Biogenesis of Iron-Sulfur Clusters and Intracellular Iron Metabolism

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LSU Doctoral Dissertations Graduate School

2009 Biogenesis of iron-sulfur clusters and intracellular iron metabolism Jacob Philip Bitoun Louisiana State University and Agricultural and Mechanical College, [email protected]

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Recommended Citation Bitoun, Jacob Philip, "Biogenesis of iron-sulfur clusters and intracellular iron metabolism" (2009). LSU Doctoral Dissertations. 670. https://digitalcommons.lsu.edu/gradschool_dissertations/670

This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. BIOGENESIS OF IRON-SULFUR CLUSTERS AND INTRACELLULAR IRON METABOLISM

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Biological Sciences

By Jacob Philip Bitoun B.S., Louisiana State University, 2004 August, 2009

ACKNOWLEDGEMENTS

In retrospect, it has become evident that I would not have been able to complete this journey through graduate school without the help of many people. I would like to thank my major professor, Dr. Huangen Ding, for his patience and mentorship. Only now, as a Ph.D candidate, do I realize how much time and commitment he has invested into me and my research.

I would like to thank my committee members: Dr. John R. Battista, Dr. Joomyeong Kim, Dr.

Yong-Hwan Lee, and Dr. Donghui Zhang for their insightful discussions.

I would like to thank other professors who impacted my time at LSU: Dr. Jacqueline

Stephens, Dr. Simon Chang, Dr. Greg Pettis, Dr. Grover Waldrop, Dr. Jayne Garno and Dr.

Megan Macnaughtan. I also would like to thank Dr. Erin Hawkins.

I would like to thank my colleagues in the Ding Lab: Binbin Ren, Juanjuan Yang, Dr.

Xuewu Duan, Guoqiang Tan, and Huang Hao for their insights into certain experiments, discussions in the lab, and friendship. Also I would like to thank the numerous graduate and undergraduate students who have helped over the years.

I would like to thank my family for their encouragement and support. Of course, I would not be in this position without the love from my mother, Ann Woods. I would like to thank my brother Joshua Bitoun for supplying me with endless white noise while conducting my experiments. I would like to thank Philip Fricano Jr., Inez Fricano, Roy Woods, and Jacques

Bitoun for all listening to me when I needed support.

I would like especially thank Abby Gandolfi for her support and encouragement.

Without her it would have been a more challenging road. Thank you for always believing in me, and challenging me.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

LIST OF SCHEMES ...... viii

LIST OF ABBREVIATIONS ...... ix

ABSTRACT ...... xi

CHAPTER 1: LITERATURE REVIEW ...... 1 IRON IN BIOLOGY...... 1 HISTORICAL PERSPECTIVE ...... 2 IRON-SULFUR CLUSTER VERSATILITY ...... 6 IRON-SULFUR CLUSTER BIOGENESIS ...... 10 IRON ACQUISTION AND STORAGE ...... 13 OXIDATIVE AND NITROSATIVE STRESS ...... 17 STATEMENT OF RESEARCH PROBLEMS ...... 22 STATEMENT OF RESEARCH OBJECTIVES ...... 24 REFERENCES ...... 25

CHAPTER 2: CHARACTERIZATION OF A HUMAN IRON-SULFUR CLUSTER ASSEMBLY PROTEIN, HISCA ...... 48 INTRODUCTION ...... 48 MATERIALS AND METHODS ...... 50 RESULTS ...... 53 DISCUSSION ...... 59 REFERENCES ...... 61

CHAPTER 3: FERRITIN A ACTS AS AN IRON RESERVOIR FOR IRON-SULFUR CLUSTER RE-ASSEMBLY AFTER OXIDATIVE STRESS ...... 66 INTRODUCTION ...... 66 MATERIALS AND METHODS ...... 68 RESULTS ...... 71 DISCUSSION ...... 83 REFERENCES ...... 85

CHAPTER 4: NSRR, A TRANSCRIPTIONAL REPRESSOR OF THE NITROSATIVE STRESS RESPONSE, RESPONDS TO NITRIC OXIDE AND CELLULAR REDOX STATUS ...... 91 INTRODUCTION ...... 91 MATERIALS AND METHODS ...... 93 iii

RESULTS ...... 99 DISCUSSION ...... 116 REFERENCES ...... 118

CHAPTER 5: CONCLUSIONS ...... 125 SUMMARY ...... 125 FUTURE RESEARCH GOALS ...... 129 REFERENCES ...... 133

APPENDIX: PERMISSION TO INCLUDE PUBLISHED WORK ...... 139

VITA...... 142

LIST OF TABLES

1.1 E. coli K-12 [Fe-S] Proteins or [Fe-S] Cluster Assembly Proteins ...... 3

2.1 Oligonucleotide Sequences of the hIscA Primers ...... 50

3.1 Oligonucleotide Sequences of the ftnA Primers...... 69

4.1 Oligonucleotide Sequences of the nsrR Primers ...... 94

4.2 Oligonucleotide Sequences of the Primers for Site Directed Mutagenesis of NsrR ...... 95

4.3 Oligonucleotide Sequences of the hmpA Primers ...... 98

LIST OF FIGURES

1.1 Iron-Sulfur Clusters ...... 7

1.2 The isc Operon of E. coli ...... 12

1.3 Reduction of Molecular Oxygen ...... 18

1.4 Reaction Catalyzed by Nitric Oxide Synthase ...... 20

2.1 hIscA is the Human Homolog of E. coli IscA ...... 53

2.2 UV-Visible Absorption Spectra of Recombinant hIscA ...... 54

2.3 hIscA Binds Iron and Iron-bound hIscA can Donate Iron to E. coli IscU for [Fe-S] Cluster Assembly ...... 55

2.4 hIscA is Able to Donate Iron to E. coli IscU in the Presence of Citrate ...... 57

2.5 hIscA is Unable to Bind Divalent Metals Other than Iron ...... 58

2.6 Locating hIscA in Murine Tissues ...... 59

3.1 IscU-[2Fe-2S] is Disrupted by H2O2...... 71

3.2 IscU-[2Fe-2S] Promotes the Production of Hydroxyl Free Radicals in the Presence of H2O2 ...... 72

3.3 E. coli FtnA can Bind Iron under H2O2 Stress Conditions ...... 73

3.4 FtnA Alleviates the Iron-mediated Production of Hydroxyl Free Radicals ...... 74

3.5 FtnA Binds the Iron Released from IscU-[2Fe-2S] in the Presence of H2O2 ...... 75

3.6 The Iron-bound FtnA is not an Efficient Iron Donor for [Fe-S] Cluster Assembly in IscU ...... 76

3.7 [Fe-S] Cluster Assembly in IscU with Different Iron Sources ...... 77

3.8 Reduced Conditions Promote FtnA to Release Iron ...... 78

3.9 Apo-IscA can Retrieve Iron from Iron-bound FtnA ...... 79

3.10 Substitution of the Conserved Cysteine Residues in IscA to Serine Abolished Iron Retrieval from FtnA ...... 80

3.11 Ascorbate Aids the Thioredoxin Reductase System in Removing Iron from FtnA ...... 81 vi

3.12 Apo-IscA Shuttles Iron from Iron-loaded FtnA to IscU for [Fe-S] Cluster Assembly .....82

4.1 Sequence Alignment of Two E. coli Transcription Factors: NsrR and IscR ...... 93

4.2 Purified E. coli NsrR is an [Fe-S] Protein ...... 101

4.3 NsrR Contains a [2Fe-2S] Cluster ...... 102

4.4 Mutation of the Conserved Cysteine Residues to Serine Residues Fails to Assemble [2Fe-2S] Clusters...... 104

4.5 Formation of DNIC in E. coli Cells Containing Wild-type NsrR and its Mutants ...... 105

4.6 NsrR Consumes Nitric Oxide More Quickly than Other [Fe-S] Proteins Resulting in DNIC Formation ...... 108

4.7 NsrR is More Preferentially Modified by NO In Vivo ...... 109

4.8 The NO-modified NsrR is Efficiently Repaired In Vivo ...... 111

4.9 [2Fe-2S] Cluster of NsrR is Redox Active with a Midpoint Potential of -345 ± 7 mV ..113

4.10 Electro-mobility Shift Analysis of NsrR Binding to phmp under Reduced and Oxidized Conditions...... 115

4.11 NsrR-[2Fe-2S]+ is Released from phmp by NO ...... 116

vii

LIST OF SCHEMES

3.1 FtnA in Relation to [Fe-S] Cluster Assembly and Disruption ...... 85

4.1 Nitric oxide Modifies a [2Fe-2S] Cluster into DNICs ...... 104

4.2 NsrR Regulation of hmp ...... 118

viii

LIST OF ABBREVIATIONS

Bfr Bacterioferritin

DEA-NO Diethylamine NONOate

DNIC Dinitrosyl Iron Complex

Dps DNA Protection during Starvation Protein

DTT Dithiothreitol

E. coli Escherichia coli

EPR Electron Paramagnetic Resonance

[Fe-S] Iron-Sulfur

FNR Fumarate and Nitrate Reduction Protein

FtnA Ferritin A

FtnB Ferritin B

Fur Ferric Uptake Regulator

GSH Glutathione

GSNO S-nitrosoglutathione

H2O2 Hydrogen peroxide

NOS Nitric Oxide Synthase

NTH Endonuclease III

IRE Iron Regulatory Element

IRP Iron Regulatory Protein

N2 Nitrogen

NO Nitric Oxide

•OH Hydroxyl Radical

• - O2 Superoxide Radical ix

O2 Oxygen

PCR Polymerase Chain Reaction phmp Promoter of Gene hmp

RNS Reactive Nitrogen Species

ROS Reactive Oxygen Species sGC Soluble Guanylyl Cyclase

SOD Superoxide Dismutase

UTR Untranslated Region

ABSTRACT

Iron-sulfur ([Fe-S]) clusters represent one of natures most diverse and ubiquitous protein prosthetic groups. [Fe-S] proteins are integral for diverse biological processes. Reconstitution of apo-proteins can occur by exogenously adding excess iron and sulfide. However, due to the toxicity of iron and sulfide, it is most likely that proteins coordinate [Fe-S] cluster assembly in cells.

The first part of this dissertation addressed the iron donor for [Fe-S] cluster assembly.

Recently, it has been shown that IscA is capable of binding iron with an association constant of

3.0 x 1019 M-1. In addition, iron-loaded IscA is capable of donating iron to the proposed scaffold

IscU for nascent [Fe-S] cluster assembly. We show that hIscA, the human homolog of E. coli

IscA, functions as an iron chaperone for the assembly of [Fe-S] clusters in E. coli IscU. hIscA’s iron binding ability is similar to E. coli IscA. Moreover, hIscA is able to donate iron to IscU in the presence of 100-fold excess citrate, a metabolite capable of binding iron. This comparison signifies that [Fe-S] cluster assembly is conserved from bacteria to humans.

The second part of this dissertation determined the participation of Ferritin A (FtnA) in

[Fe-S] cluster assembly. FtnA, the major iron storage protein in E. coli, could serve as an iron reservoir when intracellular iron is depleted. We have shown that FtnA is capable of buffering iron when oxidative stress disrupts nascent [Fe-S] clusters and alleviates the production of hydroxyl radicals. Moreover, when physiological conditions return, IscA is able to retrieve iron from FtnA for [Fe-S] cluster assembly.

The final part of this dissertation corroborated the interrelatedness of the oxidative and nitrosative stress response pathways. NsrR, a nitric oxide (NO) sensitive transcriptional repressor, is shown to coordinate a redox active [2Fe-2S] cluster with a midpoint redox potential of -346 ± 7 mV. The NsrR [2Fe-2S] cluster reacts with NO more quickly than other [Fe-S] xi proteins, signifying its role as a NO sensor. Finally, modification of the NsrR [2Fe-2S] cluster by

NO results in the formation of a protein-bound dinitrosyl iron complex, relieving its DNA binding ability as a repressor.

xii

CHAPTER 1

LITERATURE REVIEW

IRON IN BIOLOGY

Iron is the fourth most abundant element in the Earth’s crust, and one of the most versatile elements in terms of its biological uses [1]. Common with other third row transition metals, the valence electrons of iron belong to the 3d subshell [2]. In biological systems, iron predominantly exists in one of two oxidation states: Fe3+ (ferric or 3d5), or Fe2+ (ferrous or 3d6)

[3]. The ability of iron to accept or donate an electron modulates the transition from Fe3+ to Fe2+ or vice versa. This redox chemistry positions iron to have fundamental roles in major biological processes including: photosynthesis, oxidative phosphorylation, nitrogen fixation, methanogenesis, H2 production and consumption, oxygen transport, gene regulation, DNA synthesis, and citric acid cycle [1].

Iron’s functionality is strictly dependent on its incorporation into proteins. Biological iron can be found in heme, iron-sulfur ([Fe-S]) proteins, mono- or binuclear iron proteins, ferritins, hemosiderins, lactoferrins, and transferrins [3-7]. Contrary to the widespread need of iron by physiological processes, iron availability is tightly controlled to prevent the accumulation of reactive oxygen species.

Before the appearance of an oxygenated atmosphere, it is plausible to consider that biological regulation of iron was not an immediate concern. However as oxygen emerged, the availability of iron shifted from the soluble Fe2+ form to the insoluble Fe3+ form. In addition,

O2’s partially reduced products react avidly with iron [8-11]. The Fenton reaction demonstrates

2+ 3+ - the potential toxicity of unregulated iron and oxygen interaction: Fe + H2O2  Fe + OH +

•OH. The hydroxyl radical (•OH) reacts nonspecifically near the diffusion limited rate with all

biomolecules [8, 12-14]. [Fe-S] proteins are not only one of the major targets of oxidative toxicity, but they may also exacerbate the Fenton reaction using iron released from the cluster.

Thus, they have been studied extensively relative to oxidative stress [14-18].

HISTORICAL PERSPECTIVE To date, [Fe-S] clusters can be found in over 540 proteins and range in biological activities from electron transport to regulation of gene expression (Table 1.1) [19]. It is bewildering to think that such versatile prosthetic groups lay unnoticed until a half century ago especially since the importance of iron in biomolecules has been documented for centuries [20].

It took emerging biotechnology including electron paramagnetic resonance (EPR), Mössbauer spectroscopy, and enhanced cellular fractionation methods to convince obstinate minds that a new field was emerging.

The combined story of the [Fe-S] protein field arose from three independent paths of unrelated research. In the mid-1950’s, research on the photoreactions in chloroplasts led to the discovery of biological factors which could catalyze the photoreduction of heme proteins and pyridine nucleotides [21]. These factors were identified by their catalytic function such as methaemoglobin reducing factor, triphosphopyridine reducing factor, and photosynthetic pyridine nucleotide reductase reducing factor [22-24]. However, no attempt was made to classify these proteins as iron-bearing until the 1960’s when Singer et al. demonstrated they contained non-heme iron. Subcellular fractionation experiments enabled those reducing factors to be concentrated to unveil the presence of non-heme iron. Simultaneously, succinate and

NADH dehydrogenases were shown to contain non-heme iron in the fractionated particles [25-

27]. Nutritional studies of xanthine oxidase showed that it also contained non-heme iron [28].

The second line of work elucidating the field of [Fe-S] proteins was lead by Helmut

Beinert at the University of Wisconsin—Madison [29]. He used EPR to study the redox

Table 1.1 E. coli K-12 [Fe-S] Proteins or [Fe-S] Cluster Assembly Proteins

Accession ID Gene Protein Accession ID Gene Protein P36683 acnB ACON2 P0ABR5 hcaE HCAE P37127 aegA AEGA P0A6Z1 hscA HSCA P25550 aslB ASLB P0A6L9 hscB HSCB P0AE56 bfd BFD P0AAJ8 hybA HYBA P12996 bioB BIOB P0AAK1 hycB HYCB P17846 cysI CYSI P16431 hycE HYCE P0AC41 sdhA DHSA P16432 hycF HYCF P07014 sdhB DHSB P16433 hycG HYCG P69054 sdhC DHSC P0AAK4 hydN HYDN P0AC44 sdhD DHSD P23481 hyfA HYFA P18775 dmsA DMSA P77329 hyfG HYFG P18776 dmsB DMSB P77423 hyfH HYFH P0AB83 nth END3 P05791 ilvD ILVD P0ACC3 erpA ERPA P0AAC8 iscA ISCA P42593 fadH FADH P0AGK8 iscR ISCR P07658 fdhF FDHF P0C0L9 iscX ISCX P24183 fdnG FDNG P62620 ispG ISPG P0AAJ3 fdnH FDNH P62623 ispH ISPH P32176 fdoG FDOG P0A6A6 leuC LEUC P0AAJ5 fdoH FDOH P60716 lipA LIPA P64638 feoC FEOC P69739 hyaA MBHS P0A9R4 fdx FER P69741 hybO MBHT P39405 fhuF FHUF P0AEI1 miaB MIAB P68646 fixX FIXX P30745 moaA MOAA P0A9E5 fnr FNR P17802 mutY MUTY P00363 frdA FRDA P11458 nadA NADA P0AC47 frdB FRDB P33937 napA NAPA P0A8Q0 frdC FRDC P0AAL0 napF NAPF P0A8Q3 frdD FRDD P0AAL3 napG NAPG P0AC33 fumA FUMA P33934 napH NAPH P14407 fumB FUMB P09152 narG NARG P52074 glcF GLCF P11349 narH NARH P0A996 glpC GLPC P19318 narY NARY P09831 gltB GLTB P19319 narZ NARZ P09832 gltD GLTD P63020 nfuA NFUA P0ABW0 hcaC HCAC P0ACD4 nifU NIFU P75825 hcp HCP P08201 nirB NIRB P75824 hcr HCR P0A9I8 nirD NIRD

―Table 1.1 cont.‖

P32131 hemN HEMN P37596 norW NORW P0A9E9 nsr NSR P0A9N8 nrdG NRDG P0AF63 nsrR NSRR P0AAK7 nrfC NRFC P0AFC7 nuoB NUOB Q46814 xdhD XDHD P33599 nuoC NUOCD P0AAL6 ydhY YDHY P0AFD1 nuoE NUOE P77748 ydiJ YDIJ P31979 nuoF NUOF P77714 ydiT YDIT P33602 nuoG NUOG P25889 yeiA YEIA P0AFD4 nuoH NUOH P0ABW3 yfaE YFAE P0AFD6 nuoI NUOI P52102 yfhL YFHL P0AFE0 nuoJ NUOJ P64554 ygcF YGCF P0AFE4 nuoK NUOK Q46905 ygcO YGCO P33607 nuoL NUOL Q46811 ygfK YGFK P0AFE8 nuoM NUOM Q46819 ygfS YGFS P0AFF0 nuoN NUON Q46820 ygfT YGFT P76081 paaE PAAE P52062 yggW YGGW P0A9N4 pflA PFLA Q46861 ygiQ YGIQ P77243 prpD PRPD P0ADW6 yhcC YHCC P0AEI4 rimO RIMO P39280 yjeK YJEK P36979 rlmN RLMN P39288 yjeS YJES P77223 rnfB RNFB P39383 yjiL YJIL P77611 rnfC RNFC P39409 yjjW YJJW P55135 rumA RUMA P77536 ykgF YKGF P16095 sdaA SDHL P0AAL9 ykgJ YKGJ P30744 sdaB SDHM P77374 ynfE YNFE P0ACS2 soxR SOXR P77783 ynfF YNFF P77667 sufA SUFA P0AAJ1 ynfG YNFG P77522 sufB SUFB P56256 ysaA YSAA P77499 sufC SUFC P77165 yagT YAGT P77689 sufD SUFD P75764 ybhJ YBHJ P76194 sufE SUFE P75863 ycbX YCBX P77444 sufS SUFS P52636 yccM YCCM P37610 tauD TAUD P76134 ydeM YDEM P42630 tdcG TDCG P77561 ydeP YDEP P30140 thiH THIH P76192 ydhV YDHV P05847 ttdA TTDA P77375 ydhX YDHX properties of the mitochondrial membrane and of soluble iron-flavoproteins. He discovered an unusual g = 1.94 signal when any of the aforementioned non-heme iron proteins were reduced exogenously [30-32]. Later research would indicate that these g = 1.94 signals were found in 4

dissimilar mammalian proteins, microorganisms, and plants suggesting a more ubiquitous story for the g = 1.94 signal present in these organisms [30, 33, 34].

