CHARACTERIZATION OF THE PROPERTIES AND ROLES OF FE-S CENTERS IN FERREDOXINS, GLUTAREDOXINS AND RADICAL S-ADENOSYL METHIONINE ENZYMES by SOWMYA SUBRAMANIAN (Under the Direction of Michael K. Johnson) ABSTRACT The objectives of the research presented in this dissertation are to characterize the properties and to understand the role of Fe-S centers in [2Fe-2S]-ferredoxins, glutaredoxins and anaerobic sulfatase maturating enzymes (anSMEs), by using a combination of mutagenesis, analytical and spectroscopic studies. The spectroscopic techniques include UV-visible absorption, circular dichroism, variable temperature magnetic circular dichroism (VTMCD), EPR, Mössbauer, resonance Raman and MALDI-TOF MS. A detailed spectroscopic characterization of Cys-to-Ser variants of [2Fe-2S]-ferredoxin from Clostridium pasteurianum and Aquifex aeolicus were undertaken in order to characterize the electronic properties of + valence-delocalized [Fe2S2] center, which constitutes the building block of higher-nuclearity Fe-S clusters. VTMCD and magnetization studies enabled the first ever measurement of the double exchange parameter (B) for a valence delocalized [2Fe-2S]+ cluster with sulfide bridging ligands. Spectroscopic studies also revealed that the [2Fe-2S]+ cluster in the valence-delocalized S = 9/2 state at room temperature converts, either partially or fully to valence-localized S = 1/2 state upon freezing. The results are interpreted to be an outcome of changes in solvent packing and H-bonding interactions near the reducible iron site that occur upon freezing and shift the equilibrium in favor of the valence-localized form. Studies carried out to understand the nature and function of Fe-S centers in Arabidopsis thaliana GrxS16, a monothiol glutaredoxin in chloroplast, revealed that recombinant GrxS16 is able to assemble a [2Fe-2S] cluster as-purified, and [2Fe-2S] and [4Fe-4S] clusters on in vitro reconstitution. The ability of GrxS16 to transfer both [2Fe-2S] and [4Fe-4S] cluster to plausible acceptor proteins suggests a role for S16 in the maturation of Fe-S proteins in chloroplast. To elucidate the nature of Fe-S centers and the mechanism of action of anSMEs, analytical and spectroscopic studies were undertaken on the wild-type and variant anSMEs from Clostridium perfringens and Bacteriodes thetaiotamicron. The results reveal that anSMEs can bind three [4Fe-4S] clusters, and activate both Cys-type and Ser-type sulfatases via radical mechanism initiated by the reductive cleavage of S-adenosyl methionine bound to the radical SAM cluster. The two additional [4Fe-4S] clusters are proposed to play a role in the activation of substrate and/or a relay that shuttles electron during enzymatic turnover. INDEX WORDS: Valence delocalization, ferredoxin, glutaredoxin, radical SAM enzymes, electron paramagnetic resonance, resonance Raman, magnetic circular dichroism, Mössbauer. CHARACTERIZATION OF THE PROPERTIES AND ROLES OF FE-S CENTERS IN FERREDOXINS, GLUTAREDOXINS AND RADICAL S-ADENOSYL METHIONINE ENZYMES by SOWMYA SUBRAMANIAN B.Sc., S.D.N.B. Vaishnav College, Madras, India, 1997 M.Sc., Anna University, Madras, India, 1999 M.S., University of Delaware, 2003 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 2010 © 2010 Sowmya Subramanian All Rights Reserved CHARACTERIZATION OF THE PROPERTIES AND ROLES OF FE-S CENTERS IN FERREDOXINS, GLUTAREDOXINS AND RADICAL S-ADENOSYL METHIONINE ENZYMES by SOWMYA SUBRAMANIAN 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 2010 iv TABLE OF CONTENTS Page LIST OF TABLES........................................................................................................................ vii LIST OF FIGURES ......................................................................................................................viii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ....................................................1 Iron-sulfur proteins: Background............................................................................1 Brief summary of presented work...........................................................................3 Structure and coordination of Fe-S clusters............................................................4 Redox properties of Fe-S clusters...........................................................................6 Electronic properties of Fe-S clusters.....................................................................7 Valence delocalization in [2Fe-2S]+ clusters..........................................................9 Glutaredoxins: roles in Fe-S cluster biogenesis and iron homeostasis.................13 Radical SAM enzymes..........................................................................................17 Abbreviations........................................................................................................21 References............................................................................................................22 2 SPECTROSCOPIC AND REDOX STUDIES OF VALENCE- DELOCALIZED [2FE-2S]+ CENTERS IN THIOREDOXIN-LIKE FERREDOXINS.........................................................................................................54 Abbreviations........................................................................................................55 Abstract.................................................................................................................56 v Introduction..........................................................................................................58 Materials and Methods.........................................................................................61 Results..................................................................................................................62 Discussion............................................................................................................73 Acknowledgements..............................................................................................80 References............................................................................................................81 3 NATURE AND FUNCTION OF FE-S CLUSTERS ASSEMBLED ON GRXS16, A CHLOROPLASTIC MONOTHIOL GLUTAREDOXIN....................127 Abbreviations......................................................................................................128 Abstract...............................................................................................................129 Introduction.........................................................................................................130 Materials and Methods........................................................................................133 Results.................................................................................................................137 Discussion...........................................................................................................143 Acknowledgements.............................................................................................147 References...........................................................................................................148 4 ANAEROBIC SULFATASE-MATURATING ENZYMES, FIRST DUAL SUBSTRATE RADICAL S-ADENOSYLMETHIONINE ENZYMES .....168 Abbreviations......................................................................................................169 Abstract...............................................................................................................170 Introduction.........................................................................................................171 Experimental Procedures ....................................................................................173 Results.................................................................................................................179 vi Discussion...........................................................................................................187 Acknowledgements.............................................................................................191 References...........................................................................................................192 5 ANAEROBIC SULFATASE-MATURATING ENZYME: A MECHANISTIC LINK WITH GLYCYL RADICAL ACTIVATING ENZYMES?.....................................223 Abbreviations......................................................................................................224 Abstract...............................................................................................................225 Introduction.........................................................................................................226 Experimental Procedures ....................................................................................227 Results.................................................................................................................231 Discussion...........................................................................................................240 Acknowledgements.............................................................................................244 References...........................................................................................................245