STRUCTURE-FUNCTION STUDIES OF XANTHINE OXIDOREDUCTASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By James Michael Pauff The Ohio State University 2008 Dissertation Committee: Professor Virginia Sanders, Advisor on Record Approved By Professor Russ Hille, Graduate Advisor Associate Professor Charles E. Bell _______________ Professor Allan Yates Advisor Integrated Biomedical Science Graduate Program ABSTRACT Xanthine oxidoreductase (XOR) is a 290 kDa molybdenum-containing enzyme that catalyzes the final two steps in human purine catabolism, taking hypoxanthine to xanthine and then on to uric acid. The enzyme exists as a homodimer, with each monomer possessing a catalytic active site that contains one molybdenum atom coordinated to a pterin ring via an enedithiolate side chain. Each monomer also contains two [2Fe2S] clusters and one equivalent of FAD. These centers form an electron transfer chain as electrons are passed sequentially from the molybdenum to the FAD via the iron-sulfur clusters. The molybdenum center cycles from MoVI to MoIV and back during catalysis, passing through an occasional MoV intermediate state. Electrons are passed from the flavin site out of the enzyme to either NAD+ or molecular oxygen. XOR is initially expressed as a dehydrogenase (xanthine dehydrogenase, XDH) with a strong preference for reducing NAD+ to NADH, + although it can reduce O2 under conditions of low NAD concentrations. Under certain conditions, XDH can be converted by oxidation and/or limited proteolysis to an oxidase form (xanthine oxidase, XO) that utilizes O2 exclusively as the terminal electron acceptor, thereby generating superoxide and other reactive oxygen species. The oxidase is formed primarily during hypoxia or ischemia, and the corresponding increase in reactive oxygen species has lead to investigation of the enzyme’s role in the pathophysiological mechanism of ischemia-reperfusion. The production of uric acid by XOR makes the enzyme a primary target in hyperuricemia, and it is this ii therapeutic intervention for which allopurinol has been used for over 60 years. The prevalence of XOR in mechanisms leading to some human diseases and the unique chemistry by which the enzyme catalyzes the production of uric acid makes this enzyme system an interesting subject of biochemical investigation. We have sought here to understand the structure and function of xanthine oxidoreductase, focusing on the nature of the molybdenum-containing active site as well as innate functional differences between the two forms of the enzyme. Our crystallographic and spectroscopic studies provide insight into the mechanism of XOR catalysis as well as the roles of XOR in human pathology. iii DEDICATION To my parents, Neil and Deborah Pauff, my brother Frank, to my fiancé Beth, and to my entire family iv ACKNOWLEDGMENTS I would first like to thank my parents for countless hours (and dollars) spent raising, teaching, correcting, guiding, and being patient with me through the years. Many lessons that I only realized much later have helped carry me and sustain me throughout my educational training. I would like to thank Dr. Russ Hille for his support, guidance, and patience during my research training in his laboratories. My sometimes sporadic ideas were met with encouragement and a willingness to let me go my own way, even though in some instances that meant a waste of resources. Thank you for allowing me the independence to learn from my own successes and mistakes, and to grow as a researcher along the way. I would also like to thank Craig Hemann for his efforts, consultation, and advice, particularly in the early stages of my dissertation work. You remain a tremendous resource. To Dr. Silke Leimkühler, thank you for the collaboration and efforts with the xanthine dehydrogenase system. To Dr. Charles Bell, thank you for the many hours of guidance and assistance with the crystallographic work. Without your advice we would not have any structures. I would also like to thank Dr. Allan Yates for his guidance and critical evaluation of my research and training as an MD/PhD student. To the entire Hille lab past and present, thank you for the consultations and the laughter as we worked alongside one another. v VITA 1981 Born – Bluffton, OH 2004 B.S. Chemistry, B.S. Biochemistry, B.A. Zoology Miami University Oxford, OH 2004 – Present Graduate Fellow, Graduate Research Associate Medical Scientist Program The Ohio State University Columbus, OH 2007 – 2008 Junior Specialist Department of Biochemistry University of California at Riverside Riverside, CA PUBLICATIONS Pauff, J.M., Hemann, C.F., Jünemann, N., Leimkühler, S., and Hille, R. (2007) The Role of Arginine 310 in Catalysis and Substrate Specificity in Xanthine Dehydrogenase from Rhodobacter capsulatus. J. Biol. Chem. 282, 12785 – 12790. Pauff, J.M., Zhang, J., Bell, C.E., and Hille, R. (2008) Substrate Orientation in Xanthine Oxidase: CRYSTAL STRUCTURE OF ENZYME IN REACTION WITH 2-HYDROXY-6-METHYLPURINE. J. Biol. Chem. 283, 4818 – 4824. Fields of Study: Biochemistry, Enzymology, Macromolecular X-ray Crystallography Major Field: Integrated Biomedical Science Graduate Program vi TABLE OF CONTENTS Abstract………………………………………………………………………………..ii Dedication…………………………………………………………………………….iv Acknowledgments……………………………..