The last line of evidence, and possibly most seminal for [Fe-S] protein elucidation was the discovery of a small protein involved in the phosphoroclastic split of pyruvate via hydrogenase of Clostridium pasteurianium [35]. This protein was also critical for nitrogen fixation [36]. Due to its brownish color and iron content, this protein was named ferredoxin.

The connection between these three lines of research was made in subsequent years. It was easier for the research on the photosynthetic photoreactions and clostridial ferredoxins to merge. The most unifying feature was the fact that photosynthetic bacteria and algae could produce H2 gas [22-24]. In chloroplasts, H2 production required a viologen and a hydrogenase enzyme. However, when a hydrogenase from C. pasteurianium was used, there was no need for viologen, and is was found that the hydrogenase contained the clostridial ferredoxin [37]. The other reducing factors could also produce H2. Furthermore, photosynthetic pyridine nucleotide reductases from Chromatium could replace the chloroplast factor in reducing NADP+ to NADPH

[38]. Logically, the next step was to see if the clostridial ferredoxin could replace the chloroplast iron protein in photoreactions since Clostridium pasteurianium does not require light [37, 39].

The experiment was successful and it was evident that there was similarity between the clostridial ferredoxins and the reducing factors. The fact that labile sulfide was found in all of the ferredoxin like proteins suggested a new type of biological catalysts was emerging [40-43].

An early unifying theme for classification of [Fe-S] proteins was the presence of non- heme iron. Evidence of the non-heme iron contribution to the g = 1.94 EPR signal arose when

57Fe was substituted in growth media for the preparation of a non-heme iron protein from

Azotobacter vinelandii [44]. The g = 1.94 signal showed hyperfine interaction to the 57Fe atom.

However, the ferredoxins associated with mammalian and bacterial non-heme proteins did not show the g = 1.94 signal despite the fact that all proteins had labile sulfide [45, 46]. The missing link which unified this area of research with the others was the temperature dependence of the g

= 1.94 signal [47]. Temperatures lower than liquid nitrogen were required to obtain the g = 1.94 signal for the ferredoxins and is explained by broadening rapid spin relaxation [31, 48].

The combination of these results into the field of [Fe-S] cluster chemistry initiated numerous studies applying what was known of mammalian non-heme iron proteins to ferredoxins and vice versa. The amino acid sequences of many [Fe-S] proteins were determined and genetic experiments were beginning [49]. The appreciation of nature’s new catalyst brought many more people into the field with specialties ranging from spectroscopists to chemical physicists [50, 51].

IRON-SULFUR CLUSTER VERSATILITY

[Fe-S] clusters are versatile prosthetic groups found in all forms of life [52]. The rate of discovery of new proteins containing [Fe-S] clusters continues at about 10 per year with the elucidation of proteins with unknown function [19]. Table 1.1 lists [Fe-S] proteins or [Fe-S] cluster assembly proteins from E. coli K-12. The putative [Fe-S] proteins were assigned based upon their similarities to known [Fe-S] cluster folds. Based upon this table, approximately 3-5% of all proteins in E. coli contain [Fe-S] clusters. The different protein folds harboring [Fe-S] clusters signify the importance of protein environment in mediating the type of reaction catalyzed by [Fe-S] chemistry.

[Fe-S] clusters represent one of the most ancient protein prosthetic groups found in nature

[53]. Prebiotic geochemical conditions suggest the possibility of the random assembly of iron and sulfur atoms into more complex coordination, possibly versatile enough to be incorporated into the very first proteins. The most common [Fe-S] clusters, [2Fe-2S] cluster or [4Fe-4S] 6

cluster, are typically ligated to proteins via cysteine residues; however, His, Ser, Asp, or backbone amides can also coordinate clusters in specific examples (Fig. 1.1) [54]. Other types of clusters including the [3Fe-4S] cluster, [8Fe-7S] cluster, [8Fe-8S] cluster, or mixed heteroatom clusters have been identified and specialize in distinct chemical reactions [55-61].

[Fe-S] clusters may be coordinated to proteins of varying architecture. This versatility has evolved different functions for [Fe-S] clusters. Their most common feature is the ability to shuttle electrons, but they also function in other processes including: DNA synthesis and repair,

RNA modification, citric acid cycle, branched chain amino acid biosynthesis, heme biosynthesis, lipoic acid biosynthesis, thiamin biosynthesis, substrate binding and activation, iron or [Fe-S] cluster storage, structural integrity of proteins, regulation of gene expression, regulation of enzyme activity, disulfide reduction, and sulfur donation [62-81].

Nitrogenase is a heterodimeric protein found in diazotrophs functioning to reduce or fix atmospheric N2 into ammonia. Each subunit contains at least one [Fe-S] clusters, albeit, they are of different types. The Fe protein contains one [4Fe-4S] cluster and shuttles electrons to the

MoFe protein where N2 reduction occurs. The MoFe protein contains two unique [Fe-S] clusters: the P cluster ([8Fe-7S]) and the FeMo cofactor ([7Fe-Mo-9S-homocitrate-X]) [82]. Due to the 7

complex coordination, specific biosynthetic machinery is present for nitrogenase maturation.

The unique [Fe-S] chemistry found in diazotrophs supplies 60% of the human population with the demand for fixed nitrogen, while the other 40% comes from the industrial Haber-Bosch process using iron as a catalyst [83].

At least three [4Fe-4S] clusters are utilized in photosynthesis by photosystem I as electron transporters to reduce a soluble ferredoxin [84, 85]. The regeneration of NADPH by photosystem I utilizes two molecules of ferredoxin-[2Fe-2S]+ transferring one electron at a time to ferredoxin-NADP+ reductase [86]. The electron transport chain of oxidative phosphorylation includes at least twelve [Fe-S] clusters in complexes I through III [87-89].

[Fe-S] cluster coordination to protein has implications in overall architecture of the domain surrounding the [Fe-S] cluster. The E. coli DNA repair enzymes endonuclease III

(NTH) and MutY have redox inactive [4Fe-4S] clusters [90]. This signifies that these [Fe-S] clusters may function to stabilize the protein [90, 91]. Indeed, experiments have shown that peptides resembling the [4Fe-4S] cluster binding motif of NTH fold into an ordered configuration in vitro [92].

[Fe-S] clusters are posed for substrate binding in both redox active and redox inactive enzymes. Aconitase, the citric acid cycle enzyme catalyzing the isomerization of citrate to isocitrate, is the best characterized member of the dehydratase family of enzymes requiring a

[4Fe-4S] cluster for turnover [56, 93]. In the active site, a unique aqua ligand coordinates a single iron atom of the [4Fe-4S] cluster which leads to high reactivity with molecular O2 and cluster degradation. However, in the presence of citrate, the labile iron atom is coordinated by citrate and isomerization proceeds [94]. Heteroatoms incorporated into the [Fe-S] environment are required for the [Ni-4Fe-5S] cluster of CO dehydrogenases [95]. Another mechanism to change the electronic environment of an enzyme’s active site involves the attachment of a 8

substrate binding metal site to a [Fe-S] cluster. This coordination chemistry can be found in nitrite and sulfite reductases via a siroheme attachment, acetyl CoA synthase via a di-nickel center attachment, and Fe-hydrogenase via a di-iron center attachment [57, 59, 60, 96].

[Fe-S] proteins in Clostridia have been implicated in acting as a reservoir for iron or [Fe-

S] clusters during times of iron limitation. Up to 12 x [4Fe-4S] clusters have been isolated from the polyferredoxins of methanogenic archaea [71]. The hydrogenase requirement of a [4Fe-4S] cluster may promote the assembly of excess [Fe-S] clusters during times favoring biosynthetic assembly since hydrogenase genes and polyferredoxin genes can be located in the same operon

[70].

[Fe-S] clusters can regulate transcription or translation depending on cellular signals.

The best known examples of [Fe-S] proteins regulating gene expression are FNR, SoxR, and

IscR. FNR (fumarate and nitrate reduction) is considered the global regulator of genes required to switch from aerobic to anaerobic conditions in E. coli [97]. Under anaerobic conditions, dimeric FNR contains a [4Fe-4S]2+ cluster which enables FNR to associate with specific cis promoter elements and prevents the transcription of the downstream genes. The presence of O2 causes degradation of the [4Fe-4S]2+ cluster into two [2Fe-2S]2+ clusters with the concomitant loss of the dimerization interface. Monomeric FNR-[2Fe-2S]2+ dissociates from cis promoter elements and allows transcription to proceed [98].

The SoxR protein contains a [2Fe-2S]+ cluster that senses oxidative stress, particularly

• - + 2+ • - the superoxide ( O2 ) anion [99]. When SoxR-[2Fe-2S] is oxidized to SoxR-[2Fe-2S] by O2 ,

SoxR activates the transcription of soxS. In turn, SoxS stimulates the transcription of over 30 oxidative stress defense and detoxification genes [100]. Modification of SoxR-[2Fe-2S]+ by nitric oxide (NO) results in the formation of SoxR with a bound dinitrosyl iron complex (DNIC) which also activates the SoxRS regulon [101]. 9

IscR-[2Fe-2S] is the transcriptional repressor of the isc operon (iscRSUA-hscBA-fdx).

The protein products of the isc operon have been shown to constitute the de novo [Fe-S] cluster assembly pathway in E. coli [75]. Apo-IscR signals that the cell is in need of [Fe-S] clusters and allows for transcription of the isc operon. On the other hand, IscR-[2Fe-2S] signals that other

[Fe-S] proteins are momentarily satiated and blocks the transcription of the isc operon [102].

Since IscR only contains three cysteine residues, IscR likely has a decreased affinity for [2Fe-2S] clusters. IscR is only able to bind [2Fe-2S] clusters when other proteins do not require them

[75]. Conversely, upon degradation of [Fe-S] clusters due to oxidative stress, the [2Fe-2S] of

IscR is likely one of the first [Fe-S] clusters destroyed.

Translational regulation by [Fe-S] clusters has been best exemplified by the discovery that apo-form of cytosolic aconitase is the same protein as the active iron regulatory protein

(IRP) 1 in eukaryotes [103-104]. Whether the translational regulation is up-regulated or down- regulated depends on the transcript [104]. There is also evidence for this translational regulation in prokaryotes [105].

IRON-SULFUR CLUSTER BIOGENESIS

[Fe-S] clusters can be assembled in vitro by adding ferrous iron, sulfide, and a reductant to an apo-protein. However, the concentrations of free iron and sulfide required for in vitro [Fe-

S] cluster assembly are toxic to the cell [106]. The prevailing thought was that [Fe-S] cluster assembly proteins would mediate the formation of [Fe-S] clusters on target apo-proteins. The first evidence of biosynthetic [Fe-S] cluster assembly machinery was reported in the nitrogen- fixing bacterium Azotobacter vinelandii [107, 108].

Nitrogenase, a multi-subunit [Fe-S] protein capable of reducing N2 to ammonia, is encoded by three genes that translate into a heterodimeric MoFe (encoded by nifD and nifK) protein and a homodimeric Fe protein (encoded by nifH) [109, 110]. The commonality between 10

the subunits for N2 fixation is the requirement of special [Fe-S] clusters. In a microarray experiment, Dean et al. investigated a subset of genes that were upregulated during conditions that favored N2 fixation [107]. The identification of the nif (nitrogen fixation) operon containing more than 20 genes functioning in nitrogenase [Fe-S] cluster biogenesis was the result of the transcriptome analysis [107]. Knockout mutants were constructed after the nif operon was deduced to search for the major players in nitrogenase [Fe-S] cluster biogenesis. The observation that a nifU-nifS- double mutant severely diminished the activity of both the MoFe protein and Fe protein signaled the importance of those genes in [Fe-S] cluster maturation [107].

NifS (encoded by nifS) was shown to be a cysteine desulfurase requiring pyridoxal-5’- phosphate for activity [111]. The NifS catalyzed reaction is the desulfurization of L-cysteine to yield L-alanine and sulfide. The sulfide could be used for [Fe-S] cluster assembly [111-113].

Mechanistic features of NifS catalysis revealed that an active site cysteine residue (Cys325) was required for activity. A Cys325-cysteinesubstrate persulfide bond could be detected upon the incubation of NifS and cysteine in a 1:1 ratio [112]. In addition, NifS is stereospecific; it will not turnover D-cysteine [112].

With the source of sulfide in hand for [Fe-S] cluster assembly, the search was on to determine the function of NifU [114]. NifU was suggested to act as either an iron donor or an assembly scaffold for nascent [Fe-S] cluster assembly. NifU functions as a homodimer and was subsequently purified containing one [2Fe-2S]2+/+ cluster per monomer [115]. Even though NifU was purified with an intact [Fe-S] cluster per monomer, primary sequence analysis suggested that coordination of other [Fe-S] clusters was possible. NifU was dissected into three domains: the

N-terminal domain containing three conserved cysteine residues, the central domain with four conserved cysteine residues (indicating a stable [2Fe-2S] cluster), and the C-terminal domain with two conserved cysteine residues [116]. The N-terminal domain of NifU with only three 11

conserved cysteine residues was purified in truncated form and supported the formation of transient [2Fe-2S]2+ clusters when incubated with NifS, L-cysteine, ferrous iron, and a reductant

[117, 118]. Even though NifU central domain site directed mutants abolished the permanent [Fe-

S] cluster coordination, the N-terminal domain could still assemble transient [2Fe-2S]2+ clusters.

Subsequent experiments have shown that an [Fe-S] cluster loaded NifU could efficiently activate the Fe protein of nitrogenase [119].

Interestingly, the nifU-nifS- strain was viable and supported minimal nitrogenase activity.

This suggested that another [Fe-S] cluster assembly system may be present in A. vinelandii

[107]. While fishing for cysteine desulfurase activity in nifS- strains, a IscS paralog was identified [120]. The genetic organization near iscS would later be called the isc (iron sulfur cluster) operon because of similarities to the nif operon (Fig. 1.2). IscS and NifS bear great sequence similarity; however, A. vinelandii iscS- cannot be rescued by expression of nifS suggesting NifS is specific for nitrogenase maturation while IscS is the general cysteine desulfurase for de novo [Fe-S] cluster assembly [120]. IscU shears sequence similarity to the N- terminal domain of NifU and can also host transient [2Fe-2S]2+ clusters[115, 121]. One unique feature of the isc operon was the involvement of the molecular chaperone proteins HscB and

HscA [120].

With the advent of the genomics era, the isc operon has been identified in numerous organisms including E. coli. Overexpression of recombinant [Fe-S] proteins yields an

incomplete occupancy of [Fe-S] cluster in protein; however, overexpressing both the target apo- protein and the isc operon resulted in an increased occupancy [Fe-S] cluster on the target [122].

Individual E. coli knockout mutants of iscS, iscU, hscB, hscA, or fdx resulted in [Fe-S] cluster distress [123]. The E. coli iscS-iscU- double mutant was constructed because knockout of either

A. vinelandii iscS or iscU resulted in a lethal phenotype [115, 123]. Remarkably, the E. coli iscS- iscU- double mutant remained viable despite being compromised [106].

The role of IscA in [Fe-S] cluster assembly remains uncertain and two different hypotheses have been predicted. The first hypothesis states that IscA is an alternative scaffolding protein [124]. IscA can bind a [2Fe-2S] cluster but the physiological role of IscA-

[2Fe-2S] remains to be established [124, 125]. The second hypothesis defines IscA as the de novo iron donor for [Fe-S] cluster assembly [126]. IscA has an iron association constant of 3.0 x

1019 M-1 under reduced conditions and donates iron to IscU in the presence of L-cysteine [126].

In fact, in an experiment whereby total cysteine residues were calibrated between IscA and BSA, both proteins were able to form the same amounts of [2Fe-2S] clusters. This suggests that the

[2Fe-2S] cluster formation on IscA may be nonspecific [127].

The viability of the E. coli iscS-iscU- strain led to the identification of a backup [Fe-S] cluster assembly system encoded by the suf (sulfur utilization factor) operon [128, 129]. The suf operon functions under conditions of iron starvation and oxidative stress to repair or replace damaged [Fe-S] clusters. Regulation of suf is mediated by OxyR and [130, 131]. The presence of global regulators of oxidative stress and iron limitation suggest that the function of the suf operon is to activate, protect, or repair [Fe-S] proteins under those stress conditions [130-132].

IRON ACQUISITION AND STORAGE

The requirement of both iron and oxygen for aerobic life seems counter-intuitive.

Respiration requires both iron and oxygen to generate the proton motive force used in ATP 13

synthesis. On the other hand, iron and oxygen chemistry potentiates the generation of hydroxyl free radicals via the Fenton reaction which damages DNA, RNA, proteins, and lipids [133, 134].

In order for the cell to minimize oxidative stress promoted by intracellular free iron, an elegant strategy has evolved to acquire iron and sequester it in a non-toxic form.

Bacteria utilize siderophores, low molecular weight iron chelators, to obtain iron from the

3+ 30 extracellular environment [135]. Siderophores have high affinity for Fe (Kaff > 10 ) using catechols, hydroxamates, and-hydroxycaboxlyates as Fe3+ binding ligands [3]. In E. coli, the most abundant siderophore is Enterobactin [136]. Fe3+-siderophore complexes are recognized by outer membrane protein complexes (FhuE, FepA, FhuA, FecA, Cir, Fiu) with affinities ranging from 0.1 – 100 nM depending on type [3, 135, 137].

Transport of the Fe3+-siderophore into the periplasm depends on the TonB-ExbB-ExbD complex which senses the cytoplasmic membrane charge gradient. There is an induced change in the TonB structure via ExbB-ExbD which opens the outer membrane protein complexes, releasing Fe3+-siderophore into the periplasm [138]. Fe3+-siderophore transport in the periplasm utilizes binding proteins that deliver the Fe3+-siderophore to the proper permease. Internalization of Fe3+-siderophore into the cytoplasm is mediated by ABC-type permeases embedded in the cytoplasmic membrane in an ATP driven reaction [137]. Dissolution of the Fe3+-siderophore is believed to be mediated through reduction of Fe3+ to Fe2+ [137].

