INVESTIGATIONS INTO THE ENZYMATIC MECHANISM OF BACILLUS SUBTILIS OXALATE DECARBOXYLASE: AN ELECTRON PARAMAGNETIC RESONANCE APPROACH By UMAR TARIQ TWAHIR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2015 © 2015 Umar Tariq Twahir To my parents, uncle, aunt and beloved wife ACKNOWLEDGMENTS I would like to start with thanking God for providing me every opportunity, blessing and success in my life. Throughout my life there were many moments of uncertainty, not knowing what the next step would be for me. However, the one consistent thing in my life from the beginning was God, and I always knew everything would be as it should, needing only from me to give my all in everything I set out to do. During my time here at the University of Florida, the success and memories were due to a collection of individuals that included mentors, colleagues, collaborators, friends and family. First and foremost I would like to extend my deepest appreciations for my Ph.D. advisor, Professor Alexander Angerhofer. With your help, support and encouragement, I have grown into the scientist that I am today. You have always gone above and beyond to ensure that I was afforded all necessary opportunities for the advancement of my research and career. You truly are the model I look to when reflecting on the prospective future of my own career. You have encouraged me to present my research at multiple local, national and even international meetings, expanding my outlook, and for all of this I am truly grateful. I would like to also thank my committee members: Professors Gail Fanucci, David Wei, Benjamin Smith, and Yuri Sautin. I know that you each have multiple commitments and serve on many committees; however, you have taken the time to assist in my academic pursuits, and for that I am extremely thankful. I have also had the opportunity to take courses and collaborate with many of you and this has made the experience even more valuable. There has been a multitude of people that have assisted me throughout the years during my research, including a number of graduate and undergraduate students. 4 I would like to acknowledge my lab mate Justin Goodsell, for all his helpful conversations, our learning pow-wows in my office, all the cheese fry runs and coffee breaks. You have truly been a great friend through this process and extremely helpful with your brain full of facts. I have to also thank Cory Lee, and Laura Molina, undergraduate researchers, for their hard work and diligence in the lab that led to progression of the research. I would like to thank one of my really great friends, Corey Stedwell, for always being there when needed. We have shared many great moments together, both professionally and recreationally. We also had the opportunity to collaborate and publish a paper together. He has always served as a great confidant, and provided some great guidance from his experiences to help me throughout my graduate career. I would like to thank Matt Burg, a friend from day one of our graduate program together and one that will last, for his help and insight into all the biochemical techniques I annoyed him to teach me. He has been a great source of information for me throughout my career, and even harbored the start a new collaboration. I would like to also thank Adam Cismesia (lunchbox) for making sure there was always coffee and snacks every day to keep us going. I would like to thank Andrew Ozarowski, located at the National High Magnetic Field Laboratory (NHMFL), for his help and assistance in my many visits to the Mag Lab. I would like to also thank Todd Prox, and Brian Smith for all their assistance in the design and construction of all and any necessary components essential to my research. Behind all of my successes stands the team of people who have help mold me into the man I am today. I would like to give a special thanks to my parents, Mohamed and Bibi Twahir, and my uncle and aunt, Shaffieq Chace and Rajdai Singh for their unwavering 5 support through my entire education. I would like to thank them for making sure they did all they could for me to leave me short of nothing. I will forever be thankful to my wife Saudia Ally for always standing by me through this process, supporting me and keeping me grounded. She has been patient with me through all my moods, and crazy ideas, always keeping firm her willingness to follow me down any path I chose. Funding for this work was provided by the National Science Foundation under Grant CHE-1213440 and the National Institute of Health Instrumentation Grant NIH S10 RR031603. The high- field EPR spectra were recorded at the NHMFL, which is funded by the NSF through the Cooperative Agreement no. DMR-1157490, the State of Florida and the U.S. DOE. 6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 11 LIST OF SCHEMES ...................................................................................................... 15 LIST OF ABBREVIATIONS ........................................................................................... 16 ABSTRACT ................................................................................................................... 18 CHAPTER 1 BACKGROUND ...................................................................................................... 20 Introduction ............................................................................................................. 20 Oxalic Acid ....................................................................................................... 20 Bacillus subtilis Oxalate Decarboxylase Structure and Reactivity .................... 20 Research Objectives and Overview ........................................................................ 28 2 ELECTRON PARAMAGNETIC RESONANCE ....................................................... 30 Electron Paramagnetic Resonance for Studying Bacillus subtilis Oxalate Decarboxylase ..................................................................................................... 30 Spin Hamiltonian ..................................................................................................... 31 Electron and Nuclear Zeeman Effect ...................................................................... 32 Hyperfine Interaction - Nuclear Quadrupole Interaction .......................................... 34 Identification of Spin Trapped Adducts via the Hyperfine Interaction ...................... 37 Zero Field Splitting-Application to Mn(II) ................................................................. 39 High Field/Frequency EPR ..................................................................................... 42 Parallel Mode EPR-Application to Mn (III) S=2 ....................................................... 45 3 IDENTIFICATION OF SUPEROXIDE PRODUCTION UNDER TURNOVER CONDITIONS FOR Bacillus subtilis OXALATE DECARBOXYLASE ..................... 48 Introduction ............................................................................................................. 48 EPR Spin-Trapping ................................................................................................. 50 Experimental ........................................................................................................... 51 Materials ........................................................................................................... 51 Electron Paramagnetic Resonance .................................................................. 52 Mass Spectrometry .......................................................................................... 53 Hydrogen Peroxide Fluorescence Assay ......................................................... 54 Results .................................................................................................................... 54 7 Spin-trapping of Radicals Produced from Wild-Type OxDC ............................. 54 Spin trapping of Radicals Produced with T165V .............................................. 60 Quantitative Analysis of Radical Production ..................................................... 64 Discussion .............................................................................................................. 66 4 REDOX CYCLING OF THE METAL CENTERS OF Bacillus subtilis OXALATE DECARBOXYLASE: IDENTIFICATION OF Mn(III) ................................................ 73 Introduction ............................................................................................................. 73 Experimental ........................................................................................................... 76 Materials ........................................................................................................... 76 Electron Paramagnetic Resonance Studies ..................................................... 76 Enzyme Oxidation Experiments ....................................................................... 77 Results .................................................................................................................... 77 X-band Redox