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View, J Med Genet 37 (2000) 1-8 INSIGHTS ON IRON-SULFUR CLUSTER ASSEMBLY DONOR PROTEINS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Eric M. Dizin, M.S. ٭ ٭ ٭ ٭ ٭ The Ohio State University 2008 Dissertation Committee: Approved by Professor James Cowan, Advisor Professor Claudia Turro Professor Thomas J. Magliery Advisor Chemistry Graduate Program ABSTRACT Iron-sulfur clusters are an important class of prosthetic group involved in electron transfer, enzyme catalysis, and regulation of gene expression. Their biosynthesis requires a complex machinery located within the mitochondrion since free iron and sulfide are extremely toxic to the cell. This research has focused on the three central proteins dedicated to the assembly: a cysteine desulfurase, Nfs1, an iron donor protein, frataxin, and an iron-sulfur cluster scaffold protein, Isu1. Human Nfs1, a PLP dependent enzyme, catalyzes the decomposition of cysteine to alanine and forms a persulfide bond with a conserved cysteine residue. To date, Nfs1 has only been partially characterized. Furthermore, its hyperproduction relies on yeast organisnm, Pichia pastoris, which is cumbersome and leads to quite low yields. Therefore, we undertook to design a bacterial expression system by cloning and overexpressing the gene in different E. coli strain. This enabled only a partial characterization of the cysteine desulfurase. Besides being an iron donor for iron-sulfur cluster assembly, frataxin has also been implicated in heme biosynthesis, and in iron storage in the mitochondrion with reported ferroxidase activity. We decided to further investigate its ability to bind to other metals, ii such as magnesium, calcium, and zinc, and also studied its ferroxidase activity as a mature and as a truncated protein. We concluded that frataxin has negligible ferroxidase activity, comparable to iron, and quite distinct from ferritin. Moreover frataxin binds zinc besides iron, but with a different stoichiometry. iii Dedicated to Odile, Henri, Pierre, Colette, Dominique and Claude iv ACKNOWLEDGMENTS I would like to express my gratitude to my advisor, Professor James A. Cowan, without whom I could not have completed this dissertation. Prof. Cowan made it possible for me to pursue my PhD in an intellectually stimulating and cheerful environment. With his broad scientific knowledge, patience, and enthusiasm, Prof. Cowan was an excellent guide. My professional development at OSU was fostered by Professors Martin Caffrey and Dehua Pei. Prof. Caffrey taught me thoroughness, and Prof. Pei was a model of scientific inquiry. Thanks to both of them, and to all my teachers in the Department of Chemistry. Thanks also to Valerie Wright and Prof. Thomas Clanton for their attentive help with the oximetry device, to Prof. Timothy Stemmler for his help with the EXAFS samples and protein NMR, to the CCIC for the mass spectrometry, and to Dr. Gordon Renkes for the circular dichroism. And thanks to the Chemistry Department for supporting me all these years. I would also like to thank my labmates: Wen-I Luo, Jeff Joyner, Dr. Chun-An Chen, Dr. Nikhil Gokhale, and Dr. Manunya Nuth. v Last but not least, I thank for their warm support my parents, Claude and Dominique, my grandparents, Henri and Odile, Anne-Sophie, Bruce, Christopher, Nicolas and Rob. vi VITA September 29, 1976 Born – Sèvres, France 2000-2001 Technician, Industrial Coating Department, Cognis GmbH, Düsseldorf 2003 M.S. Chemistry, ESCPE, Lyon, France 2001 – present Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Kodapalli, K.C.; Dizin, E.; Cowan, J.A.; Stemmler, T.L. “Letter to the Editor: 1 H, 13 C and 15 N resonance assignments for full length mature human frataxin”, manuscript in preparation for J. Biomol. NMR. 2. Huang, J.; Dizin, E.; Cowan, J. A. Mapping Iron Binding Sites on Human Frataxin. Implications for Cluster Assembly on the ISU Fe-S Cluster Scaffold Protein. J. Biol. Inorg. Chem. 2008, 13, 0000. 3. Yoon, T.; Dizin, E.; Cowan, J. A. N-terminal iron-mediated self cleavage of human frataxin: regulation fo iron binding and complex formation with target proteins. J. Biol. Inorg. Chem. 2007, 12(4), 535-42. 4. Zhu, J.; Hu, X.; Dizin, E.; Pei, D. Catalytic mechanism of S- ribosylhomocysteinase (LuxS): Direct observation of ketone intermediates by 13C NMR spectroscopy. J. Am. Chem. Soc. 2003, 125(44), 13379-81. vii 5. Zhu, J.; Dizin, E.; Hu, X., Wavreille, A.-S.; Park, J.; Pei, D. S- Ribosylhomocysteinase (LuxS) is a mononuclear iron protein. Biochemistry. 2003, 42(16), 4717-26. FIELDS OF STUDY Major Field: Chemistry Specialization: Biological Chemistry viii TABLE OF CONTENTS Page Abstract .......................................................................................................................... ii Dedication ..................................................................................................................... iv Acknowledgments ...........................................................................................................v Vita .............................................................................................................................. vii List of Tables............................................................................................................... xiii List of Figures ..............................................................................................................xiv Abbreviations ............................................................................................................. xvii Chapters: 1. Introduction 1 1.1 Iron.....................................................................................................................2 1.1.1 Reactivity ................................................................................................2 1.1.2 Distribution .............................................................................................5 1.1.3 Uptake ....................................................................................................5 1.1.4 Transport ................................................................................................8 1.1.5 Import .....................................................................................................9 1.1.6 Storage ....................................................................................................9 1.1.7 Regulation of iron metabolism .............................................................. 12 1.1.8 Iron-sulfur clusters cofactors ................................................................. 14 1.2 Iron-sulfur clusters biogenesis .......................................................................... 19 ix 1.2.1 Iron import in mitochondria .................................................................. 19 1.2.2 Frataxin ................................................................................................. 19 1.2.3 Mitochondrial ferritin ............................................................................ 23 1.2.4 Cysteine desulfurase Nfs1 ..................................................................... 25 1.2.4.1 Sulfide toxicity and Activation ..................................................... 25 1.2.4.2 PLP cofactor ................................................................................. 26 1.2.4.3 Mechanism ................................................................................... 27 1.2.4.4 Interacting partners ....................................................................... 31 1.2.5 Isu1: the scaffold protein ....................................................................... 32 1.2.6 Other scaffolding proteins ..................................................................... 33 1.2.7 Molecular chaperones ........................................................................... 34 1.2.8 Export machinery .................................................................................. 36 2. Cysteine desulfurase 38 2.1 Introduction ...................................................................................................... 38 2.2 Experimental procedures .................................................................................. 40 2.2.1 Nfs constructs ....................................................................................... 40 2.2.2 Expression and Purification ................................................................... 41 2.2.2.1 Full length and (1-31) HS-Nfs1 .................................................. 42 2.2.2.2 (1-58) HS-Nfs1 .......................................................................... 43 2.2.3 Mass spectrometry ................................................................................ 44 2.2.3.1 In Gel Digestion ........................................................................... 44 2.2.3.2 Protein identification .................................................................... 45 2.2.3.3 Database search ............................................................................ 46 2.2.4 Western blots ........................................................................................ 46 2.2.5 Factor Xa digestion ............................................................................... 47 x 2.2.6 U.V. Characterization............................................................................ 47 2.2.7 Activity Assays ....................................................................................
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