Ferritins constitute a broad superfamily of iron storage proteins that have been identified in all forms of life except lactobacilli [139]. Bacterioferritins also belong to this ferritin superfamily but are unique to bacteria and identifiable by the presence of heme [140]. The main function of the ferritin family is to sequester intracellular ferrous iron in a non-toxic form. Under high iron concentrations, ferritins will bind the excess iron to sequester it as an inert mineral inside the peptide core. Under iron limiting conditions, ferritins will release iron upon reduction 14

of the core for the incorporation into [Fe-S] proteins or hemes [141-149]. Homozygous deletion of a ferritin gene in mice is lethal, and human diseases have been associated with ferritin mutations [150-153].

Ferritins and bacterioferritins usually consist of homopolymers of 24 subunits for the maxi-ferritins and 12 subunits for the mini-ferritins [145, 154, 155]. Usually, individual polypeptides are ~20 kD and contain a four helical bundle motif [156]. The assembly of 24-mers is best described as a hollow sphere with an outer diameter of 120 Å and an inner diameter 80 Å capable of accommodating up to 4500 iron atoms [157, 158]. The symmetry associated with oligimerization has been called 432; the 3-fold axes contain the channels that connect the inner and outer part of the molecule.

Mammalian ferritin subunits come in two different varieties: the H-chain and the L-chain

[144]. Any plausible assembly of H and L subunits is possible as long as they assemble into 24- mers; however, higher H-chain content implies higher ferroxidase activity while higher L-chain content implies greater iron binding capacity [144]. H-rich ferritins are more predominant in the heart and the brain, L-rich ferritins are more predominant in the liver and the spleen [145, 159-

162].

The mechanism by which iron is sequestered inside the maxi-ferritins can be divided into three distinct steps. The first step involves ferrous iron binding at the ferroxidase center [157,

158]. The ferroxidase center is a di-iron binding site composed of conserved glutamate and histidine residues although the different types of ferritins constitute different arrangements [163-

165]. The second step involves catalytic oxidation of ferrous iron to ferric iron using molecular

O2 as the oxidant whereby a diferric-peroxo intermediate is formed along with the production of

H2O2 [166-170]. The final step involves entry of the diferric-oxy mineral precursors into the ferritin’s core [171-177]. 15

In E. coli, there are at least four genes (ftnA, ftnB, bfr, and dps) that belong to the ferritin superfamily. The proteins encoded by those genes are Ferritin A (FtnA), Ferritin B (FtnB),

Bacterioferritin (Bfr), and DNA protection during starvation protein (Dps) [3, 178]. FtnA is expressed during the log phase and functions as the main iron storage protein [179, 180]. The role of FtnB has not been elucidated in E. coli but it lacks many of the conserved amino acids comprising the ferroxidase center [3, 178]. Bfr is a stationary phase protein that is believed to attenuate oxidative stress since either O2 or H2O2 functions as the oxidant in the mineralization

[136, 180-182]. Dps, using H2O2 as the oxidant, protects DNA from hydroxyl radical damage in stationary phase [183-186].

Regulation of the ferritin genes is necessary to provide the cell the balance between iron- mediated toxicity and the iron necessary for metabolism. In E. coli, the ftnA and bfr genes are regulated using an elegant mechanism under iron replete or iron deplete conditions [136, 180,

187]. Both genes are upregulated by the Fur-Fe2+ repressor relaying the signal that iron is abundant in the cell. On the other hand, most iron acquisition genes are negatively regulated by

Fur-Fe2+ [156, 187-189]. One such negatively regulated gene is a small RNA called rhyB [187,

190-192]. When Fe2+ dissociates from Fur, transcription of rhyB is activated [187]. RhyB targets the mRNA of ftnA, bfr, and many [Fe-S] proteins for degradation in a reaction depending on s sRNA chaperone protein Hfq [187].

In order to peel away all the layers of the ferritins’ gene regulation, it is necessary to examine the regulation of Fur. Upstream of the fur gene is a CAP binding site which links fur expression to general metabolism [193]. In addition, fur expression is correlated to an oxidative stress response mediated by two additional transcription factors: namely OxyR and SoxRS

[194]. There is an OxyR binding site in the fur promoter which activates transcription under conditions of H2O2 stress [194]. OxyR senses intracellular accumulation of H2O2 via the 16

formation of a disulfide bond [195]. Other OxyR upregulated genes include: katG, ahpF, ahpC, gorA, grxA, dps, and oxyS [196]. The products of these genes are suited to detoxify H2O2 or repair H2O2 mediated damage. The SoxRS activation of fur is more indirect and a consequence of being genetically positioned downstream of the fldA gene. The fldA gene has a SoxS

• - recognition motif in its promoter and is thus induced under O2 stress [194].

Dps is one of the most abundant proteins in stationary phase and forms biocrystals with the chromosome through nonspecific binding [183, 197]. Upon the transition from exponential phase to stationary phase, dps is induced in a sigma factor dependent manner [197].

Biocrystalization is thought to confer the cell with properties including gene regulation and iron detoxification [198]. Unlike the indirect activation of ftnA or bfr due to oxidative stress, direct activation of dps can occur in exponential phase via oxidized OxyR [197, 199].

As mentioned previously, translational regulation of the ferritin proteins has been established in eukaryotes. Ferritin mRNA contains a 5`-untranslated region (UTR) containing an iron regulatory element (IRE) to which IRPs can bind [200-204]. Binding of the IRPs to the IRE prevents the mRNA from associating with the ribosome and inhibits ferritin synthesis. IRP1 is the same polypeptide as cytosolic apo-aconitase thus linking [Fe-S] cluster availability to iron storage [103, 201, 205]. When aconitase contains a [4Fe-4S] cluster, steric hindrance prevents binding to the IRE [74]. IRP2 binding to the IRE is not linked to iron status but rather the iron- mediated oxidative status of the cell [74, 206, 207]. The formation of a disulfide bond in IRP2 prevents its association to the IREs [208]. Unsurprisingly, the translation of ferritin mRNA is linked with iron status and oxidative stress in eukaryotes.

OXIDATIVE AND NITROSATIVE STRESS

Oxidative stress is an excessive production of pro-oxidants or reactive oxygen species

(ROS) inside the cell [209]. As an evolutionary necessity, microbes developed multi-layered 17

defense mechanisms to combat these ROS. During aerobic respiration, ROS can accumulate and damage DNA, RNA, lipids, proteins, hemes, and [Fe-S] clusters [210, 211]. Moreover, the presence of Fe2+, as found in [Fe-S] clusters, can exacerbate oxidative stress via the Fenton reaction.

ROS can either be formed endogenously by enzyme autooxidation or when the respiratory chain leaks partially reduced O2 intermediates (Fig. 1.3) [212, 213]. In addition, cells face exogenous ROS derived from redox-cycling compounds, competing bacteria, or phagosomal NADPH oxidases [214]. Exogenously derived ROS likely exceed the cell’s capacity to cope with the stress using basal mechanisms and has forced the evolution of SoxRS

• - and OxyR. SoxR senses intracellular O2 and OxyR senses H2O2 through mechanisms previously described.

Preventative maintenance of ROS is established through small molecule antioxidants or enzymatic scavengers. The enzymatic defenses of ROS include the actions of superoxide dismutase (SOD), catalase, glutathione synthetase, and glutathione reductase. SOD contains an

• - oxidized active site metal, and catalyzes the dismutation of two molecules of O2 into one molecule of O2 and one molecule of H2O2 regenerating the oxidized active site metal [215]. The

H2O2 generated by SOD is rapidly consumed by catalase in a reaction independent of ATP or reducing equivalents [216]. 18

• - Glutathione (GSH), a highly concentrated intracellular antioxidant, reacts with H2O2, O2 , or •OOH generating a glutathione radical (GS•) [217]. Two GSH radicals dimerize to form

GSSG. Lastly, with the aid of reducing equivalents, glutathione reductase regenerates 2GSH from GSSG [217]. Other nonenzymatic systems capable of attenuating ROS are the thioredoxin and glutaredoxin proteins and the lipophilic antioxidants including ubiquinone and menaquinone

[218].

Nitric oxide (NO), a cell permeable free radical gas, is highly reactive with transition metal centers including heme and [Fe-S] clusters of proteins [219]. NO can be formed in mammalian cells via nitric oxide synthases (NOS), and there are at least three different types of

NOS: neuronal, endothelial, and inducible. They catalyze the reaction of L-arginine, NADPH,

+ and O2 to NO, L-citrulline, and NADP . NOS depends on numerous cofactors including FAD,

FMN, heme, BH4, as well as calmodulins for activity [220].

The most well established role for NO is the activation of soluble guanylate cyclase

(sGC) by binding to a heme moiety. sGC is a heterodimeric enzyme that converts guanosine 5`- triphosphate to cGMP [221]. cGMP is a second messenger acting upon a wide range of physiological processes such as smooth muscle relaxation, clot formation, and neurotransmission

[222, 223].

Low molecular weight thiols including cysteine and glutathione and free radicals

• - including O2 can also react with NO to from reactive nitrogen species (RNS). Low intracellular concentrations of NO typically favor a reaction with transition metals whereas higher concentrations of NO promote the generation of nitrosating and oxiding agents including

- peroxynitrite (OONO ) and N2O3 [219]. Reactions of NO with biomolecules trigger physiological effects including changes in gene expression, changes in enzyme activity, signal transduction, immune response, and vasodilatation [219]. 19

In their natural habitats, bacteria are exposed to different sources of NO and have adapted regulatory mechanisms to control NO mediated stress. Under anaerobic conditions, denitrifying bacteria can dissimilate nitrate into N2 during which NO is formed as a product of nitrite reductase [224, 225]. Non-denitrifying bacteria in close proximity to denitrifying bacteria may be exposed to the NO generated as a result of this anaerobic respiration. Pathogenic bacteria invading mammalian cells may encounter NO generated by iNOS in host macrophages as an attempt to destroy the pathogen (Fig. 1.4) [226, 227].

NO accumulation resulting from denitrification or as part of the oxidative burst of macrophages renders bacteria vulnerable to NO induced toxicity. As a result, bacteria have evolved multiple mechanisms to evade the bactericidal effects of NO and RNS. The most evident response to NO is a change in gene expression. Many transcriptional repressors have been identified with NO-sensing properties including: FNR, SoxR, Fur, NorR, and NsrR.

FNR is the global regulator in the switch from oxic to anoxic metabolism [97, 228].

Upon shift to oxic conditions, FNR changes from an active state to inactive state in terms of transcriptional regulation. Under anaerobic conditions, FNR maintains a stable [4Fe-4S]2+ cluster and suppresses the transcription of hundreds of genes. Under aerobic conditions,

2+ 2+ molecular O2 degrades the [4Fe-4S] of FNR to a [2Fe-2S] and the associated change in conformation upon [Fe-S] cluster degradation prevents dimerization of FNR thereby disabling association with target DNA sequences. Interestingly, under anaerobic conditions NO will also react with the [4Fe-4S]2+ of FNR resulting in the formation of a dinitroysl iron complex (DNIC);

FNR-DNIC also losses affinity for target DNA sequences [229].

As mentioned previously, SoxR responds to NO via formation of SoxR-DNIC which also activates the SoxRS regulon [101]. The presence of a [2Fe-2S] cluster in SoxR is required to illicit this response, and the SoxR-DNIC is stable in vitro [101]. However, in vivo data shows that SoxR-DNIC is rapidly repaired through an unknown mechanism [101]. Other nitrosylated

[Fe-S] proteins have been repaired in vitro using IscS [230].

Fur is a well-characterized transcriptional regulator that functions as an iron-dependent repressor controlling the expression of iron related genes. Fe2+-Fur can be modified by NO in vitro, forming a Fur-DNIC [231]. Fur-DNIC does not bind DNA and in vivo treatment using an

NO donor shows derepression of Fur-regulated promoters. Microarray experiments of the Fur regulon shows shifts in gene expression after exposure to S-nitrosoglutathione (GSNO) or acidified nitrite [232]. However, other microarray experiments indicate that Fur may be a functional NO responsive regulator under conditions of iron limitation [233, 234].

NorR controls the transcription of norVW encoding flavorubredoxin (NorV) and its oxidoreductase (NorW) capable of reducing NO to N2O at the expense of NADH under anaerobic conditions [235]. NorR is a ζ54 dependent enhancer binding protein. NorR activates the transcription of norVW upon exposure to NO, nitroprusside, GSNO, or acidified nitrite [236].

NorR senses NO through a mononuclear iron center that is modified to a mononitrosyl iron complex upon NO exposure [237]. Activated NorR does not dissociate from the promoter, rather it promotes the opening of the norVW transcription bubble via ATP hydrolysis [237-239]. 21

A novel NO-sensitive regulator, NsrR, was recently discovered by microarray analysis when some E. coli genes were induced in response to NO in a norR-fur-soxRS-oxyR- background

[232]. The most highly regulated genes by NsrR are hmp, ygbA, ytfE, hcp-hcr, and members of the nrf operon [240-242]. Notably, all of these genes with known functions play a role in mediating resistance to NO-related stress. Hmp or flavohemoglobin is responsible for the NO- oxygenase activity [235, 243]. This enzyme has a koff/kon for NO 0.000008 µM << koff/kon for O2

0.012 µM and a Km for O2 of 30-100 µM [244, 245]. YtfE is a member of the binuclear iron proteins, and is implicated in mediating RNS damaged [Fe-S] cluster repair [246-251]. The members of the nrf operon constitute a periplasmic nitrite reductase [241, 252, 253]. hcp encodes the hybrid cubane protein and may be required in hydroxylamine assimilation or detoxification [241, 254-256].

STATEMENT OF RESEARCH PROBLEMS

Recent research in E. coli has elucidated the functions of key proteins needed for [Fe-S] cluster biosynthesis. It is widely accepted that IscS is a cysteine desulfurase that catalyzes the desulfurization of L-cysteine providing sulfur to IscU [257]. IscU is a scaffolding protein where nascent [Fe-S] clusters are assembled as evidenced by spectroscopic methods [123]. The involvement of the heat shock cognate proteins HscA and HscB mediate the transfer of nascent

[Fe-S] clusters from IscU to target apo-proteins in an ATP dependent reaction [258-260]. The function of IscA remains controversial. IscA can bind transient [Fe-S] clusters; this has led some researchers to believe that IscA is a secondary [Fe-S] assembly scaffold [261-263]. However, the [Fe-S] cluster on IscA may be nonspecific and a result of having solvent exposed cysteine residues in close proximity. Indeed, BSA hosts the same transient [Fe-S] cluster as IscA when the cysteine residues of the two proteins were calibrated [127]. Another hypothesis suggests that

IscA is the iron donor for the assembly of [Fe-S] clusters on IscU [126, 264-271]. This 22

hypothesis is supported by the fact that IscA contains iron after purification without any measurable sulfide. The association constant between ferrous iron and IscA was determined to be 3.0 x 1019 M-1 using a citrate based comparison [126]. In fact, it takes 100-fold excess citrate to remove half of the iron from IscA [126, 271].

If IscA is the iron donor for [Fe-S] cluster assembly on IscU, then from where does the iron originate? The labile iron pool may not be enough to accommodate robust assembly after onslaught by oxidative stress. When reducing conditions return to a depleted labile iron pool,

IscA must look to other sources to facilitate [Fe-S] cluster assembly. One of these sources may be the ferritins, a group of iron storage proteins conserved across all domains of life [3].

Despite the abundant information available on iron entry into the ferritins, iron exit from these nanocages is less understood, and some have called the iron stored in ferritin as biologically unavailable. If this was the case, shouldn’t evolution have provided a robust iron specific permease to remove excess iron from the cellular milieu rather than a beautifully designed storage protein? Recently, small peptides have been shown to aid the release of iron from ferritin [272]. Release of iron from ferritin may be peptide specific but it also may need the right balance of reductants and chelators. One reason that iron release from ferritin is somewhat mysterious is that the intracellular redox potential has not been appropriately approximated in the test tube.

Under H2O2 stress conditions, solvent accessible [Fe-S] proteins may be a culprit to promoting the generation of the hydroxyl radical. Thus, disruption of [Fe-S] proteins by ROS would result in enhanced, more vehement ROS. However, there have not been any studies tracing iron released from [Fe-S] proteins to the ferritins. In the presence of H2O2, IscA loses its iron binding capacity, likely through the formation of a disulfide. Since the ferritins bind iron

through negatively charged carboxyl groups in amino acids, H2O2 should not impact ferritin’s iron binding ability.

Like ROS, NO has the ability to modify [Fe-S] proteins. One of the major targets of NO cytotoxicity appears to be IlvD, a [4Fe-4S] protein involved in the branched chain amino acid biosynthesis [273]. The NO-response network contains genes geared to combat nitrosative stress evidenced by three different microarray studies [232-234]. NO-mediated induction of this network occurs through a signal received by NsrR. By sequence comparison to IscR, NsrR is predicted to contain a [2Fe-2S] cluster and is further supported by the upregulation of the NO response network when cells were exposed to 2`-2` dipyridyl, an iron chelator [242]. However, in vitro characterization of this protein has not yet been attempted.

In addition, the extent to which [Fe-S] clusters mediate genomic instability in eukaryotes is perplexing. Most of the [Fe-S] cluster assembly proteins are nuclear encoded and have N- terminal targeting peptides for allocation to different organelles. Mitochondrial genomic instability has been addressed in regards to oxidative stress generated by [Fe-S] clusters, and just recently it has been shown that all genomes are targets of [Fe-S] cluster mediated stress [274,

275].

STATEMENT OF RESEARCH OBJECTIVES

The purpose of the research presented here is three-fold unified by the delicate need to minimize oxidative and nitrosative stress by balancing iron between a biologically active form and a biomineral form. The first aim is to elucidate the function of hIscA, the human homolog of

E. coli IscA. The similarities are striking, including primary amino acid sequence to iron specificity. hIscA expression patterns from different mammalian tissues using anti-hIscA polyclonal antibody would increase understanding of compartmentalized [Fe-S] cluster assembly. 24

The second aim is to determine the role of FtnA, the main iron storage protein in E. coli, in [Fe-S] cluster assembly. Specifically, is FtnA an iron buffer capable of alleviating the formation of the hydroxyl radicals when solvent exposed [Fe-S] clusters are subjected to H2O2 stress conditions? Once the H2O2 stress is alleviated, is the iron in FtnA available for IscA to mediate the reassembly of the disrupted [Fe-S] clusters? Since IscA has such a high iron binding affinity, all that would seem necessary is to reduce iron atoms of FtnA’s core one at a time.

The final objective is to elucidate the role of NsrR as an NO-sensitive transcriptional repressor. Does NsrR coordinate an [Fe-S] cluster and of what geometry and oxidation state is it? The possibility exists and will be referenced within that a special duality may exist between

• - the oxidative and nitrosative stress responses. The primary signal for SoxR is O2 , though NO elicits the same response. The primary signal for NsrR appears to be NO, yet redox potential may elicit a similar response.

The interplay of iron and oxygen is a tightly regulated process. Due to cytotoxicity of oxygen activated intermediates, there are many details of [Fe-S] cluster biogenesis left unanswered. Understanding the objectives set forth above can only improve the current state of knowledge in [Fe-S] protein biochemistry. Oxidative or nitrosative stress will likely cause a shift from [Fe-S] cluster biogenesis to stress related repair mechanisms. The utilization of the ferritins as an iron supplier upon this shift in cellular redox status has not yet been addressed.

