STRUCTURE AND FUNCTION OF IRON-SULFUR CLUSTER BIOSYNTHESIS PROTEINS AND THE INFLUENCE OF OXYGEN LIGATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sheref S. Mansy, B.S. The Ohio State University 2003 Dissertation Committee: Dr. James A. Cowan, Adviser Dr. Ming-Daw Tsai Dr. Venkat Gopalan Adviser Dr. Mark P. Foster Ohio State Biochemistry Program ABSTRACT Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU is believed to be involved in iron-sulfur cluster delivery to apo iron-sulfur proteins. However, most of the evidence supporting the function of IscU stems from genetic and cellular biological studies. Therefore, we set out to biochemically characterize human, yeast, and prokaryotic IscU proteins. A variety of spectroscopic techniques were used to evaluate IscU including, UV-visible absorption, Mössbauer, near- and far- UV circular dichroism, mass spectrometry, atomic absorption, and nuclear magnetic resonance. Herein we demonstrate that IscU proteins coordinate reductively labile [2Fe-2S]2+ centers and are capable of mediating delivery of intact cluster to apo protein targets. Furthermore, extensive structural and dynamic data of a hyperthermophilic homologue, Thermotoga maritima IscU, revealed that IscU adopts a mobile molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. Such a dynamic molecule may allow for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and ii recognition and delivery of that cluster to a target protein. Additionally, we utilized X- ray crystallography to elucidate a high resolution structure of an oxygen ligated [4Fe-4S] high potential iron protein. iii To my mother and father, Wafeya and Samir iv ACKNOWLEDGMENTS I thank my adviser, J. A. Cowan, for expert intellectual guidance and for providing a highly stimulating and professional research environment. I am also grateful to those that have served my on dissertation committee, Ming-Daw Tsai, Venkat Gopalan, Donald H. Dean, and Mark P. Foster. One of the enjoyable aspects of research is the opportunity to collaborate with other laboratories and researchers. I thank Russ Hille and Craig Hemann for EPR and resonance Raman experiments, Kari Green-Church for mass spectrometry, Jon-David Sears and Don Ordaz for fermentations, In-Ja L. Byeon for NMR training, M. Sundaralingam and Yong Xiong for X-ray crystallography, Kristene K. Surerus for Mössbauer spectroscopy (University of Wisconsin, Milwaukee), and Marco Sola for electrochemistry (University of Modena and Reggio Emilia, Italy). My time in Italy would have been far less productive and enjoyable had it not been for Cristina Del Bianco, Rainer Küemmerle, Mariapina D'Onofrio, Karel Kubicek, and Fiorenza Cramaro. Also, I value the opportunities to have worked with people in the Cowan laboratory, including Anjali Patwardhan, Gong Wu, Shu-pao Wu, Taejin Yoon, Matthew W. Foster, Chun-An Chen, Manunya Nuth, and Sreedhara Alavattam. v I greatly appreciate the funding that I received from the Chemistry-Biology Interface Program, the Ohio State Biochemistry Program, and the Ohio State University. vi VITA February 17, 1975 Born, Eugene, Oregon, USA 1997 B.S. Microbiology, Ohio State University 1997 American Heart Association Undergraduate Student Summer Fellowship, Ohio State University 1997-1998 Graduate Research Associate, Ohio State University 1998-2001 NIH Chemistry - Biology Interface Training Grant, Ohio State University 2001-2002 Graduate Research Associate, Ohio State University 2003-present Presidential Fellowship, Ohio State University PUBLICATIONS 1. Sheref S. Mansy, John S. Olson, Gonzalo Gonzalez, and Marie A. Gilles- Gonzalez (1998) Imidazole is a Sensitive Probe of Steric Hindrance in the Distal Pockets of Oxygen-Binding Heme Proteins. Biochemistry 37, 12452-12457. 2. Weimin Gong, Bing Hao, Sheref S. Mansy, Gonzalo Gonzalez, Marie A. Gilles- Gonzalez, and Michael K. Chan. (1998) Structure of a Biological Oxygen Sensor: A New Mechanism for Heme-Driven Signal Transduction. Proc. Natl. Acad. Sci. USA 95, 15177-15182. vii 3. Matthew W. Foster, Sheref S. Mansy, Jungwon Hwang, James E. Penner-Hahn, Kristene K. Surerus, and J. A. Cowan. (2000) A Mutant Human IscU Protein Contains a Stable [2Fe-2S]2+ Center of Possible Functional Significance. J. Am. Chem. Soc. 122, 6805-6806. 4. Sheref S. Mansy, Yong Xiong, Craig Hemann, Russ Hille, M. Sundaralingam, and J. A. Cowan. (2002) Crystal Structure and Stability Studies of C77S HiPIP: A Serine Ligated [4Fe-4S] Cluster. Biochemistry 41, 1195-1201. 5. Gong Wu, Sheref S. Mansy, Shu-pao Wu, Kristene K. Surerus, Matthew W. Foster and J. A. Cowan. (2002) Characterization of an Iron-sulfur Cluster Assembly Protein (ISU1) from Schizosaccaromyces pombe. Biochemistry 41, 5024-5032. 