM Low molecular mass Mrs3/4 cells WT Wild type NHHS Nonheme high-spin FTS Flow-through solution LC-ICP-MS Liquid chromatography with on-line detection by an inductively coupled plasma mass spectrometer LIP Labile iron pool OXPHOS Oxidative phosphorylation TCA Tricarboxylic acid (cycle) vi ROS Reactive oxygen species ODE Ordinary differential equation vii TABLE OF CONTENTS Page ABSTRACT ………………………………………………………………………………...ii ACKNOWLEDGMENTS.………………………………………………………………….iv CONTRIBUTORS AND FUNDING SOURCES………………………………………..….v NOMENCLATURE………..…………………………………………………………..…...vi TABLE OF CONTENTS……………………...……………………………………..…....viii LIST OF FIGURES………………………………………………………………..……....xiv LIST OF TABLES………………………………………………………………...……...xviii CHAPTER I INTRODUCTION ………………………..……………………………………1 Introduction and Instrumentation……………………………………………………..1 Labile Metal Pools……………………………………………………………………4 Dangers of Fe and the Need for Regulation…………………………………………..5 Principles of Mössbauer Spectroscopy………………………………………………..8 Coordination Chemistry of Fe……………………………………………………….10 Types of Fe evident in biological samples…………………………………………...11 Aggregated Ferric ions……………………………………………………………….13 Online and Offline LC-ICP-MS……………………………………………………...14 Mathematical Modeling………………………………………………………………15 Transition to Projects…………………………………………………………………16 Modeling Efforts to Determine Regulation Species in S. cerevisiae…………………17 Recovery of Mitochondrial Iron Contents in Mrs3/4ΔΔ Yeast Cells…………………21 viii Multi-Tiered Model…………………………………………………………………..22 Yeast cell wall acts to store iron for metabolically deactivating cells……………….24 E. coli possess a respiratory shield similar to yeast mitochondria…………………...24 Additional Projects…………………………………………………………………...28 CHAPTER II MATERIALS AND METHODS……………………………………………..29 Fe stock prep and Media components………………………………………………..29 Minimal media……………………………………………………………………….30 Rich Media…………………………………………………………………………...31 Cell Plates…………………………………………………………………………….31 Cell Stocks……………………………………………………………………………32 Cell Growth…………………………………………………………………………...33 Mitochondria Isolation………………………………………………………………..34 Mössbauer Spectroscopy……………………………………………………………...36 Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry………….39 Electron Paramagnetic Resonance (EPR) Spectroscopy……………………………...40 Mathematical Modeling……………………………………………………………….41 CHAPTER III MITOCHONDRIAL IRON-SULFUR CLUSTER ACTIVITY AND CYTOSOLIC IRON REGULATE IRON TRAFFIC IN SACCHAROMYCES CEREVISIAE…………………………………………………………………………………42 Summary………………………………………………………………………………42 Introduction……………………………………………………………………………43 Materials and Methods………………………………………………………………...52 Results…………………………………………………………………………………63 ix Discussion……………………………………………………………………………..70 Author Contributions…………………………………………………………………..74 Acknowledgement……………………………………………………………………..74 Supplemental Material…………………………………………………………………74 CHAPTER IV RECOVERY OF MRS3ΔMRS4Δ SACCHAROMYCES CEREVISIAE CELLS UNDER IRON-SUFFICIENT CONDITIONS AND THE ROLE OF FE 580……………………………………………………………………..…90 Summary……………………………………………………………………………….90 Introduction…………………………………………………………………………….91 Experimental Procedures……………………………………………………………….94 Results………………………………………………………………………………….99 Discussion……………………………………………………………………………..123 Acknowledgements……………………………………………………………………129 CHAPTER V A MATHEMATICAL MODEL OF IRON IMPORT AND TRAFFICKING IN WT AND MRS3/4 YEAST CELLS…………………………….....130 Summary……………………………………………………………………………..130 Introduction…………………………………………………………………………..131 Methods and Results…………………………………………………………………139 Conclusions…………………………………………………………………………..168 Funding……………………………………………………………………………….171 Acknowledgements…………………………………………………………………..171 Supplemental Material………………………………………………………………..171 x CHAPTER VI FERRIC IONS ACCUMULATE IN THE WALLS OF METABOLICALLY INACTIVATING SACCHAROMYCES CEREVISIAE CELLS AND ARE REDUCTIVELY MOBILIZED DURING REACTIVATION……..…....173 Summary……………………………………………………………………………….173 Introduction…………………………………………………………………………….174 Methods………………………………………………………………………………...178 Results and Discussion…………………………………………………………………181 Conclusions…………………………………………………………………………….223 Acknowledgements…………………………………………………………………….228 CHAPTER VII CHARACTERIZATION OF THE LABILE IRON POOL IN ESCHERICHIA COLI, AND A “RESPIRATION SHIELD” THAT PROTECTS IT FROM O2 DAMAGE………………………………………………………….….………......229 Summary……………………………………………………………………………....229 Introduction…………………………………………………………………………....230 Results…………………………………………………………………………………235 Discussion……………………………………………………………………………..261
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