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CHAPTER 2

CHARACTERIZATION OF A HUMAN IRON-SULFUR CLUSTER ASSEMBLY PROTEIN, HISCA

INTRODUCTION

Iron-sulfur ([Fe-S]) cluster biogenesis requires at least six proteins (IscS, IscU, IscA,

HscB, HscA, and Fdx) in bacteria and eukaryotic organisms [1-5]. IscS is a cysteine desulfurase which removes sulfur from L-cysteine and provides it to IscU [6-12]. IscU is the scaffold protein where nascent [Fe-S] clusters are assembled. IscU usually forms [2Fe-2S] clusters but can form [4Fe-4S] clusters via reductive coupling of two [2Fe-2S] clusters [13]. The [Fe-S] clusters on IscU are subsequently transferred to targets including apo-Fdx in the presence of

HscA, HscB, and ATP [14, 15].

The function of IscA remains controversial. One hypothesis states that IscA is an alternative [Fe-S] cluster scaffold [16-19]. Evidence supporting this hypothesis is demonstrated by the fact that IscA can host transient [2Fe-2S] clusters and those clusters can be transferred to target apo-proteins [19-23]. The other hypothesis states that IscA is an iron-binding protein that functions as de novo the iron donor for [Fe-S] cluster assembly on IscU [24-26]. This hypothesis is supported by the fact that IscA binds iron very tightly with an apparent iron association constant of 3.0 x 1019 M-1 in reducing conditions. Iron-loaded IscA can transfer its iron to IscU for [Fe-S] cluster assembly under physiologically relevant reducing conditions [27-30].

The controversy over the iron donor for [Fe-S] cluster assembly has not been settled. In addition to IscA, eukaryotic frataxin has been implicated as a potential iron donor [31-33].

Decreases in mammalian frataxin expression have been linked with Freidreich’s ataxia, a disease associated with neurodegeneration and cardiomyopathy [34, 35]. S. cerevisiae knockouts of frataxin homolog YFH1 have deficiencies in [Fe-S] cluster maturation, increased mitochondrial

iron localization, and increased oxidative stress [36, 37]. However, the S. cerevisiae YFH1- strain is viable suggesting the interplay of other iron donors for [Fe-S] cluster biogenesis [38,

39]. In accordance with the yeast knockout, deletion of CyaY, frataxin’s homolog in E. coli, does not cause an overt phenotype [40].

Recently, it has been suggested that CyaY and IscA exist as dual iron donors depending on oxidative stress levels [41]. IscA is believed to be the iron donor for [Fe-S] cluster assembly under physiologically relevant reducing conditions, whereas CyaY is thought to mediate [Fe-S] cluster assembly during periods of enhanced oxidative stress [41]. Since CyaY uses a solvent exposed patch of acidic amino acids to bind iron, enhanced oxidative stress will not impede its iron binding ability [42]. On the other hand, IscA uses a ―cysteine pocket‖ to coordinate iron, and exposure of those thiols to oxidative stress may generate a disulfide in the iron binding pocket [43]. The fact that both of these protein architectures exist in organisms ranging from bacteria to humans implies the importance of having two iron donors for [Fe-S] cluster biogenesis.

In an attempt to determine if the iron binding ability is unique to E. coli IscA, we cloned and purified human IscA (hIscA). hIscA is located on chromosome 9 and contains an N-terminal mitochondrial targeting sequence. The sequence of the cloned hIscA homolog was identical to that recently identified from a human brain cDNA expression library with serum from a patient suffering from the autoimmune Sjorgen’s syndrome [44]. Our results show that recombinant hIscA is an iron binding protein with an iron association constant of 1.0 x 1020 M-1 in reducing conditions and that binding of iron in hIscA is specific. hIscA maximally binds one iron per dimer. Substitution of the conserved cysteine residues in hIscA with serine residues abolished the iron binding. In addition, we found that hIscA can provide iron for the [Fe-S] cluster assembly in E. coli IscU in the presence or absence of citrate. 49

A polyclonal antibody raised in rabbit was used to determine the expression of hIscA in different murine tissues. The murine IscA (mIscA) is 100% identical in amino acid sequence to hIscA. mIscA is most highly expressed in murine tissue extracts of brain, heart, and kidney.

Bovine heart mitochondria also reveal contain a high level of hIscA, confirming the functionality of the N-terminal signal [44].

MATERIALS AND METHODS

Cloning and Purification of Human IscA Homolog

A cDNA clone (DKFZp547G027Q) containing a full length human IscA homolog

(MGC4276) was obtained from the German Cancer Center Research Center (RZPD)

(http://www/rzpd.de). The clone was from a human fetal brain (16-23 weeks) cDNA library.

The full length human IscA (hIscA) contains 129 amino acid residues with a mitochondrial targeting signal peptide in the N-terminus [44]. The primers listed in Table 2.1 were used for amplifying human IscA cDNA such that the mitochondrial targeting signal sequence was omitted. The amplified hIscA gene was digested with NdeI and BlpI and ligated into pET28b+. pT-hIscA was then introduced into E. coli strain BL21(DE3) (Novagen). The His-tagged hIscA was expressed and purified as described previously [28]. The N-terminal His-tag was removed from hIscA by incubation with 0.65 units/mL of thrombin (Pierce) overnight. The protein concentration of apo-hIscA was calculated based on the absorption peak at 280 nm using an extinction coefficient of 4.2 mM-1cm-1.

Iron Binding Analysis in hIscA

The iron-depleted hIscA (apo-hIscA) was prepared by incubating purified hIscA with 10 mM EDTA and 2 mM dithiothreitol (DTT) at 37°C for 60 minutes, followed by passing the sample through a 5 mL Hi-Trap desalting column (GE Healthcare) equilibrated with buffer containing 200 mM NaCl and 20 mM Tris (pH 8.0). For the iron-binding experiments, apo- hIscA (50 µM) was incubated with increasing concentrations of Fe(NH4)2(SO4)2 in the presence of 2 mM DTT in open-to-air microfuge tubes for 15 minutes. The protein samples were then passed through a HiTrap desalting column to remove unbound iron and DTT.

The total iron content of protein samples was measured either by inductively coupled plasma mass spectroscopy (Dept. of Chemical Engineering, LSU) or a colorimetric assay with

Ferrozine®. For the colorimetric assay the protein samples were heated to 85°C for 15 minutes with 500 µM Ferrozine® and 2 mM L-cysteine. After incubation, the protein samples were centrifuged briefly at 10,000 rpm to remove the precipitate. The concentration of the iron-

Ferrozine® complex was measured at 564 nm using an extinction coefficient of 27.4mM-1cm-1.

Both techniques resulted in similar iron determinations.

The total sulfide content of protein samples was measured using a method by Siegel [45].

Protein samples were incubated at room temperature for 20 minutes with 2 mM N,N-dimethyl-p- phenylene-diamine sulfate (DPD) and 3 mM FeCl3. At the same time, a standard curve was generated using Na2S. The sulfide concentration was determined by extrapolating the absorbance values at 669 nm to the standard curve of Na2S. For every iron and sulfide measurement, the Endonuclease III-[4Fe-4S] was used as an internal control.

Iron Association Constant Determination for hIscA

For iron association constant determinations, the iron-loaded hIscA (100 µM) was incubated with increasing concentrations of sodium citrate at room temperature for 30 minutes 51

before the samples were re-purified through the HiTrap desalting column. Re-purification allows for the excess citrate and citrate-Fe3+ to be removed from the sample. The remaining iron binding in hIscA was analyzed from the absorption at 315 nm as described previously [27].

Iron-Sulfur Cluster Assembly in E. coli IscU

The E. coli IscU and IscS were prepared as described previously [27, 46]. In a typical experiment E. coli IscU (50 µM) was incubated with IscS (1 µM), DTT (2 mM), NaCl (200 mM), and Tris (20 mM) (pH 8.0). The reaction mixture was purged with argon gas and pre- incubated at 37°C for 5 minutes before L-cysteine (1 mM) was added to initiate [Fe-S] cluster assembly. The [Fe-S] cluster assembly in IscU was monitored in a Beckman DU640 UV-Visible absorption spectrophotometer equipped with a temperature controller. The absorption peak at

456 nm was used to calculate the amount of [2Fe-2S] clusters in IscU using an extinction coefficient of 5.8 mM-1cm-1 [6].

Western Blot

In order to detect hIscA from tissues, a custom polyclonal antibody against hIscA was raised in rabbit (Affinity Bioreagents). Murine tissue samples from mice were a kind gift provided by Dr. Joomyeong Kim (Louisiana State University). The proteins from the murine tissues were extracted with T-Per Tissue Protein Extraction Reagent (Pierce) supplemented with protease inhibitor cocktail (Pierce). Total protein extracted from the murine tissues was quantified using the Bradford method with BSA as a standard. Total protein (10 µg) from each tissue extract was loaded onto a 12% polyacrylamide gel and subjected to SDS-PAGE for 80 minutes at 180 V. The protein samples were then transferred to an Immun-Blot PVDF

Membrane (Bio-Rad) in an upright transfer apparatus for 45 minutes at 100 mA. PVDF membranes were blocked with a 3% BSA and 0.05% Tween-20 solution for 60 minutes. Then, the custom anti-hIscA antibody (1:1000) was directly added to the blocking solution and allowed 52

to incubate for another 60 minutes. The membranes were then washed with PBS buffer five times for 5 minutes each. To the last wash, the goat-anti-rabbit HRP conjugate (1:2500)

(Affinity Bioreagents) was added and incubated for 45 minutes. Again the membranes were washed with PBS buffer five times for 5 minutes each. Finally, Immun-Star™ WesternC™

Chemiluminescent (Bio-Rad) was added according to the manufacturer’s instructions.

RESULTS

Fig. 2.1 represents a BLAST search using NCBI. The search revealed a hypothetical

human protein MGC4276 with homology to E. coli IscA dubbed hIscA. hIscA is located on human chromosome 9 and is predicted to have 149 amino acids with an extra 22 at the N- terminus relative to the E. coli homolog (Fig. 2.1A). The additional 22 amino acids constitute a

N-terminal mitochondrial targeting sequence [44]. hIscA bears 30% sequence identity and 70% sequence similarity to E. coli IscA. All three cysteine residues are conserved in both proteins

(Fig. 2.1B).

Recombinant hIscA was expressed in E. coli cells, and purified as described in Materials and Methods. Overexpression and purification of hIscA from E. coli yielded a > 95% pure protein (Fig. 2.2A). As shown in Fig. 2.2B, the UV-Vis absorption spectra of as-purified hIscA sulfide content of purified hIscA were analyzed. On average, there were 0.15 ± 0.05 irons per

hIscA monomer and 0.03 ± 0.01 sulfides per hIscA monomer. The small amount of sulfide may be due to a contaminant protein since apo-hIscA showed 0.02 ± 0.01 sulfide per hIscA monomer.

The iron center in hIscA was stable and resistant to oxygen; however, incubation of hIscA with the iron chelator EDTA almost completely removed the absorption peak at 315 nm

(Fig. 2.2B, trace c). On the other hand, the absorption peak at 315 nm was almost completely restored when apo-hIscA was incubated with equal concentrations of ferrous iron and 2 mM

DTT (Fig. 2.2B, trace a). The iron content analysis indicated that hIscA binds 0.50 ± 0.10 iron per monomer. This stoichiometry is similar to that found for E. coli IscA [27].

The Iron Association Constant of hIscA is Comparable with Human Transferrin

To further determine the iron binding activity of hIscA, apo-hIscA (50 µM) was incubated with increasing concentrations of Fe(NH4)2(SO4)2 in the presence of 2 mM DTT. As shown in Fig. 2.3A, there is a linear increase in the iron to protein ratio as a function of iron

between 0 and 15 µM. At concentrations of iron above 20 µM, hIscA is apparently saturated which is similar to the E. coli IscA iron binding curve [27]. The stoichiometry determined from

Fig. 2.3A implies that hIscA is only able to bind one iron per dimer.

To determine the iron association constant of hIscA, sodium citrate was used as an iron competitor [48]. When iron-loaded hIscA was incubated with increasing concentrations of sodium citrate, the absorption peak at 315 nm gradually decreased. At equilibrium, 75% of the absorption amplitude at 315 nm of iron-loaded hIscA was removed after the protein was incubated with 100 mM sodium citrate. Using the iron association constant of citrate (1.0 x 1017

M-1), we estimated that the apparent iron association constant of hIscA is 1.0 x 1020 M-1 [48].

This value is close to the reported value for E. coli IscA of 3.0 x 1019 M-1 and human transferrin

4.7 x 1020 M-1 [27, 49]. These results suggest that like IscA, hIscA is an iron binding protein.

Iron-loaded hIscA can Provide Iron for the Iron-Sulfur Cluster Assembly on E. coli IscU

Previously, we reported that iron-loaded IscA can provide iron for the [Fe-S] cluster assembly on E. coli IscU [27, 28]. Since hIscA is homologous to E. coli IscA, it is plausible that iron-loaded hIscA may provide iron for the [Fe-S] cluster assembly on E. coli IscU.

When iron-loaded hIscA was used as the iron source for [Fe-S] cluster assembly on E. coli IscU, the characteristic absorption peak at 456 nm appeared [6, 27, 28]. Using an extinction coefficient of 5.8 mM-1cm-1 for the [2Fe-2S] on IscU, we determined that 12 µM [2Fe-2S] cluster was assembled on IscU [6]. This suggests that over 90% of the iron can be transferred from hIscA to E. coli IscU (Fig. 2.3B).

Intracellular metabolites with well positioned carboxyl groups are able to bind iron [28].

In an attempt to mimic intracellular conditions, citrate (5 mM) was included in the [Fe-S] cluster assembly reaction. As seen in Fig. 2.4A, the addition of citrate inhibits the [Fe-S] cluster assembly on IscU when Fe(NH4)2(SO4)2 (40 µM) is used as an iron donor. However, when 56

purified hIscA (80 µM protein) was included in the incubation solution, [Fe-S] clusters can be assembled on IscU (Fig. 2.4B). This result demonstrates the ability for hIscA to donate iron for

[Fe-S] cluster assembly in IscU in the presence of over 50-fold excess citrate.

In order to determine if hIscA’s ability of iron binding is specific, we measured hIscA’s ability to bind other divalent metals under the same incubation conditions. After incubating 100

µM apo-hIscA, 50 µM divalent metal, and 2 mM DTT for 20 minutes at 37°C, we measured the

UV-Visible absorption spectra of re-purified hIscA (Fig. 2.5A). It is clear that none of these other divalent metals except iron gives the re-purified hIscA the absorption peak at 315 nm. In another experiment we incubated 100 µM apo-hIscA, 50 µM Fe2+, 50 µM divalent metal, and 2 mM DTT for 20 minutes at 37°C. After incubation, we again re-purified hIscA and monitored

the UV-Visible spectra (Fig. 2.5B). It can be seen that hIscA incubation with iron and either zinc or copper results in an overall decrease in the absorption at 315 nm. It cannot be ruled out that these two metals bind hIscA; their binding might be immeasurable by UV-Visible spectroscopy.

However, direct confirmation would only come from the utilization of other analytical techniques including inductively coupled plasma mass spectroscopy.

We have also determined the hIscA expression from various murine tissues. As shown in

Fig. 2.6A, only murine brain tissue extract was probed. The presence of a signal in the brain extract was promising, and led us to check other tissues for expression of hIscA (Fig. 2.6B).

Amongst the tissues tested, hIscA is most highly expressed in brain, heart, and kidneys. The signals from the tissue extracts are higher in molecular weight than purified hIscA. The purified 58

hIscA protein is missing the N-terminal mitochondrial targeting sequence which suggests that the native protein should migrate slightly slower if the signaling peptide is cleaved in mitochondria.

One reason for the difference in mobility is that there could be a covalent modification in hIscA dimers. Another explanation could be that hIscA is part of a large complex, possibly an SDS resistant [Fe-S] cluster assembly complex.

DISSCUSSION

It has been established that sulfide in [Fe-S] clusters is provided by catalytic removal of sulfur from L-cysteine via cysteine desulfurases in both bacteria and eukaryotic organisms [6, 9-

12, 50, 51]. However, the iron source for [Fe-S] cluster biogenesis remains controversial. Since an elevated level of intracellular free iron content will promote the generation of hydroxyl free

radicals via the Fenton reaction, the intracellular free iron concentration is likely to be much less than what has been used for in vitro [Fe-S] cluster reconstitution experiments [52]. Accordingly, it has been speculated that specific protein partners must be involved in mediating iron and sulfide delivery for the [Fe-S] cluster biogenesis in cells [2].

It has been reported that human frataxin may act as an iron donor to ISU, a human IscU homolog [33]. However, deletion of frataxin orthologs in S. cerevisiae and E. coli suggest that they are non-essential for [Fe-S] cluster assembly. Deletion of the IscA homologs in S. cerevisiae demonstrates accumulation of mitochondrial iron and deficiency of [Fe-S] cluster proteins [26]. In previous studies, IscA has been implicated in mediating the iron transfer to

IscU [27, 28]. The complete network of iron bioavailability has yet to be determined, but could include a more precise role for the frataxin family under oxidative stress conditions and the IscA family under reducing conditions [41].

hIscA, the human homolog to E. coli IscA, is located on human chromosome 9 (Fig. 2.1), and is targeted to mitochondria via an N-terminal signal peptide. Targeting of hIscA to mitochondria may suggest that [Fe-S] cluster assembly in mitochondria utilizes a different pathway than cytosolic [Fe-S] cluster assembly. On the other hand, there is another IscA homolog in humans encoded by the ISCA2 gene, but it remains uncharacterized. The protein products from both human genes contain conserved cysteine residues which could coordinate iron. hIscA may help chaperone iron so that it does not encounter the oxidative stress in mitochondria.

The results shown here suggest that hIscA has a very similar iron binding activity to E. coli IscA, and that the iron-loaded hIscA can provide iron for the assembly of [Fe-S] clusters on

E. coli IscU. This observation is consistent with a previous report showing that hIscA is able to complement the deficiency of IscA homologs in Saccharomyces cerevisiae [44]. The iron 60

binding ability of hIscA is specific in terms of UV-Visible characterization. However, the possibility exists that both zinc and copper may also bind hIscA.

The expression of hIscA in different tissues corroborates a previous study whereby hIscA mRNA levels were highest in the brain, heart, and kidneys [44]. It seems appropriate for this localization pattern since these organs demand high energy. Collectively, these results suggest that IscA may act as an iron donor for the [Fe-S] cluster assembly from bacteria to humans.