6. Gong Wu, Sheref S. Mansy, Craig Hemann, Russ Hille, Kristene K. Surerus, and J. A. Cowan. (2002) Iron-Sulfur Cluster Biosynthesis: Characterization of Schizosaccaromyces pombe Isa1 J. Biol. Inorg. Chem. 7, 526-532. 7. Sheref S. Mansy, Gong Wu, Kristene K. Surerus, and J. A. Cowan. (2002) Iron- Sulfur Cluster Biosynthesis: Thermotoga maritima IscU is a Structured Iron-Sulfur Cluster Assembly Protein. J. Biol. Chem. 277, 21397-21404. 8. Ivano Bertini, J. A. Cowan, Cristina Del Bianco, Claudio Luchinat, and Sheref S. Mansy. (2003) Thermotoga maritima IscU. Structural Characterization and Dynamics of a New Class of Metallochaperone. J. Mol. Biol. 331, 907-924. FIELD OF STUDY Major Field: Biochemistry viii TABLE OF CONTENTS Abstract........................................................................................................................... ii Dedication....................................................................................................................... iv Acknowledgments......................................................................................................... v Vita................................................................................................................................. vii List of Tables................................................................................................................. xiv List of Figures................................................................................................................ xv List of Abbreviations..................................................................................................... xix Chapters: 1 Introduction..........................................................................................................1 1.1 Iron-Sulfur Cluster Proteins..........................................................................1 1.1.1 Types of Protein Bound Iron-Sulfur Clusters...............................1 1.1.2 Importance of Iron-Sulfur Cluster Proteins..................................3 1.1.3 Redox Function.............................................................................4 1.1.4 Enzymatic.....................................................................................4 1.1.5 Sensing.........................................................................................5 1.2 Metallochaperones.......................................................................................6 1.2.1 Metallochaperone Function.........................................................6 1.2.2 Copper Metallochaperone Structure............................................7 1.2.3 Non-Copper Metallochaperones..................................................9 1.2.4 Necessity of Iron-Sulfur Cluster Metallochaperones...................9 1.3 Iron-Sulfur Cluster Assembly.....................................................................10 1.3.1 Nitrogen Fixation........................................................................10 1.3.2 Identification of Bacterial ISC Proteins......................................11 ix 2 Characterization of Human and Yeast ISC Proteins........................................13 2.1 Introduction...............................................................................................13 2.2 Materials and Methods..............................................................................16 2.2.1 General Chemicals....................................................................16 2.2.2 Protein Expression....................................................................16 2.2.2 Human ISU Purification...........................................................17 2.2.3 S. pombe ISU1 and ISA1 Purification and Reconstitution.......18 2.2.4 Wild Type S. pombe Ferredoxin Purification...........................19 2.2.5 Cys to Ser Substituted S. pombe Ferredoxin Purification.........20 2.2.6 Human Ferredoxin Purification................................................20 2.2.7 Mutagenesis..............................................................................21 2.2.8 UV-Visible Absorption Spectroscopy......................................24 2.2.9 Circular Dichroism...................................................................24 2.2.10 Native Polyacrylamide Gel Electrophoresis.............................24 2.2.11 EDC Cross-Linking..................................................................25 2.3 Results and Discussion..............................................................................25