………………………………………v Vita……………………………………………………………………………………vi List of Figures…………………………………………………………………………x List of Tables…………………………………………………………………………xii Abbreviations………………………………………………………………………..xiii References…………………………………………………………………………..154 Chapters: 1. Introduction………………………………………………………………………..1 1.1. General Background………………………………………………………….1 1.1.1. Molybdenum…………………………………………………………..1 1.1.2. Molybdenum in biology and molybdenum-containing enzymes….......2 1.1.3. Xanthine oxidoreductase………………………………………………4 1.2. Xanthine oxidoreductase in physiology………………………………………5 1.3. Xanthine oxidoreductase in pathology………………………………………..7 1.3.1. Inhibition of XOR……………………………………………………..9 1.4. Structure of xanthine oxidoreductase………………………………………..12 1.5. Mechanism of catalysis and the oxidation-reduction chemistry chemistry of XOR…………………………………………………………………………15 1.5.1. The molybdenum-containing active site……………………………..17 1.5.2. Oxidative and reductive half-reactions of XOR catalysis……………22 1.5.3. Active site residues in the molybdenum site…………………………25 1.5.4. The pterin cofactor, iron-sulfur clusters, and the flavin site…………29 vii 1.6. Preparation and Isolation of Xanthine Oxidoreductase in the Present Studies 32 1.6.1. Bovine xanthine oxidase and xanthine dehydrogenase………………32 1.6.2. Xanthine dehydrogenase from Rhodobacter capsulatus……………..34 1.6.3. Activity-to-flavin determination and percentage of active enzyme….34 1.6.4. Inactive and inactivating XOR……………………………………….35 2. The Role of Arginine 310 in the Active Site of Xanthine Dehydrogenase from Rhodobacter capsulatus………………………………………………………….37 2.1. Introduction………………………………………………………………….37 2.2. Materials and Methods………………………………………………………42 2.2.1. Preparation of wild-type and mutant xanthine dehydrogenase from Rhodobacter capsulatus………………………………………………...42 2.2.2. Rapid-reaction experiments…………………………………………..43 2.2.3. Data acquisition and analysis………………………………………...43 2.3. Results and Discussion………………………………………………………44 3. Substrate Orientation in Xanthine Oxidase from Bos taurus, Crystal Structure with 2-hydroxy-6-methylpurine………………………………………………….50 3.1. Introduction………………………………………………………………….50 3.2. Materials and Methods………………………………………………………51 3.2.1. Materials……………………………………………………………...51 3.2.2. Preparation and isolation of bovine xanthine oxidase………………..51 3.2.3. Crystal growth, diffraction, and data acquisition…………………….52 3.2.4. Data processing and refinement……………………………………...55 3.3. Results and Discussion………………………………………………………56 3.3.1. Overall structure of xanthine oxidase with 2-hydroxy-6-methylpurine………………………………………...56 3.3.2. 2-hydroxy-6-methylpurine in the active sites of xanthine oxidase…..60 4. Xanthine and Lumazine in the Active Site of Xanthine Oxidase from Bos Taurus………………………………………………………………….64 4.1. Introduction………………………………………………………………….64 4.2. Materials and Methods………………………………………………………68 4.2.1. Materials……………………………………………………………...68 4.2.2. Preparation of bovine xanthine oxidase and the desulfo-form……….68 4.2.3. Crystal growth, diffraction, and data acquisition…………………….69 4.2.4. Data processing and refinement……………………………………...70 4.3. Results and Discussion………………………………………………………71 4.3.1. Xanthine oxidase with lumazine……………………………………..71 4.3.2. Desulfo-xanthine oxidase with xanthine……………………………..75 5. Activity of Xanthine Dehydrogenase versus Xanthine Oxidase in the Range of Physiological pH…………………………………………………………………91 5.1. Introduction………………………………………………………………….91 viii 5.2. Materials and Methods………………………………………………………96 5.2.1. Preparation of bovine xanthine oxidase and bovine xanthine dehydrogenase…………………………………………………………..96 5.2.2. Steady-state kinetics and construction of the pH profile for bovine XDH…………………………………………………………………….98 5.2.3. Consumption of oxygen by XDH in the absence of NAD+………….99 5.3. Results and Discussion……………………………………………………..100 5.3.1. pH profiles for the two forms of XOR……………………………...100 5.3.2. Oxygen consumption by XDH……………………………………...105 6. Inhibition Studies of Xanthine Oxidase………………………………………...109 6.1. Introduction………………………………………………………………...109 6.2. Materials and Methods……………………………………………………..119 6.2.1. Compounds and reagents……………………………………………119 6.2.2. Isolation