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CHAPTER 3: FERRITIN A ACTS AS AN IRON RESERVOIR FOR IRON- SULFUR CLUSTER RE-ASSEMBLY AFTER OXIDATIVE STRESS*

INTRODUCTION

Iron-sulfur ([Fe-S]) clusters represent one of the most versatile and diverse iron containing cofactors found in biology. Due to their broad structural and electronic features, evolution has utilized [Fe-S] clusters in wide range of physiological processes including nitrogen fixation, oxidative phosphorylation, photosynthesis, sugar metabolism, the branched chain amino acid biosynthesis, RNA modification, DNA synthesis and repair, regulation of gene expression, and heme, biotin, and lipoic acid biosynthesis [1-6]. Despite the fact that [Fe-S] clusters may assemble spontaneously in vitro given a reducing environment with high concentrations of free iron and sulfide, recent evidence has shown that in vivo [Fe-S] cluster biosynthesis requires multiple proteins. Homologs of these proteins have been discovered in organisms ranging from archaea to humans [1, 4 ,6]. In E. coli, at least two gene clusters have been identified: iscRSUAhscBAfdx and sufABCDSE [7-9]. They contain proteins with properties essential to de novo [Fe-S] cluster biogenesis or repair [7-9]. Deletion of the isc operon from E. coli results in a reduced [Fe-S] cluster assembly activity; however a double deletion of the isc operon and the suf operon results in an inviable phenotype [8].

The biochemical function of many of the [Fe-S] cluster assembly genes has been elucidated. IscS is a cysteine desulfurase that contains pyridoxal-5’-phosphate cofactor [10, 11].

IscS catalyzes the transfer of sulfane sulfur from cysteine to IscU via an active site cysteine forming a persulfide intermediate [12-16]. IscU is regarded as the [Fe-S] cluster assembly

______

*Reprinted from Biometals, Vol 21(6), J.P. Bitoun, G. Wu, and H. Ding, Escherichia coli FtnA acts as an iron donor for re-assembly of iron-sulfur clusters in response to hydrogen peroxide stress, Pages 693-703, Copyright 2008, with permission from Springer.

scaffold where nascent [2Fe-2S] clusters or [4Fe-4S] clusters are built and then transferred to target apo-proteins [17,18]. HscB and HscA directly interact with IscU and stimulate the transfer of [2Fe-2S] clusters to apo-ferredoxin upon ATP hydrolysis [19-21]. SufS and IscS are paralogs with 30% sequence identity; however, the cysteine desulfurase activity of SufS is relatively low

[22, 23]. SufS activity can be stimulated by SufE and the SufBCD complex, and SufS transfers sulfane sulfur to SufB via SufE [24]. IscA and SufA are also paralogs sharing 46% sequence identity. Both proteins are capable of hosting labile [2Fe-2S] clusters, so it was first suggested that A-type proteins were an alternative [Fe-S] cluster assembly scaffold [25-28]. One unique property of A-type proteins is that they are capable of binding iron very tightly with an association constant of 2.0 x 1019 M-1 under reducing conditions. Iron-loaded IscA and iron- loaded-SufA are efficient iron donors for [Fe-S] cluster assembly on IscU [30-34]. Taken together, these results suggest that one physiological property of the A-type proteins is to act as an iron chaperone for the biogenesis of [Fe-S] clusters [33].

Paradoxically, some [Fe-S] clusters are highly sensitive to reactive free radicals [35, 36].

It is thought that [Fe-S] clusters were one of nature’s first catalyst evolving well before the appearance of atmospheric O2 by photosynthesis [37]. The appearance of O2 and its reduced intermediates has possibly stifled evolution of [Fe-S] clusters [37]. The disruption of [Fe-S] clusters changes the activity of the proteins and the iron released upon disruption will further potentiate the formation of the hydroxyl free radical via Fenton chemistry [3, 38].

The full mechanism of iron toxicity after [Fe-S] cluster disruption has not been fully understood. One possible outcome is that upon disruption of [Fe-S] clusters, the iron may be sequestered by an iron storage protein. Ferritin A (FtnA), the main iron storage protein in E. coli could sequester the iron, and may attenuate the production of hydroxyl free radicals in the presence of H2O2. Upon restoration of reducing conditions, iron from FtnA may be mobilized. 67

Here, we report that IscU-[2Fe-2S] is degraded into apo-IscU in the presence of H2O2.

The iron released from the disrupted [2Fe-2S] can generate hydroxyl free radicals (•OH) via

Fenton chemistry. However, the addition of FtnA will alleviate the •OH formed as a result of

• IscU-[2Fe-2S] incubation with H2O2. FtnA alleviates OH by sequestering the iron released from the disrupted [2Fe-2S] clusters. Even though, FtnA becomes iron-loaded, it is unable to donate iron for [Fe-S] cluster assembly on IscU. However, upon the return of reducing conditions, IscA can retrieve iron from FtnA and shuttle the iron to IscU for nascent [Fe-S] cluster assembly.

MATERIALS AND METHODS

Cloning and Purification of E. coli FtnA

The polymerase chain reaction (PCR) was used to amplify a DNA fragment from E. coil’s genomic DNA harboring the open reading frame containing ftnA. PCR was carried out using the primers FtnA1 and FtnA2 (Table 3.1) to incorporate the appropriate restriction sites flanking the amplified ftnA gene. The amplified ftnA was digested with the restriction enzymes

NcoI and HindIII and subsequently ligated into the expression vector pET28b+ to yield pT-FtnA containing a hexameric histidine tag to the carboxyl terminus of the FtnA polypeptide. pT-FtnA was transformed into E. coli BL21(DE3) cells, and colonies harboring the pT-FtnA plasmid were isolated on kanamycin selection LB plates. Direct confirmation of the plasmid was carried out via DNA sequencing using the T7 primer (Table 4.1).

An overnight E. coli culture containing pT-FtnA was diluted 100-fold into 500 mL of fresh Luria-Bertaini medium supplemented with 100 µg/mL of ampicillin. The cells were allowed to reach an optical density of 0.6 at 600nm by aerobic incubation in a 37°C incubator when 200 µM IPTG was added to the culture to induce FtnA synthesis [39]. After three hours of induction, the cells were harvested and concentrated 25-fold in buffer A (20 mM Tris, pH 8.0,

500 mM NaCl). The cells were disrupted by passage through a French Press twice and then 68

centrifuged to remove cellular debris. The supernatant was applied to a Ni-agarose column (0.5 mL) (Qiagen) attached to an ÄTKA-fast liquid chromatography system (Amersham

Biosciences). The column was washed with 3 column volumes of buffer A followed by 3 column volumes of buffer B (15 mM imidazole in buffer A). The FtnA protein was the eluted with buffer C (250 mM imidazole in buffer A). The eluted FtnA was then applied to a Hitrap- desalting column (5.0 mL) (Amersham Biosciences) equilibrated in buffer A to remove imidazole from the protein samples. Protein concentration was determined by either the

Bradford protein assay kit (Bio-Rad) using bovine serum albumin as a standard or by the absorbance at 280 nm with the extinction coefficient of 22.0 mM-1cm-1 corresponding to apo-

FtnA. Gel filtration analyzes showed that recombinant FtnA was a homopolymer of 24 subunits with a molecular weight of 440 kD as reported by others [40]. The E. coli thioredoxins reductase and thioredoxin were prepared as previously described [41, 42]. The purity of all proteins was determined to be greater than 95% judging from its electrophoresis on a 15% polyacrylamide gel containing SDS followed by staining with Coomassie™ Blue.

Hydroxyl Radical Determination

Hydroxyl free radicals were measured by the procedure describe by Halliwell et al [43].

Hydroxyl free radicals generated in a solution degrade 2-deoxyribose to form a malondialdehyde-like compound that reacts with thiobarbituric acid to form a chromogen. In the experiments, IscU-[2Fe-2S] or freshly prepared Fe(NH4)2(SO4)2 was incubated with potassium 69

phosphate buffer K2PO4 (10 mM, pH 7.4), NaCl (60 mM), 2-deoxyribose (4 mM) at 37°C for 10 minutes before H2O2 was added to initiate the Fenton reaction. The reactions were continued for an additional 25 minutes. Then, a developing solution containing 1% thiobarbituric acid and

2.8% trichloroacetic acid was mixed with the incubation solutions in 0.67-times volume excess for 15 minutes at 100°C. The mixtures were centrifuged for 10 minutes, and then the amounts of chromogen in the supernatants were quantified using the emission wavelength of 553 nm and the excitation wavelength of 532 nm in a Perkin-Elmer LS-3 Fluorormeter.

Iron Binding Analysis and Iron Determination

The iron binding in the proteins was analyzed after the proteins were incubated with freshly prepared Fe(NH4)2(SO4)2 in the presence of thioredoxin (5 µM), thioredoxins reductase

(0.5 µM), and NADPH (500 µM). The proteins were re-purified using a Mono-Q column (0.98 mL). Briefly, samples were loaded on to the Mono-Q column pre-equilibrated with a buffer containing 20 mM Tris (pH 8.0), and eluted with a linear gradient of NaCl (0–1 M) within 10 column volumes at a flow rate of 1 ml · min−1. All solutions were purged with pure argon gas before use. Each eluted fraction was immediately transferred to an anaerobic cuvette and analyzed in a Beckman DU-640 UV–visible absorption spectrometer. The eluted samples were then subjected to SDS/PAGE analysis. Re-purification of the proteins did not affect the iron binding in FtnA, as over 95% of the total iron content remained in the protein after re- purification [30,31,34]. The iron content was determined as previously described.

Iron-Sulfur Cluster Assembly on IscU

To assemble [2Fe-2S] clusters on apo-IscU, IscU (50 µM) was incubated with IscS (1

µM) and Fe(NH4)2(SO4)2 (400 µM) in the presence of thioredoxin (5 µM), thioredoxins reductase (0.5 µM), and NADPH (500 µM). When indicated, iron-bound IscA or iron-bound

FtnA was used as the iron donor instead of Fe(NH4)2(SO4)2. Initiation of [Fe-S] cluster assembly 70

was carried out by adding L-cysteine (1 mM) into the above reaction. Kinetic monitoring of [Fe-

S] cluster assembly on IscU was carried out by measuring the amplitude of the λmax at 456 nm using a Beckman DU640 spectrometer. Otherwise, IscU-[2Fe-2S] was re-purified using a

Mono-Q column [31].

RESULTS

Hydrogen Peroxide Disrupts IscU-[2Fe-2S] and Promotes the Production of Hydroxyl Free Radicals

IscU is a well characterized scaffold protein for the biogenesis of [Fe-S] clusters.

Purified E. coli IscU-[2Fe-2S] cluster had an absorption peak at 456 nm (Fig. 3.1A) [12, 18].

The [2Fe-2S] cluster in IscU was relatively stable in the presence of the thioredoxin reductase system [12, 31]. However, when hydrogen peroxide was added to the incubation solution, the

absorption peak at 456 nm of IscU-[2Fe-2S] cluster was quickly eliminated (Fig. 3.1B). The total iron and sulfur content analyses of the re-purified IscU confirmed that IscU-[2Fe-2S] was converted to apo-IscU by hydrogen peroxide.

The iron released from the disrupted [Fe-S] clusters could potentially promote the production of hydroxyl free radicals via the Fenton reaction [44]. To test this idea, we utilized the 2-deoxyribose method to measure the production of hydroxyl free radicals in solutions as described in the Materials and Methods [43]. Fig. 3.2 shows that the production of hydroxyl free radicals was almost linearly proportional to the concentration of IscU-[2Fe-2S] in the incubation

solution. The amount of hydroxyl free radicals generated by IscU-[2Fe-2S] was also close to that when an equivalent amount of ferrous iron were used in the incubation solutions, suggesting that both iron released from IscU-[2Fe-2S] by hydrogen peroxide contributed to the production

of hydroxyl free radicals. Taken together, these results showed that hydrogen peroxide disrupts

IscU-[2Fe-2S] and that the iron released from the disrupted [Fe-S] clusters promotes the production of hydroxyl free radicals in the presence of the thioredoxin reductase system.

FtnA Alleviates the Production of Hydroxyl Free Radicals by Scavenging the Iron Released from the IscU [2Fe-2S] Cluster

In order to fully access the function of FtnA in response to H2O2 stress, it was necessary to confirm that FtnA could bind iron under these stress conditions (Fig. 3.3) [45, 46]. Unlike

IscA which binds iron through thiol groups of cysteine residues, FtnA binds iron through surface negatively charged carboxyl groups of amino acids. In order to determine if FtnA binds iron

under hydrogen peroxide stress conditions, apo-FtnA was incubated with increasing concentrations of Fe(NH4)2(SO4)2 in the presence of 2 mM H2O2. As can be seen in Fig. 3.3A,

FtnA binds iron with similar affinity under hydrogen peroxide stress conditions. Fig. 3.3B 73

shows the characteristic UV-Vis spectra of FtnA iron binding, also consistent with FtnA iron binding without hydrogen peroxide stress [47].

To avoid the excessive production of deleterious hydroxyl free radicals, the iron released from the disrupted [Fe-S] clusters must be efficiently sequestered. Although IscA is an iron binding protein and provides iron for the biogenesis of [Fe-S] clusters under physiologically relevant conditions, IscA fails to bind any iron in the presence of hydrogen peroxide [30, 31, 33].

Indeed, we found that addition of apo-IscA had no effect on IscU-[2Fe-2S] mediated production of hydroxyl free radicals in the presence of hydrogen peroxide (Fig. 3.4) [48]. Thus, other cellular proteins likely exist to scavenge the iron from oxidatively disrupted [Fe-S] clusters.

Increasing evidence indicated that ferritins, a large family of iron storage proteins found in bacteria and mammalian cells, may have an important role in cellular defense against

oxidative stresses [45, 47, 49-57]. In E. coli, there are at least four ferritin-like proteins: FtnA,

Ferritin B (FtnB), Bacterioferritin (Bfr), and DNA protection during starvation protein (Dps)

[49]. To examine whether ferritins could scavenge the iron released from the disrupted [Fe-S] clusters under oxidative stress conditions, we have used FtnA, the major iron storage protein in

E. coli cells [40, 54].

To further test whether the iron released from IscU-[2Fe-2S] was sequestered by apo-

FtnA, both FtnA and IscU were re-purified after incubation with hydrogen peroxide using an anion exchange Mono-Q column. Fig. 3.5A shows that the re-purified FtnA had a significant

increase of the absorption peak at around 310 nm, indicative of iron binding in the protein [47].

The total iron content analyses of the re-purified FtnA showed that over 90% of the iron in

IscU-[2Fe-2S] was transferred to apo-FtnA after incubation with hydrogen peroxide (Fig. 3.5B).

These results demonstrate that apo-FtnA is able to scavenge the iron released from the disrupted

[Fe-S] clusters and alleviates the production of hydroxyl free radicals in the presence of hydrogen peroxide.

The Iron-bound FtnA is Not an Efficient Iron Donor for the Iron-Sulfur Cluster Assembly in IscU

As a major iron storage protein in E. coli, it is possible that FtnA may act as an iron donor for the biogenesis of [Fe-S] clusters. As shown in Fig. 3.6, the iron-bound FtnA (the ratio of iron to the FtnA monomer was about 4) was incubated with IscU, cysteine desulfurase IscS and L-cysteine in the presence of the thioredoxin reductase system at 37°C anaerobically.

Fig. 3.6A shows that little or no [Fe-S] clusters were assembled in IscU after 30 min incubation. Increase of the iron-bound FtnA concentration by 5 fold in the incubation solution did not significantly increase the [Fe-S] cluster assembly in IscU (data not shown). In contrast, when the iron-bound IscA was used as the iron donor, an absorption peak at 456 nm of the IscU

[2Fe-2S] cluster quickly appeared as we reported previously (Fig. 3.6B) [31]. Thus, unlike the iron-bound IscA, the iron-bound FtnA is not an efficient iron donor for the [Fe-S] cluster assembly in IscU.

In Fig. 3.7, we monitor the [Fe-S] cluster assembly on IscU kinetically using three different iron sources that have been calibrated in terms of iron concentration: Fe(NH4)2(SO4)2

(squares), iron-bound IscA (circles), and iron-bound FtnA (diamonds). As depicted by the steep increase in absorbance at 456 nm, Fe(NH4)2(SO4)2 and iron-bound IscA are equally capable of donating iron to IscU for [Fe-S] cluster assembly [12]. However, when iron-bound FtnA is used as the iron donor, IscU does not generate the characteristic peak at 456 nm indicating that no [Fe-

S] cluster assembly on IscU has occurred.

IscA can Retrieve Iron from the Iron-bound FtnA

In the presence of the thioredoxins reductase system IscA acts as a strong iron binding protein with an iron association constant of 2.0 x 1019 M-1 [29, 30, 34]. This led us to speculate that IscA may be able to compete with FtnA for iron binding under normal physiological conditions.

First, it was necessary to determine if the iron binding ability of FtnA was specific and not an artifact of adventitious iron binding. In order to assay the amount of iron released from

FtnA, we employed Ferrozine, an iron chelator [58]. As seen in Fig. 3.8 (black trace) FtnA does not release any significant amount of iron without the addition reducing agent. The small

amount of iron released is likely caused by the iron chelator. In the presence of NADPH (green trace), FtnA still does not release substantial iron signifying that NADPH alone cannot reduce iron core of FtnA. However, upon addition of NADPH and thioredoxin reductase (blue trace) or

NADPH, thioredoxin reductase, and thioredoxin (red trace), the amount of iron released from 78

FtnA is dramatically increased. It is possible that either the FADH moiety of TrxB or the active site thiols of TrxA are capable of reducing the iron center of FtnA.

We then postulated that IscA could bind the iron that was being released from the reduction of the FtnA core. Apo-IscA was incubated with the iron-bound FtnA (the ratio of iron to the FtnA monomer was about 4) in the presence of the thioredoxin reductase system at 37°C for 1 hour to establish the iron binding equilibrium. IscA and FtnA were then re-purified using a

Mono-Q column (Fig. 3.9A). The total iron content analyses showed that a significant amount of iron was transferred from the iron-bound FtnA to apo-IscA after the incubation (Fig. 3.9B).

Under the same experimental conditions, IscU failed to retrieve any iron from the iron-bound

FtnA in the presence of the thioredoxin reductase system (data not shown), suggesting that IscA, but not IscU, can acquire the iron from the iron-bound FtnA under relevant reducing conditions. 79

The non-denaturing polyacrylamide electrophoresis and the gel filtration chromatography studies indicated that IscA and the iron-bound FtnA did not form any stable protein complexes

(data not shown), indicating that the iron-bound FtnA and apo-IscA did not form a stable protein complex to facilitate the iron transfer. The iron mobilization from ferritins requires reduction of ferric iron to ferrous iron, and IscA scavenges free ferrous iron only in the presence of the thioredoxin reductase system [51]. We propose that the iron transfer from the iron-bound FtnA to apo-IscA is through the binding competition for ferrous iron in the presence of the thioredoxin reductase system [30].

It is believed that IscA coordinates iron through three conserved cysteine residues via a cysteine pocket motif [29, 59]. As seen in Fig. 3.10, we determine the role of the conserved

cysteine residues in retrieving iron from FtnA, we mutated the cysteine residues to serine residues in IscA. Fig. 3.10 shows the same experiments as Fig. 3.9 except IscA C101S replaced wildtype IscA. IscA C101S cannot retrieve iron from FtnA. This result is consistent with the notion that cysteine residues are responsible for iron binding in IscA. Similar results were obtained for IscA C35S and IscA C99S (data not shown).

As shown in Fig. 3.11, ascorbate greatly enhances the thioredoxin reductase system in

mobilizing the iron core of FtnA, allowing IscA to bind to saturation. As demonstrated in Fig.

3.9, IscA is capable of recruiting iron from iron-loaded FtnA. However, increasing the concentration of apo-IscA does not result in a substantial increase in iron retrieval using the thioredoxin reductase system. In an attempt to retrieve more iron out of FtnA, we added other natural reducing reagents like GSH and ascorbate into the thioredoxin reductase system [60, 61].

