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Roles of Glutaredoxin and Thioredoxin-Like Nfu Proteins in Iron

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Roles of Glutaredoxin and Thioredoxin-Like Nfu Proteins in Iron ROLES OF GLUTAREDOXIN AND THIOREDOXIN-LIKE NFU PROTEINS IN IRON- SULFUR CLUSTER BIOGENESIS by SIBALI BANDYOPADHYAY (Under the Direction of Michael K. Johnson) ABSTRACT Iron-sulfur (Fe-S) clusters are one of the most ancient, ubiquitous and functionally versatile prosthetic groups in nature. Based on the organization of genes in bacterial operons, three highly conserved types of machinery for the biogenesis of Fe-S clusters have emerged, namely the NIF (Ni trogen Fixation), the ISC (Iron-Sulfur Cluster) and the SUF (Su lfur Mobilization) systems. However, recent studies have implicated roles in Fe-S cluster biogenesis for accessory proteins such as glutaredoxins and thioredoxin-like Nfu proteins that are not encoded by genes in nif , isc , or suf operons. This research project was designed to elucidate the role of dithiol and monothiol glutaredoxins and thioredoxin-like Nfu proteins in Fe-S cluster biogenesis. The approach involved structural and/or spectroscopic characterization of clusters assembled on plant dithiol and monothiol glutaredoxins and Azotobacter vinelandii NfuA protein coupled with in vitro studies of cluster transfer to physiologically relevant acceptor proteins and in vivo assessment of function via gene knock out or complementation studies. The A. vinelandii NfuA protein was shown to assemble a subunit-bridging [4Fe-4S] 2+ cluster which is capable of activating apo aconitase at physiologically relevant rates. Taken together with in vivo studies, which indicate impaired maturation of Fe-S proteins in strains inactivated for nfuA , the results indicate a role of NfuA as a class of intermediate [Fe-S] cluster carrier proteins in bacteria. Spectroscopic, mutagenesis and crystallographic studies of the cytosolic poplar dithiol glutaredoxin C1 (CGYC active site) have demonstrated that dithiol glutaredoxins can assemble a novel glutathione-ligated [2Fe-2S] 2+ cluster at the subunit interface of the homodimer and have established the active-site sequence requirement for Fe-S cluster incorporation. The inability to transfer the [2Fe-2S] 2+ clusters to apo forms of acceptor Fe-S proteins suggests that the cluster in dithiol glutaredoxins may play a role in sensing oxidative stress. The chloroplastic poplar monothiol glutaredoxin S14 (CGFS active site) was also shown to assemble a similar glutathione-ligated [2Fe-2S] 2+ cluster at the subunit interface of the homodimer. However, in contrast to dithiol glutaredoxins, kinetic studies indicate that the [2Fe-2S] 2+ cluster on glutaredoxin S14 is rapidly and stoichiometrically transferred intact to apo Synechocystis ferredoxin, a physiologically relevant acceptor protein . Moreover, i n vivo studies showed that glutaredoxin S14 can complement the Fe-S cluster assembly deficiency in yeast mitochondrial monothiol glutaredoxin 5 (CGFS active-site) deletion mutants. These results indicate that monothiol glutaredoxins play a role as scaffold proteins involved in the assembly and/or delivery of [2Fe-2S] 2+ clusters in mitochondria and chloroplasts. INDEX WORDS: Iron-sulfur clusters, Cluster transfer, Aconitase assays, Cluster carriers, Scaffold proteins, Glutaredoxins, Glutathione, Dithiol, Monothiol, Poplar, Ferredoxins. ROLES OF GLUTAREDOXIN AND THIOREDOXIN-LIKE NFU PROTEINS IN IRON- SULFUR CLUSTER BIOGENESIS by SIBALI BANDYOPADHYAY M.Sc. Chemistry, University of Calcutta, Calcutta, India, 1999 B.Sc. Chemistry, Scottish Church College, University of Calcutta, Calcutta, India, 1997 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2007 © 2007 Sibali Bandyopadhyay All Rights Reserved ROLES OF GLUTAREDOXIN AND THIOREDOXIN-LIKE NFU PROTEINS IN IRON- SULFUR CLUSTER BIOGENESIS by SIBALI BANDYOPADHYAY Major Professor: Michael K. Johnson Committee: Michael W. W. Adams Robert A. Scott Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2007 DEDICATION To my loving parents Dr. Monoranjan Banerjee and Mrs. Shanta Banerjee iv ACKNOWLEDGEMENTS My deepest appreciation goes to my advisor and mentor Prof. Michael K. Johnson who introduced me to the field of research in spectroscopy and protein biochemistry and guided me through the course of graduate studies. I also thank my committee members Prof. Michael W.W. Adams, Prof. Robert A. Scott and Dr. Marly Eidsness for their valuable suggestions and guidance during my PhD. A big thanks goes to our collaborators Prof. Jean-Pierre Jacquot, Prof. Vincent Huynh, Prof. Dennis D. Dean, Dr. Nicolas Rouhier, Dr. Patricia Dos Santos and Dr. Sunil Naik for their tremendous help. Thanks to all former and present members of the Johnson lab for teaching me the ways of life. I would specially thank Kala for helping me design a lot of experiments and being with me in difficult situations. It would not be possible for me to complete my PhD without the constant affection and support of my husband Ritesh. I also appreciate the support I received from my friends Nilanjana and Abhijit who helped me settle down in this new country. I have no words to express my love and gratitude for my parents without whom I would not be where I am today. They have constantly encouraged and supported me in pursuing my dreams and taught me good moral values. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .....................................................1 Iron-Sulfur Proteins: Background and Importance ...................................................1 Structure, Spin States and Redox Properties of Simple Fe-S Centers……………...3 Functions of Biological Fe-S Centers .......................................................................8 Three Fe-S Cluster Assembly Systems ...................................................................12 Accessory Proteins Involved in Fe-S Cluster Biogenesis .......................................21 Summary of Presented Work ..................................................................................25 References ...............................................................................................................26 2 GENETIC, BIOCHEMICAL AND SPECTROSCOPIC ANALYSIS OF THE AZOTOBACTER VINELANDII NFUA GENE PRODUCT ....................................54 Abstract ...................................................................................................................55 Introduction .............................................................................................................56 Experimental Procedures.........................................................................................59 Results .....................................................................................................................64 Discussion ...............................................................................................................72 Acknowledgements .................................................................................................76 vi References ...............................................................................................................76 3 CHLOROPLAST MONOTHIOL GLUTAREDOXINS ACT AS SCAFFOLD PROTEINS FOR THE ASSEMBLY AND DELIVERY OF [2FE-2S] CLUSTERS…………………………………………………………………….…96 Abstract ...................................................................................................................97 Introduction .............................................................................................................98 Materials and Methods ..........................................................................................101 Results ...................................................................................................................106 Discussion .............................................................................................................116 Acknowledgements ...............................................................................................120 References .............................................................................................................121 4 CONCLUSION AND FUTURE WORKS ................................................................149 References .............................................................................................................155 APPENDIX Functional, structural and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1...........................................................162 vii LIST OF TABLES Page Table 1-1: Functions of biological Fe-S clusters ...........................................................................42 Table 3-1: Primers used in this study for the production of recombinant proteins......................126 Table 3-2: New plasmids employed in the work .........................................................................127 Table 3-3: Yeast strains employed in the work ...........................................................................128 viii LIST OF FIGURES Page Figure 1-1: Crystallographically
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