The effect from GSH was not as pronounced though (data not shown).

Apo-IscA Mediates the Iron-Sulfur Cluster Assembly in IscU when the Iron-bound FtnA is Used as the Iron Source In Vitro

Iron-bound IscA can provide iron for the [Fe-S] cluster assembly in IscU, and apo-IscA can retrieve iron from the iron-bound FtnA (Fig. 3.6), we reasoned that apo-IscA may promote the [Fe-S] cluster assembly in IscU when the Fe-FtnA was used as the iron source (Fig. 3.12).

Indeed, addition of apo-IscA significantly increased the [Fe-S] cluster assembly in IscU under the experimental conditions. Re-purification of IscU from the incubation solution further revealed that the [Fe-S] clusters were assembled in IscU, but not in IscA (data not shown). Thus, the iron stored in FtnA can be mobilized by IscA for the [Fe-S] cluster assembly in IscU in the presence of the thioredoxin reductase system. The absorbance associated with IscU-[2Fe-2S] is not as intense as Fig. 3.6 due to equilibrium establishment.

DISSCUSSION

Ferritins are a large group of iron storage proteins found in diverse organisms [49-53]. In mammals, ferritins consist of 24 subunits of two types, H and L [51]. Under aerobic conditions, ferrous iron is rapidly oxidized to the mineral ferrihydrite by the built-in di-iron ferroxidase center in ferritin H subunits [52]. Up to 4500 iron atoms can be stored inside an approximately 8 nm nanocage of the ferritin polymer [49-52]. Over-expression of human H subunit ferritin in cultured HeLa cells and eryhtroid cells renders the cells resistive to hydrogen peroxide [55, 56].

In vitro electron paramagnetic resonance (EPR) spin trapping study also indicates that human H subunit ferritin can detoxify the iron-mediated production of hydroxyl free radicals. In mitochondria and microbes, ferritins exist as a homopolymer composed of 24 identical H-type subunits [49, 50, 53]. In bacteria, genetic studies suggest that ferritins also have an important role in the cellular defense against oxidative stresses [54, 57].

Here, we report that purified FtnA, a major iron storage protein in E. coli, is able to scavenge the iron released from the disrupted [Fe-S] clusters and alleviates the iron-mediated production of hydroxyl free radicals in the presence of hydrogen peroxide in vitro. The results provide biochemical evidence showing that ferritins can effectively prevent the production of hydroxyl free radicals due to the disruption of [Fe-S] clusters under hydrogen peroxide stress.

While the mechanism by which iron enters ferritins has been well characterized, much less is known on how the iron is released from ferritins [51]. At least two models have been proposed for the iron release from ferritins. In the first model, the iron-bound ferritins are degraded in lysosomes, resulting in iron release to cytoplasm [62, 63]. However, the questions as how ferritins enter lysosomes and how iron is released to cytoplasm still remain to be addressed. Also, there are no lysosomes for possible degradation of ferritins in bacteria. In the second model, the ferritin pores formed in the interface of the subunits may reversibly unfold and fold to allow the iron release from ferritins [64, 65]. Recent studies further revealed that some specific peptides are able to promote the iron release from ferritins, supporting the notion that the iron release from ferritins may be regulated by other proteins [66].

Here we show that in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential, the iron bound in FtnA can be retrieved by an iron binding chaperon IscA without any degradation of FtnA [67]. Since IscA only binds ferrous iron, we propose that ferric iron stored in FtnA is first reduced to ferrous iron by the thioredoxin reductase system, making the iron accessible for IscA to bind [30, 34]. Nevertheless, we were unable to mobilize all iron from FtnA for the iron binding in IscA under the experimental conditions. It is likely that additional protein partners are required to completely release iron from ferritins [66].

The finding that IscA can retrieve the iron from the iron-bound FtnA provides new evidence for the notion that IscA is a physiological iron donor for the assembly or repair of [Fe-

S] clusters [30-32, 34]. We show here that IscA and FtnA have very different iron binding properties. Under normal physiological conditions (e.g. in the presence of the thioredoxin reductase system), IscA is a strong iron binding protein that can retrieve iron from the iron storage protein FtnA. However, under oxidative stress conditions (e.g. in the presence of hydrogen peroxide), IscA fails to bind any iron, whereas FtnA becomes a potent iron binding 84

protein to scavenge the iron released from the disrupted [Fe-S] clusters [48]. The iron binding in

FtnA effectively prevents the production of hydroxyl free radicals, likely because the build-in ferroxidase of FtnA converts ferrous iron to mineral ferric iron and alleviates the Fenton reaction

[47]. When normal physiological conditions are re-established, IscA retrieves the iron from

FtnA for the re-assembly of the disrupted [Fe-S] clusters in proteins. Scheme 3.1 summarizes the interplay of IscA and FtnA for the iron binding under normal physiological and oxidative stress conditions. The dynamic iron binding equilibrium between IscA and ferritins is expected to prevent the iron-mediated production of hydroxyl free radicals under oxidative stress conditions and yet ensure the iron supply for the biogenesis and/or repair of [Fe-S] clusters under normal physiological conditions.

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CHAPTER 4 NSRR, A TRANSCRIPTIONAL REPRESSOR OF THE NITROSATIVE STRESS RESPONSE, REPSONDS TO NITRIC OXIDE AND CELLULAR REDOX STATUS INTRODUCTION

Endogenously generated nitric oxide (NO) may accumulate in cells via nitrate reduction, homolytic cleavage of S-nitrosoglutathione (GSNO), or as part of the immune response of mammalian macrophages [1-5]. NO is freely diffusible across cellular membranes and highly reactive with transition metal centers located in hemes, mono or binuclear iron proteins, and iron-sulfur ([Fe-S]) proteins [6-8]. In E. coli, transcription factors bearing NO-reactive transition metals include FNR-[4Fe-4S], SoxR-[2Fe-2S], IscR-[2Fe-2S]; however, NO is not thought to be their primary stimulus [9-11]. Two additional iron bearing transcription factors, NorR and NsrR, are believed to exclusively sense NO [12, 13]. NorR contains a mononuclear iron center and NO modification to a mononitrosyl iron complex results in activation of NorR [14]. NsrR regulates the expression of more genes than NorR and is found in more bacterial species thus, it has been called the global NO regulator.

When NO levels exceed the basal threshold, gene expression will be modulated in an effort to combat nitrosative stress [15-17]. Despite the differences in medium choice, growth conditions, and nitrosative stress implementation, three transcription units were upregulated in all of the NO transcriptome studies: nrdH, norVW, and hmpA. NrdH is related in sequence to glutaredoxins and in function to thioredoxins, and acts as a reductant for class Ib ribonucleotide

• - reductase (NrdEF). The nrdHIEF operon is also induced by superoxide ( O2 ) and hydrogen peroxide (H2O2) in an OxyR and SoxRS independent fashion. As of yet the regulator controlling nrdH expression upon NO exposure has not been revealed. NorV is a flavorubredoxin, and

NorW is its associated flavorubredoxin reductase; they function together to reduce NO to nitrous 91

oxide (N2O) and are controlled by NorR [16, 18, 19]. hmp encodes a flavohemoglobin capable

2- of either oxidizing NO to nitrate (NO3 ) or reducing NO to N2O [20, 21]. hmp expression is complex with transcriptional regulation occurring via FNR, MetR, NsrR, and possibly Fur [9, 12,

22, 23].

In vivo microarray experiments have shown that E. coli NsrR regulates the transcription of more than 20 genes and that a majority of these genes play a role in detoxification of NO or other RNS species [24]. Additionally, NsrR target’s promoters fused to lacZ identify direct in vivo significance of the NsrR mediated NO response. The most highly induced gene ytfE was shown to have 30-times more transcript when NO was present in the system. Interestingly, ytfE encodes a nitrosative stress sensitive [Fe-S] cluster repair protein which may imply NsrR needs

YtfE to repair itself after NO exposure. Other highly inducible genes include hcr-hcp encoding hybrid cluster protein-NADH oxidoreductase, ygbA encoding a protein of unknown function, napFDAGHBC encoding periplasmic nitrate reductase, and nrfABCDEFG encoding periplasmic nitrite reductase [24].

NsrR orthologs are found in a wide range of proteobacteria except δ-proteobacteria which have NnrR, a protein with putatively similar function [25]. As of now, organisms with a demonstrated role of NO sensing by NsrR include: E. coli, B. subtillus; Nitrosomonas europaea;

Salmonella enteric serovar Typhimurium; Neisseria gonorrheae; Neisseria meningitidis [12, 26-

30].

NsrR shows 58% sequence similarity and 33% sequence identity to IscR, the transcriptional repressor of the [Fe-S] assembly operon (iscRSUAhscBAfdx) in E. coli (Fig. 4.1).

NsrR and IscR are the only Rrf2–type transcription repressors of the winged helix superfamily in

E. coli [25, 27]. Dimeric IscR represses the isc operon when each monomer has a [2Fe-2S] cluster bound, suggesting a feedback loop of [Fe-S] cluster biogenesis. When IscR loses its 92

[2Fe-2S] cluster, the affinity of IscR with the isc promoter diminishes and transcription of the isc operon begins [31]. Due to similarity between NsrR and IscR, it is hypothesized that NsrR contains an [Fe-S] cluster that senses NO via the modification of the [Fe-S] cluster to a dinitroysl iron complex (DNIC).

MATERIALS AND METHODS

Cloning and Purification of Wildtype E. coli NsrR

The polymerase chain reaction (PCR) was used to amplify a DNA fragment from E. coil’s genomic DNA harboring the open reading frame containing nsrR. PCR was carried out using the primers NsrR1 and NsrR2 (Table 4.1) to incorporate the appropriate restriction cut sites flanking the amplified nsrR gene. The amplified nsrR was digested with the restriction enzymes

NcoI and HindIII and subsequently ligated into the expression vector pBAD-Myc-His A to yield pBAD-NsrR containing a hexameric histidine tag to the carboxyl terminus of the NsrR polypeptide. pBAD-NsrR was transformed into electrocompetent E. coli MC4100 cells, and colonies harboring the pBAD-NsrR plasmid were isolated by growing the cells on LB-agar

plates supplemented with 100 µg/mL of ampicillin overnight. Direct confirmation of the plasmid was carried out via DNA sequencing using the pBAD primer (Table 4.1).

An overnight E. coli culture containing pBAD-NsrR was diluted 100-fold into 500mL of fresh Luria-Bertaini medium supplemented with 100 µg/mL of ampicillin. The cells were allowed to reach an optical density of 0.6 at 600nm by aerobic incubation in a 37°C incubator when 0.02% of arabinose was added to the culture to induce NsrR synthesis [32]. After two hours of induction, the cells were harvested and concentrated 25-fold in buffer A (20 mM Tris, pH 8.0, 500 mM NaCl, 2.5 mM DTT, 10% glycerol). The cells were disrupted by passage through a French Press twice and then centrifuged to remove cellular debris. The supernatant was applied to a Ni-agarose column (0.5 mL) (Qiagen) attached to an ÄTKA-fast liquid chromatography system (Amersham Biosciences). The column was washed with 3 column volumes of buffer A followed by 3 column volumes of buffer B (15 mM imidazole in buffer A).

The NsrR protein was the eluted with buffer C (700 mM imidazole in buffer A). The eluted

NsrR was then applied to a Hitrap-desalting column (5.0 mL) (Amersham Biosciences) equilibrated in buffer A to remove imidazole from the protein samples. The fast protein liquid chromatography system was controlled by the UNICORN software (Amersham Biosciences) that allows a reproducible protein purification profile. Protein concentration was determined by either the Bradford protein assay kit (Bio-Rad) using bovine serum albumin as a standard or by the absorbance at 280 nm with the extinction coefficient (ε) of 8.04 mM-1cm-1 corresponding to 94

apo-NsrR. The purity of purified NsrR was determined to be greater than 90% judging from its electrophoresis on a 15% polyacrylamide gel containing SDS followed by staining with

Coomassie™ Blue. Iron and sulfide content were determined as previously described.

Site Directed Mutagenesis of Conserved Cysteine Residues of NsrR

The site direct mutagenesis of E. coli NsrR was carried out with QuikChange™ mutagenesis kit (Stratgene) and the pBAD-NsrR plasmid according to manufacturer’s instructions. The primer pairs used to incorporate individual missense mutations were: NsrR-

C91SF and NsrR-C91SR; NsrR-C96SF and NsrR-C96SR; and NsrR-C102SF and NsrR-C102SR (Table

4.2). Transformation, selection, and sequencing of these mutants were carried out identically to wildtype.

EPR Analysis

The X-band EPR spectra were obtained using a Bruker model ESR-300 spectrometer equipped with an Oxford Instruments 910 continuous-flow cryostat. Typical EPR parameters were: microwave frequency, 9.47 GHz; microwave power, 10.0 mW; modulation amplitude, 1.2 mT; sample temperature, 20 K; receive gain, 105 [33]. Quantification of the protein-bound

DNIC was calculated from the EPR signature at g = 2.04 from freshly prepared glutathione- bound DNIC as described in Vanin [34].

In Vivo NO Exposure of E. coli Anaerobically

Overnight cultures of E. coli containing recombinant plasmids of different [Fe-S] proteins were diluted 1:100 in fresh LB medium and incubated at 37°C with aeration at 250 rpm until the

O.D. reached 0.8. L-arabinose was added to the cultures at a final concentration of 0.02% and induction was carried out for two hours. Cells were harvested and resuspended to an O.D. of 2.0 in minimal medium. E. coli cells were transferred to 125 mL Erlenmeyer flasks with a stir bar and sealed with rubber septa with 4 needles representing two inlets and two outlets. The cell suspensions were treated with argon gas with gentle stirring for 10 minutes to ensure removal of oxygen before gaseous NO was applied to the cell suspensions. Argon was continuously passed through the cell suspensions in the presence of NO gas. The NO gas was first passed through a soda-lime column to ensure the removal of NO2 and higher nitrogen oxides before being connected to Silastic tubing with an inner diameter of 0.635 mm and an outer diameter of 1.1938 mm. The length of Silastic tubing was adjusted to ensure that a rate of 100 nM/sec was being exposed to the cell suspensions. After NO treatment for 4 minutes, the cell suspensions were continually degassed for an additional 2 minutes to purge dissolved NO.

Samples of the cell suspensions were immediately taken and placed into EPR tubes, transferred to a dry ice and ethanol mixture, and finally transferred to liquid nitrogen for storage.

All of the samples were subtracted from that of an untransformed MC4100 subjected to the same procedure. The EPR spectra of the cell suspensions were monitored as described in the previous section.

Otherwise, cells were collected via centrifugation, resuspended in 20 mM Tris, pH 8.0,

500 mM NaCl, 2.5 mM DTT, 10% Glycerol, and lysed via the French Press. Then cellular 96

debris was removed by centrifugation and the soluble cell extract was purified as described above.

Redox Midpoint Potential Determination

In order to determine the redox midpoint potential of the [2Fe-2S] cluster on NsrR, the method from Ding and Demple was used [35]. Briefly, a specially designed anaerobic cuvette was used for the redox titrations [36] that had an inlet and outlet used for maintaining anaerobic conditions while monitoring the UV-Vis spectrum. The NsrR solution and the cuvette were purged of O2 with argon for 50 minutes before titration of reducing equivalents. A small magnet was placed under the cuvette to maintain homogeneity and argon flow was maintained throughout the experiment. The redox potential was adjusted by injecting micro liter volumes of degassed 10 mM sodium dithionite in 1 M Tris (pH 8.0) with a gas-tight 10 µL Hamilton syringe through a rubber septum. The redox potential was monitored with a redox microelectrode.

The redox state of the [2Fe-2S] cluster of NsrR was monitored by measuring the absorbance at 416 nm, the λmax for the oxidized protein. The redox mediator safranine O was added to a final concentration of 3 µM. Since there is no isosbestic point for reference between reduced and oxidized [2Fe-2S] of NsrR, a basic integral was used to compare the area under the spectrum between the wavelengths of 438 nm and 394 nm. Once calculated, the integrals were normalized between 0 (fully reduced) and 1 (fully oxidized).

Radioactive Labeling of the Promoter of hmp

The promoter of hmp (phmp) was amplified by PCR using the primers hmpA1 and hmpA2

(Table 4.3). The amplified hmp promoter was purified twice via gel purification kit (Qiagen) according to manufacturer’s instructions. Radioactive labeling of the hmp promoter was carried out by using 10 U T4 Polynucleotide Kinase in 70 mM Tris-HCl pH 7.6, 10 mM MgCl2,

5 mM dithiothreitol, 2 µCi [γ32P]-ATP (Perkin Elmer), and 0.9 pmol of the hmp promoter in a 15 97

µL reaction. The labeling reaction was incubated for 45 minutes at 37°C. Following incubation, the sample volume was adjusted to 50 µL with sterile water and applied to an Illustra

ProbeQuant™ G-50 Micro Column (GE Healthcare) to remove unincorporated [γ32P]-ATP. The collected radioactive labeled hmp promoter was diluted to 10 nM using sterile water and saved at room temperature for two weeks.

Electro Mobility Shift Analysis

The ability of NsrR to bind the promoter of hmpA was carried out using electro mobility shift assays. A 4.5% polyacrylamide gel containing 2.7 mM sodium acetate (pH 7.5), 8.2 mM

Tris (pH 8.0), and 2.1% glycerol was used for the separation of protein-DNA complex from

DNA probe. The running buffer contained 10 mM Tris (pH 8.0) and 3.3mM sodium acetate (pH

7.5). The samples to be loaded on the gel typically contained a buffer consisting of 15 mM KCl,

2% glycerol, 3 mM MgCl2, and 2 mM Tris (pH 7.5) [37]. 1 nM of the radioactive hmpA promoter was included in each sample. NsrR was added between 1-100 ng. Reactions were allowed to carry out for 10 minutes at room temperature. In some instances, 10 mM sodium dithionite dissolved in 1 M Tris (pH 8.0) was used. In these experiments, extreme caution was taken to remove oxygen from all of the solutions by degassing with argon. The lanes of the gel were pre-washed with running buffer or running buffer with 10 mM sodium dithionite. The gel was pre-run for 10 minutes at 100 V. The samples were loaded and the gel was run for 40 minutes at 100 V. After electrophoresis, the gel was transferred to filter paper, dried, and x-ray 98

film was exposed overnight in cassette. Standard protocols were used to develop the autoradiograph 12 hours after insertion into the cassette.

In Vitro NO Consumption

A NO sensitive electrode (ISO-NOP) connected to an Apollo-4000 Free Radical

Analyzer was used to monitor NO concentration in a sealed vial with gentle stirring. The response of the NO electrode was less than 5 seconds and the observed current using the NO electrode was linearly proportional to the NO concentration from 0 to 500 µM. A NO releasing compound diethylamine NONOate (DEA-NO) was used as a NO donor in vitro. The concentration of diethylamine NONOate was measured from the absorption peak at 250 nm with an extinction coefficient of 6.5 mM-1cm-1. The anaerobic reaction vials were prepared by degassing with pure argon gas. Pre-degassed freshly prepared diethylamine NONOate dissolved in 20 mM Tris, pH 10.5 was injected into the sealed vial of 3 mL of pre-degassed 20 mM potassium phosphate buffer, pH 7.4. When the NO released from the diethylamine NONOate reached a plateau, pre-degassed protein sample was injected into the sealed vial anaerobically using a gas tight Hamilton syringe. The injection of an equal volume of degassed phosphate buffer was used as a reference [33].

RESULTS

Characterization of Purified NsrR

Overexpression of E. coli NsrR was initially carried out using a pET28b+ expression system. After induction with IPTG, the cell pellet was dark brown, an indication that expressed

NsrR may contain iron. However, after lysing the cells, > 90% of NsrR precipitated into the cellular debris pellet as evidenced by the SDS-PAGE analysis (data not shown). From this observation, we can infer that in high concentrations NsrR tends to aggregate. It is very important to try to exclude all O2 from the NsrR preparation. Interestingly another [Fe-S] 99

protein, MutY showed similar aggregation properties when one of its four conserved cysteine residues was mutated to an alanine [38].

In an attempt to obtain soluble NsrR, gene nsrR was subcloned into the pBAD-Myc-His

A expression vector. After induction with 0.02% arabinose, the cell pellets had a similar brownish color. The color remained in supernatants after cell lysis and subsequent centrifugation. NsrR was purified from the cell extract via two steps. The cell extract was first loaded onto a Ni-NTA column, and the eluate was then desalted using a HI-Trap desalting column. UV-Visible spectral analysis of purified NsrR revealed features comparable with other

[Fe-S] proteins (Fig. 4.2A). One unique absorption feature of NsrR is a peak with λmax at 416 nm and shoulder peaks around 460 nm and 580 nm (Fig. 4.2B).

The ratio of 416nm: 280nm varied slightly with preparation but was 0.25±.03 (n=3).

Iron and sulfide content analysis revealed 1.70 ± 0.21 moles of iron per mole of sulfide per mole of protein. It is important to note that NsrR did not maintain its structural integrity during the freeze-thaw process and was sensitive to atmospheric O2 during the purification procedure. Thus purified NsrR was maintained close to 0°C containing 40% glycerol and 2.5 mM DTT in degassed buffer.

Protein determination revealed that only 25% of NsrR is occupied with a [2Fe-2S] cluster which is consistent with the overexpression of other [Fe-S] proteins [39]. One explanation for this result may be that the [Fe-S] cluster assembly machinery is unable to keep pace with overexpressing NsrR Occupancy could also be an underestimate since NsrR does not have any tryptophan residues and there were contaminating proteins of higher molecular weight [40].

Other studies in Bacillus subtillus have suggested that the extinction coefficient should be 32 mM-1cm-1 for the [2Fe-2S] NsrR instead 8.04 mM-1cm-1 as calculated based on primary structure alone [40]. Using this extinction coefficient, occupancy would be close to 100%, which has not 100

been observed for overexpression of other [Fe-S] proteins. The SDS gel shows that purified

NsrR is > 90% pure (Fig. 4.2C). The molecular weight of untagged NsrR would be 15.59 kD, and the incorporation of the linker and histidine tag introduce an additional 2.66 kD to generate the recombinant NsrR of 18.24 kD as seen on the SDS gel (Fig. 4.2C).

EPR Analysis Suggests NsrR Contains a [2Fe-2S] Cluster

EPR has been routinely used for the classification of [Fe-S] proteins since their discovery in 1960 [41]. EPR is a technique analogous to NMR except the magnetic moment of 101

the electron is of interest instead of the nucleus. Typical [2Fe-2S] proteins coordinated by cysteines have an EPR signature signal upon reduction with dithionite called the g = 1.94 value.

Re-oxidation of the [2Fe-2S] cluster can be mediated by ferriccyanide more quickly than by atmospheric oxygen. Multiple [2Fe-2S] proteins including human ferrochelatase, plant ferredoxins, IscR, and SoxR have been identified via EPR [31, 42-44].

In order to determine the type of [Fe-S] cluster in NsrR we used EPR analysis. Freshly prepared NsrR (500 µM) did not generate an EPR signature (Fig. 4.3A). This is expected for an antiferromagnetically coupled [2Fe-2S]2+ . Upon the addition of excess sodium dithionite to the as-purified NsrR sample, there is an EPR signal at g = 1.94 [45].

A novel method for probing [Fe-S] proteins utilizes an in vivo approach. This method employs MC4100 E. coli strain with nonpolar deletions of iscA and sufA paralogs in combination.

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The iscA-sufA- double mutant strain is unable to grow aerobically in minimal media due to branched chain amino acid auxotrophy but shows substantial growth in rich media [32]. The iscA-sufA- double mutant fails to assembly [4Fe-4S] clusters in dihydroxyacid dehydratase, ThiC, aconitase B, and endonuclease III. On the other hand, the [2Fe-2S] clusters of SoxR, Fdx, and

FhuF are assembled in the iscA-sufA- double mutant [32].

pBAD-NsrR was introduced into both MC4100 and the iscA-sufA- double mutant. NsrR was expressed for 2 hours in each of the hosts, and then purified as previously described. The

UV-Visible spectrum for NsrR expressed from both hosts is nearly identical. Iron and sulfide are present in a 1:1 ratio and the [2Fe-2S] cluster occupancy of each sample is 18%. The presence

- of the λmax at 416 nm can be clearly seen in the proteins purified from MC4100 and the iscA

/sufA- double mutant, indicating that iscA and sufA are disposable in the synthesis of the [2Fe-2S] cluster in NsrR (Fig. 4.3B). Bacillus subtillus NsrR has recently been shown to be a [4Fe-4S] protein, and it was suggested that the existence of the [2Fe-2S] cluster is a degradation product

[40]. However, our EPR spectra and our evidence from the iscA-sufA- double mutant suggests a

[2Fe-2S] cluster on NsrR.

The Conserved Cysteines in NsrR are Involved in Ligating the [2Fe-2S] Cluster

Primary sequence similarity between IscR and NsrR indicates that the [2Fe-2S] cluster is ligated by three conserved cysteine residues and another unknown ligand [25]. To test the functional significance of theses cysteine residues, site-directed mutagenesis was carried out to substitute each cysteine with serine. UV-Visible spectra indicate that NsrR mutants are missing the unique λmax at 416 nm, indicative of the [2Fe-2S] cluster (Fig. 4.4). In addition, there is little detectable iron or sulfide present in these preparations. From this experiment, we concluded that each of the three conserved cysteine residues is involved in ligating the [2Fe-2S] cluster to NsrR.

The identification of the unknown ligand for the [2Fe-2S] cluster in NsrR will be challenging 103

until a crystal structure of NsrR is solved [46]. The use of other ligands to [Fe-S] clusters can modulate the electronic environment which may be crucial in NsrR’s ability to sense NO.

In Vivo Modification of NsrR by NO

[Fe-S] proteins are one of the major targets of NO cytotoxicity [47]. Upon reaction of

[Fe-S] clusters with NO, the iron center is directly modified and forms stable bonds with NO displacing sulfide concomitantly (Scheme 4.1). The resulting structure is a dinitrosyl iron complex (DNIC). DNICs are easily observed via EPR at g = 2.04 [48-50] . DNICs are important signals which may alert the cell to engage the oxidative stress response [10].

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DNICs were first reported for [Fe-S] proteins in Clostridium botulinum [50]. Since the initial discovery, DNIC’s have been reported in numerous purified [Fe-S] proteins, activated macrophages as a consequence of inducible nitric oxide synthase (iNOS), and in tumor cells cultured with activated macrophages [10, 33, 51, 52]. The expanding repertoire NO modification of [Fe-S] proteins implies a fundamental sensory mechanism that may have been exploited by the earliest proteobacteria [53].

Foregoing results suggest that NsrR contains a [2Fe-2S] cluster coordinated via three conserved cysteine residues and another unidentified ligand. Now it is important to understand the mechanism by which NsrR senses NO. MC4100 cells transformed with either the wild-type pBAD-NsrR or one of the cysteine to serine NsrR mutants were grown to mid log phase and then induced with 0.002% arabinose for 90 minutes. The cells were concentrated in minimal media to an O.D. of 40.0. The whole cell suspensions were subjected to gaseous NO for 4 minutes anaerobically, and aliquots were immediately frozen for EPR analysis as seen in Fig. 4.5.

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The whole cells transformed with wild-type NsrR showed a drastic increase in DNICs when compared to any of the mutants. The g = 2.04 amplitude for each sample was subtracted from a baseline sample of NO exposed MC4100 cells without plasmid. Evidently, NsrR is able to form DNIC in vivo. The presence of DNIC in the mutants may reflect other iron bearing proteins that are upregulated via an unknown mechanism involving apo-NsrR (which may mimic the NsrR-DNIC) [29]. Interestingly, one of the major upregulated genes upon NO exposure, ytfE, encodes the di-iron protein YtfE involved in [Fe-S] cluster repair under nitrosative stress

[54, 55]. Overexpressing the NsrR mutants may trigger the cell to produce YtfE constitutively.

Thus, the DNIC signal seen in the whole cell NsrR mutants may reflect an NsrR regulated iron protein.

Reactivity of NsrR with NO In Vitro

In order to determine the hierarchy of NO modification of [Fe-S] proteins, we compared the reactivity of NO with recombinant [Fe-S] proteins: NsrR-[2Fe-2S], Fdx-[2Fe-2S], and IlvD-

[4Fe-4S]. The reaction kinetics of these [Fe-S] proteins were investigated using the NO donor

DEA-NONOATE which releases 1.5 mol NO with a half life of 16 minutes at room temperature and pH 7.2. NO concentration in a pre-degassed sealed vial was monitored using an NO sensitive electrode until a steady state was reached. At steady state NO concentration, pre- degassed NsrR-[2Fe-2S], Fdx-[2Fe-2S] or IlvD-[4Fe-4S] were injected into the vial to a final iron concentration of 7 µM using a gas tight Hamilton syringe. As shown in Fig. 4.6A (blue trace), NsrR-[2Fe-2S] reacts rapidly with NO [56, 57]. IlvD-[4Fe-4S] has been previously shown to react with NO with a rate constant of 7.0 ± 2.0 x 106 M-2 s-1 [33]. However, the reaction of NO with Fdx [2Fe-2S] is much slower than the reaction of NO with NsrR. In regard to the similar reactivity of NO with IlvD-[4Fe-4S] and NsrR-[2Fe-2S], it can be corroborated that [Fe-S] proteins may be one of the major targets of NO cytotoxicity. Since NsrR was 106

proposed to function as a NO sensor, it is unsurprising that it has the most robust NO reactivity.

The reactions outlined in Fig. 4.6A were also monitored via EPR spectroscopy. The easily identifiable g = 2.04 signal of DNICs appears very quickly (within 30 seconds) after injecting

NsrR-[2Fe-2S] into the anaerobic NO solution (Fig. 4.6A). The signal intensity increases in samples taken at later times (Fig. 4.6C) which indicates progression of the reaction. However,

EPR analysis of Fdx-[2Fe-2S] does not reveal the g = 2.04 DNIC signature at any of the time points examined (Fig. 4.6D). The small (~ 10 µM) consumption as evidenced by the NO electrode on the reaction of NO with Fdx-[2Fe-2S] after 180 seconds may be attributed to secondary reactions including the modification of thiols [58]. Approximately the same concentration of NO (~ 10 µM) was consumed by NsrR within 30 seconds and the EPR signature clearly indicates the presence of DNICs. A comparison of wild-type NsrR and NsrR C91S shows the differential NO reaction rates (Fig. 4.6B). Due to the absence of substantial iron in NsrR

C91S, the total protein in each sample was calibrated. NsrR C91S does not react with NO unlike wild-type NsrR, and like in accordance with the Fdx-[2Fe-2S] results, EPR does not reveal

DNIC formation in NsrR C91S (data not shown).

Reactivity of NsrR with NO, In Vivo

The in vitro NO modification of [2Fe-2S] proteins suggests hierarchical control with

NsrR being highly reactive. Of course, this is the expected result if NsrR is a NO sensor protein.

Moreover, it would be of interest to determine if NsrR was more reactive with NO in vivo. To test this idea, recombinant NsrR and Fdx were overexpressed, concentrated in minimal medium to an O.D. of 2.0, and treated with NO anaerobically using Silastic tubing at a flow rate of 100 nmol/sec. At each time point, 25 mL of the cell suspension was withdrawn from NO treatment and placed on ice. Purification of the recombinant proteins from the 25 mL suspensions was carried out as previously described. 107

108

In vivo NO treatment of NsrR should be less sensitive than the in vitro approach because

NO reacts with other targets. EPR analysis of purified protein indicates in vivo NO treatment of

NsrR results in the formation of DNICs within the first minute (Fig. 4.7A). Similar treatment of

Fdx did not discern any noticeable DNIC after 4 minutes (Fig. 4.7B). It was important to show that the recombinant proteins expressed at similar levels (Fig. 4.7C) in order to exclude the possibility that there was not enough Fdx present to facilitate DNIC formation.

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Recovery of NsrR-DNIC Does Not Involve Nascent Protein Synthesis

The ability of an [Fe-S] protein to sense NO forming DNICs underpins one pathway of signal transduction. However, like a kinase needs a phosphatase, a protein bound DNIC needs to be repaired. Otherwise, there would be unregulated consequences of the signal. In this case, once NO levels decrease to the basal threshold, NsrR-DNIC must revert to NsrR-[2Fe-2S] to repress transcription of NO detoxification and defense genes.

It seems more plausible that [Fe-S] cluster machinery would repair the DNIC rather than protein degradation and resynthesis. The repair process would use proteins already present saving energy reserves. To test this idea, we designed an in vivo experiment to determine if nascent protein synthesis was needed to facilitate repair.

MC4100 transformed with pBAD-NsrR was expressed as previously described. Cells were concentrated to an O.D. of 2.0 in minimal media supplemented with 33 µg/mL of chloramphenicol and split into three flasks. One 500 µL aliquot from unmodified cells was collected and immediately frozen in an EPR tube (Without treatment). The remaining two samples were subjected to anaerobic NO treatment for 4 minutes. After NO treatment, a second

500 µL aliquot was collected and immediately frozen in an EPR tube (+ NO treatment). The cells from the remaining flask were collected, resuspended in pre-warmed fresh LB supplemented with 33 µg/mL of chloramphenicol, and incubated at 37°C and 250 rpm aerobically for an additional 45 minutes. After the incubation period, the cells were concentrated to O.D. 2.0 in minimal media, and a 500 µL aliquot was collected and immediately frozen in an

EPR tube (+ NO treatment, + Recovery). The remaining cells from all three flasks were lysed via French Press and purified as described previously.

In accordance with our previous data, overexpressed NsrR in MC4100 did not show an

EPR signature (Fig. 4.8A). After 4 minutes of gaseous NO treatment to overexpressed NsrR in 110

MC4100, the characteristic DNIC signal appeared (Fig. 4.8A). The DNIC signal from the NO- treated cells was abolished in vivo by simply incubating the cells in growth media (Fig. 4.8C).

Since chloramphenicol was included in the media to inhibit nascent protein synthesis, the removal of the protein bound DNICs occurs via a mechanism that is already expressed. In vitro, the removal of protein bound DNIC’s require IscS. 111

After purification of the bulk cell extract derived from the same cells as Fig. 4.8A, it appears evident that NO modification causes a small change in the UV-VIS spectrum of NsrR with a decreased absorbance at λmax 416 nm and λmax 320 nm. After recovery in LB medium, the

UV-Visible spectrum of NsrR-DNIC was restored to that of unmodified NsrR (Fig. 4.8B) Iron and sulfide analysis of untreated and recovered NsrR reveals a similar 1:1 ratio observed previously; however, NO treated NsrR had an iron to sulfide ratio of 7:1. The presence of any sulfide may be due to incomplete conversion of NsrR-[2Fe-2S] to NsrR-DNIC or other protein contamination. Analysis of the SDS gel (Fig. 4.8C) reveals that NsrR is not a target of protease in the DNIC form since the amount recovered from all samples is similar. This line of evidence points to a repair mechanism that does not require new protein synthesis whether it be NsrR or

[Fe-S] cluster repair proteins.

NsrR-[2Fe-2S] is Redox Active with an Estimated Midpoint Potential of -346 mV

The ability of [Fe-S] clusters to shuttle electrons is a feature exploited by both membrane bound and soluble proteins. The appearance of the g = 2.04 EPR signal for reduced as-purified

NsrR potentiated our interest in determining if the [2Fe-2S] cluster of NsrR is a redox active transcription factor. Redox potential modulating the activity of transcription factors has only been demonstrated in SoxR [35].

The most identifying feature of the [2Fe-2S] cluster of NsrR is the λmax at 416 nm which can be seen in Fig. 4.9A (black trace). It is now clear that this λmax at 416 nm is attributed to an oxidized [2Fe-2S]2+ cluster. Upon the addition of excess dithionite anaerobically (red trace), the

λmax at 416 nm disappears (Fig. 4.9A). This spectral change can be due to the conversion of

[2Fe-2S]2+ to [2Fe-2S]+. A hallmark of redox active proteins is their ability to interconvert between redox states, so we tried to rescue the oxidized spectrum by opening the cuvette to air and monitoring the kinetics of [2Fe-2S]+ re-oxidation. As seen in Fig. 4.9A (blue trace), after 112

one hour of exposure to atmospheric O2 there is a notable reappearance of the λmax at 416 nm. It is noted in Fig. 4.9B that after one hour of re-oxidation, we could only restore 40-50% of the initial oxidized [2Fe-2S]2+ cluster. A possible explanation for this result is that this experiment

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was carried out at room temperature which facilitates the precipitation of NsrR. The heightened overall absorbance due to the presence of precipitated protein would skew the results since the area under the curve would be hidden from the integration.

The addition of micro volumes of sodium dithionite allowed for reproducible tracing of the [2Fe-2S] cluster absorption versus redox potential. As judged from the Nernst plot in Fig.

4.9C, the redox midpoint potential can be determined as the inflection point on the curve. Fitting analysis suggests that the [2Fe-2S] cluster of NsrR has a redox midpoint potential of -345.7 ± 7 mV. E. coli maintains an intracellular redox potential of approximately -380 mV during exponential growth [59]. This suggests that the [2Fe-2S] cluster of NsrR is likely in a delicate balancing act between the [2Fe-2S]2+ cluster and the [2Fe-2S]+ cluster. The [2Fe-2S]2+ observed during purification procedures indicates that oxidation is an inevitable process of this protein.

Redox Potential Influences NsrR’s Ability to Bind the Promoter of hmpA

NsrR has been implicated in regulation of hmpA by several in vivo studies and the presence of a consensus NsrR binding motif upstream of hmpA [12, 25, 60]. Despite the complex regulation of hmpA by FNR, MetR, and possibly Fur, transcription of hmpA under anaerobic conditions is not carried out fully unless there is a source of NO present [61].

In order to access the functionality of NsrR [2Fe-2S]+,2+ on DNA binding, electro mobility shift analysis was performed. PCR was used to amplify the region of the hmpA promoter containing the NsrR binding site. The amplified product was 100 bp in length and was purified and radiolabeled with [γ32P]-ATP.

NsrR [2Fe-2S] was incubated with the hmp promoter (phmp) under different redox potentials. The autoradiograph shown in Fig. 4.10 shows differential DNA binding under different redox potentials. As determined by quantification, 30 ng of the reduced NsrR-[2Fe-

2S]+ binds radiolabeled phmp at least 4-fold better than the oxidized NsrR-[2Fe-2S]2+. For 114

comparison, 30 ng of NsrR in a 10 µL final volume corresponds to approximately 160 nM. The tighter repression of phmp under reduced conditions may be due to a more stable phmp-NsrR-

[2Fe-2S]+ complex. In order to access NsrR-[2Fe-2S]+ binding target promoters across a range of redox potentials, a different method must be used to more strictly control the redox potential.

Redox Potential Modulates NsrR’s DNA Binding Ability in the Presence of NO

The redox mediated differential DNA binding complicates the function of NsrR as a repressor. It was shown that under anaerobic conditions hmp needs a NO stimulus for complete transcription [61]. NsrR controls the expression of NO detoxification and defense genes, some of which are produced only anaerobically or under extremely micro-oxic conditions like nrfA encoding a periplasmic nitrite reductase [24, 62]. Taken together, it seems as though NsrR mainly operates as a transcriptional switch under anaerobic conditions where intracellular conditions are in the reduced state. Most likely, NsrR is still a transcriptional repressor in oxidative environments only exerting less of a repressor effect as shown in Fig. 4.10.

NO sensing by NsrR [2Fe-2S]+ under reduced conditions would likely be the most important for anaerobiosis. As demonstrated in Fig. 4.11A, NsrR [2Fe-2S]+ binds tightly to the 115

hmp promoter without NO treatment. However, when an in vitro NO-modified NsrR was used, binding to the hmp promoter was abolished in a concentration dependent manner. However, carrying out the same experiment at a higher reduction potential yielded different results. NsrR-

[2Fe-2S]2+ still binds to the hmp promoter but NO modification does not signal for NsrR-[2Fe-

2S]+ to release hmp (data not shown).

DISSCUSSION

Taken together, our results elucidate the complexity by which Rrf2-type transcriptional repressors regulate gene expression. E. coli NsrR is a transcriptional repressor of genes involved in cellular detoxification of NO [63]. NsrR utilizes a [2Fe-2S] cluster ligated by three conserved cysteine residues and another unknown ligand. It is possible that the unknown ligand could be histidine as suggested by primary sequence or a water ligand leading to the instability of the [Fe-

S] cluster. NsrR senses NO via modification of its [2Fe-2S] cluster to a DNIC as demonstrated with both in vitro and in vivo evidence. Modification of NsrR-[2Fe-2S] to NsrR-DNIC removes transcriptional repression of a target promoter.

IscR, the transcriptional repressor of the isc operon, is another member of the Rrf2 protein family. The similarity in protein structure and possible functionality suggests another link between [Fe-S] cluster biogenesis and nitrosative stress response [25]. Once NO destroys an

[Fe-S] cluster, NsrR and IscR may act in concert to eliminate NO stress and repair [Fe-S]

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clusters, respectively. One important difference between NsrR and IscR has emerged. IscR is functional in both apo and holo forms; however, to date NsrR has only been demonstrated to be functional in the holo form. In addition, there has been minimal success finding NsrR activated genes [64].

NO modification of [Fe-S] cluster containing transcriptional repressors was first identified for SoxR and later for FNR [10]. SoxR responds to NO as a secondary stimulus to superoxide but enhances soxS mRNA to similar levels. The [2Fe-2S] cluster of NsrR is not only modified by NO, but it is also redox active with an estimated midpoint potential of -345 ± 7 mV.

The reduced NsrR-[2Fe-2S]+ shows at least 4-fold better affinity for the target promoter than the oxidized NsrR-[2Fe-2S]2+. Purification of NsrR under oxic conditions could shift NsrR-[2Fe-2S] to the oxidized state. It would be interesting to determine if NsrR senses superoxide as a secondary stimulus in NO’s absence. The regulons of SoxR and NsrR are different, but both should be active under nitrosative and oxidative stress conditions.

A representation of the NsrR mediated regulation of hmp is given in Scheme 4.2. NsrR exerts its most potent repressor effect under reduced conditions when NO is absent (Scheme

4.2A). It would be a waste of cellular energy to activate genes under non-stress conditions.

NsrR [2Fe-2S]+ exhibits at least 3-fold tighter binding to hmp than NsrR [2Fe-2S]2+ (Scheme

4.2C). Acting as a secondary sensor of oxidative stress, NsrR [2Fe-2S]2+ only partially upregulates the NO stress response [65]. This activation may be a precautionary measure in case of accompanying nitrosative challenge. Derepression of hmp occurs when NsrR [2Fe-2S]+ senses NO forming NsrR-DNIC (Scheme 4.2B). When cellular NO concentrations reach the basal threshold, NsrR-DNIC can be efficiently repaired into NsrR [2Fe-2S] with existing cellular machineries likely including the proteins encoded by the isc operon and YtfE [66, 67].

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CHAPTER 5

CONCLUSIONS

SUMMARY

The experiments presented here boost the current state of knowledge of iron-sulfur ([Fe-

S]) cluster biogenesis and intracellular iron metabolism. With the advent of the genomics era, the field of [Fe-S] protein chemistry has moved into its adolescence with the discovery of novel

[Fe-S] requiring proteins. Using sequence alignment, we were able to define functionality of the human homolog of E. coli IscA, duly named hIscA. We were able to show how intracellular iron is trafficked from disrupted [Fe-S] clusters to ferritins under oxidative stress conditions.

Moreover, upon reestablishment of intracellular reducing conditions, we have shown that iron is mobilized out of the Ferritin A (FtnA) core and employed for reassembly of [Fe-S] clusters [1].

Lastly, in unveiling [Fe-S] proteins as major cellular targets of nitric oxide (NO), we have elucidated the biophysical properties of the global nitric oxide (NO) sensitive transcriptional repressor NsrR.

The functions of the [Fe-S] cluster assembly proteins from humans are not as well characterized as their homologs in bacteria and lower eukaryotes. Humans have two homologs of Escherichia coli (E. coli) IscA, encoded by the genes ISCA1 and ISCA2. hIscA contains an N- terminal mitochondrial targeting peptide [2]. We have cloned, expressed, purified, and characterized the protein hIscA encoded by ISCA1. The results indicate that hIscA is a specific iron binding protein with an apparent iron association constant of 3.0 x 1020 M-1, similar to what has been reported for E. coli IscA [3]. hIscA’s iron center can be mobilized by L-cysteine and transferred to E. coli IscU for [Fe-S] cluster assembly. Furthermore, when iron-loaded horse spleen ferritin was incubated with apo-hIscA in the presence of the thioredoxin reductase system,

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hIscA became an iron-loaded species signifying the recruitment of iron by hIscA from horse spleen ferritin as seen for the E. coli proteins (data not shown). It is important to note the [Fe-S] cluster assembly process in E. coli does not differentiate between IscA and hIscA. This finding is significant in that the [Fe-S] cluster assembly process functions using proteins derived from different species. This finding suggests that the overall mechanism of [Fe-S] cluster biogenesis is similar in bacteria and humans.

To determine mammalian expression of hIscA, we used murine tissues (kindly provided by Dr. Joomyeong Kim) since murine IscA (mIscA) is 100% identical in amino acid sequence to hIscA. We found that mIscA is expressed at different levels amongst the tissues tested with the highest expression in the murine brain, heart, and kidneys. The role of hIscA in repair of [Fe-S] clusters has not been explored in mammalian cells, but the localization patterns indicate hIscA expression correlates to tissues that may be susceptible to oxidative stress. Interestingly, the extract derived from murine lungs did not show any hIscA localization. This may indicate that

O2 tensions are too high for hIscA to bind iron and is not used as the iron donor for [Fe-S] cluster biogenesis in the lungs. Since ISCA2 has yet to be studied, it is impossible to rule out tissue specific expression with the most suitable hIscA functioning as the iron donor in human [Fe-S] cluster assembly. Indeed, there are potential post-translational events that can alter hIscA’s function in eukaryotic cells that may lead to differential localization.

One of the major questions addressed by this research was a simple one. Where does the iron come from? It has been known for almost 10 years that the inorganic sulfide in [Fe-S] clusters is the product of cysteine desulfurase activity on L-cysteine [4]. Advances in the field have demonstrated the involvement of [Fe-S] cluster assembly scaffolding proteins (IscU,

SufBCD), folding chaperone proteins (HscB, HscA), reducing factors (Fdx), and multiple apo- protein targets [5-8]. 126

Gene knockout analysis has identified phenotypes associated with deletions of members of the isc operon. Most notably are deficiencies in the [Fe-S] cluster assembly process. The most severe deletion is iscS- strain [9]. In addition to being the more robust cysteine desulfurase for [Fe-S] cluster assembly, IscS also participates in other sulfur and selenium transferase reactions including the biosynthesis of 4-thiouridine, thiamine, biotin, molybdopterin, and 2- selenouridine [10-15]. Overexpressing SufS, which has not been implicated in the other sulfur transferase pathways, cannot complement iscS- strain.

With so much information regarding the exact nature of sulfur incorporation into biomolecules, it seems surprising that an iron donor for [Fe-S] cluster assembly has still been elusive. In E. coli there are at least two candidates to fulfill the role of the iron donor: IscA and

CyaY [16-21]. Interestingly, no one had considered the possibility of multiple iron donors until

Ding et al. demonstrated a significant role for both IscA and CyaY under different cellular conditions [22]. IscA binds iron specifically and tightly, albeit with low occupancy (0.5 Fe: monomer). On the other hand, CyaY accumulates iron through carboxylate ligands on its surface and then oligimerizes [23]. CyaY has been compared to the ferritin superfamily in regard to its ferrous and ferric iron binding, detoxification properties, and oligimerization. In vitro, CyaY has a higher occupancy (up to 26 Fe/monomer) than IscA, but much lower occupancy than the ferritins (187 Fe/monomer). The iron association constant for CyaY is 1014-fold less than IscA under physiologically relevant reducing conditions [24]. Hypothetically, if CyaY could bind 107

Fe / monomer, there would still need to be 103.5 more functional copies of CyaY to begin competing with one functional copy of IscA in terms of iron binding.

Since parallels were drawn between CyaY and the ferritins, we carried out the same experiments outlined in Chapter 3 substituting CyaY for FtnA. Unsurprisingly, the results were nearly identical (data not shown). CyaY could inhibit the hydroxyl radical formation from 127

disrupted [Fe-S] clusters under H2O2 stress conditions, and CyaY was found to directly bind the iron released from the disrupted [Fe-S] clusters. In addition, when iron-loaded CyaY was incubated with apo-IscA in the presence of the thioredoxin reductase system, iron would be transferred from CyaY to IscA.

Under H2O2 stress conditions, IscA is incapable of binding iron. The proposed mechanism for this abolishment in iron binding is that IscA ligates iron through cysteine thiols that are oxidized to a disulfide in the presence of H2O2 [22]. Since CyaY uses an acidic amino acid patch to bind iron, H2O2 stress does not impact iron binding. Dual iron donors represent a self check mechanism in the event that one is lost, but there is no doubt that there is a preference to use one under different cellular conditions.

The ability of FtnA to buffer iron released from disrupted [Fe-S] clusters is novel. Free ferrous iron in cells would cause drastic damage via the Fenton reaction. Upon oxidative challenge by H2O2, the nascent [Fe-S] clusters on IscU are destroyed. FtnA sequesters the released iron in a non toxic form until the oxidative burst has subsided. Then upon the return of reducing conditions, IscA recruits iron from FtnA.

[Fe-S] clusters are not only targets of oxidative stress, but also they are modified by the gaseous free radical signaler nitric oxide (NO). The auto-oxidation of NO in the presence of O2 is 2.0 x 106 M-2 s-1 whereas NO reacting with certain [Fe-S] proteins occurs at rates of 7.0 ± 2.0 x

107 M-2 s-1 [25, 26]. Thus, the rate of NO reacting with [Fe-S] clusters is at least 3-fold greater than its auto-oxidation. This high reactivity of NO with [Fe-S] clusters has been exploited by the transcription factor SoxR and newly characterized NsrR. NsrR uses a [2Fe-2S] cluster as an NO sensing mechanism. When an NO stimulus is presented, NsrR-[2Fe-2S] is modified to NsrR-

DNIC. NsrR-DNIC loses affinity for its target promoters thereby allowing transcription. The de

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novo transcription of the gene nsrR would provide pivotal in determining the optimal conditions for regulation.

Characterization of NsrR from Streptomyces coelicolor has recently been reported.

Interestingly, the addition of up to 20 mM sodium dithionite had little effect on the UV-visible absorbance spectrum of NsrR indicating that the cluster is stable in the presence of dithionite

[27]. Tucker et al. concluded that the [2Fe-2S] cluster of NsrR from S. coelicolor had a reduction potential too low to be effectively reduced by this powerful reductant [27]. However, we have found that E. coli NsrR is redox active with differential DNA binding affinity between the reduced [2Fe-2S]+ cluster and the oxidized [2Fe-2S]2+ cluster. It is no surprise that oxidation of the [2Fe-2S] of NsrR promotes gene expression. Cellular targets of reactive oxygen species

(ROS) and reactive nitrogen species (RNS) are in many cases the same molecules, and the reactions between ROS with RNS superposes their respective response network crosstalking

[28]. Flavohemoglobin is the major NO detoxification system, yet only functions in the presence of O2. As mentioned previously SoxR-[2Fe-2S] can sense NO to stimulate the SoxRS regulon

[29]. Similarly, here we have shown that NsrR senses redox potential to stimulate its own

• - regulon. It would be interesting to determine if superoxide ( O2 ) or H2O2 could directly stimulate the NsrR regulon.

FUTURE RESEARCH GOALS

In regards to human [Fe-S] cluster biogenesis, there is still much to be explored.

Homologs of E. coli IscA, IscS, and IscU have been mapped to different chromosomes in human genome. These genes are named ISCA1, ISCA2, NFS1, and NFU2. The NFS1 and NFU2 proteins are differentially spliced to generate various isoforms targeted to the mitochondria, cytosol, and nucleus [30-33]. The iron donors in the cytosol and nucleus have yet to be identified but could be post translational modified hIscA or human frataxin. Interestingly, 129

nuclear targeting of the human cysteine desulfurase NFS1 and [Fe-S] cluster assembly scaffold

NFU1 appear to be a repair mechanism since mRNA translation occurs in the cytosol. Other

[Fe-S] assembly proteins have also been identified in eukaryotes including humans. These proteins have specifically been implicated in cytosolic and nuclear [Fe-S] cluster assembly:

CFD1, IOP1, CIA1, NBP35, and NAR1 [34-38].

In vitro systematic comparisons of the potential human iron donors with NFU1 and NFS1 could shed some light on human [Fe-S] cluster assembly. Co-localization experiments using the anti-hIscA antibody could enable more tissue specific pathways. Specific ABC transporter proteins like the Atm1 and Atm2 identified in yeast are likely to be involved with maturation of

[Fe-S] proteins [39-41].

Further experiments involving the aforementioned iron trafficking in E. coli could detail more systematically the affinities of FtnA and CyaY for iron derived from disrupted [Fe-S] clusters. Logically, it would seem more beneficial for CyaY to bind iron first since it could directly participate in [Fe-S] cluster assembly on IscU under oxidative stress conditions.

Reduction of the FtnA core involves recycling the NADPH/NADP+ pool which would require more energy [42, 43]. However, the relative amounts of CyaY and FtnA in E. coli would be a major player in the hydroxyl radical detoxification scheme.

The redundancy of the ferritin genes in E. coli potentiates more complex iron regulation than revealed. For instance, Dps may be the preferential iron storage protein in stationary phase cells when it is abundantly expressed by sigma factor [44, 45]. The ability of Dps to nonspecifically bind DNA and the fact that it uses H2O2 as the oxidant for iron mineralization suggests a different oxidative stress defense system whereby the DNA is crystallized like eukaryotic chromosomes [46, 47]. Dps provides a physical buffer between DNA and cellular milieu. It would be of interest to determine the most efficient iron storage system in regard to 130

[Fe-S] cluster biogenesis. Can IscA retrieve iron more readily from bacterioferritin (Bfr)? In addition, the ability of Bfr to reduce NO to N2O upon oxidation of ferrous iron suggests a role in the NO stress response [48].

Recently, it has been revealed that an E. coli iscA-sufA- strain of is unable to grow in minimal media under aerobic conditions [49]. Supplementation with thiamine and branched chain amino acids restores aerobic growth. The only detectable phenotype in rich media is the slow doubling time. Interestingly, incorporation of [Fe-S] clusters into overexpressed proteins in the iscA-sufA- strain only occurs for [2Fe-2S] proteins. Apparently, in iscA-sufA- there is an aberration in [4Fe-4S] cluster biogenesis suggesting differential maturation for [2Fe-2S] clusters and [4Fe-4S] clusters. [49].

Ideally, the triple mutant iscA-sufA-erpA- should be generated because ErpA shares sequence homology to IscA and SufA. Could ErpA be the iron donor of last resort for the [2Fe-

2S] assembly? ErpA is functionally involved in isoprenoid biosynthesis; moreover, deletion of erpA results in a null phenotype if grown in the presence of O2. Anaerobic analysis of ErpA may provide better answers into [2Fe-2S] assembly [50]. As of September 2008, almost 70% of all known [Fe-S] folds harbor [4Fe-4S] clusters. This staggering percentage signifies the importance of IscA and SufA in [Fe-S] cluster biogenesis [51].

Using site directed mutagenesis, we observed all cysteine to serine mutants resulted in

NsrR devoid of any detectable iron and sulfide. There has been no in vivo functional assignment to apo-NsrR; however, it merits further research since apo-IscR coordinates gene regulation in E. coli [52-54]. Apo-NsrR may resemble NsrR-DNIC in function allowing the constitutive expression of NO stress related defense and detoxification genes. A crucial feature of pathogenic bacteria invading mammalian cells is their ability to withstand the oxidative burst of activated macrophages [55, 56]. In this case, evolution may have exerted selective pressure on 131

those networks constitutively expressing the NO defense genes using apo-NsrR or no repressor at all.

The full extent of the NsrR regulon has not yet been fully established; there are at least 20 genes identified in NO-mediated microarray studies, but many of these genes are of unknown function [57, 58]. Their NsrR regulation should start functional studies. Interestingly, one of the most highly upregulated genes upon NO exposure is ygbA, a putatively small protein with 4 well positioned cysteine residues raising the possibility of the identification of another [Fe-S] protein

[59].

Another NO specific transcription factor, NorR, has been identified in E. coli; however, it only regulates one operon containing two genes [60, 61]. NorR uses mononuclear iron instead of a [2Fe-2S] cluster to sense NO. [2Fe-2S] clusters, like the one present in NsrR, may provide more flexibility in the control of gene regulation evidenced by the extent of the NO responsive regulators [62-66]. The sheer number of genes regulated by NsrR signifies that something about it makes it use more ubiquitous.

Little information is available regarding the Rrf2 family of transcription factors to which

NsrR and IscR are members [59, 67, 68]. Crystallization studies on these proteins would provide great insight into their gene regulation, but they have not been successful to date. Their tertiary and quaternary structures could provide a detailed model of the [Fe-S] cluster binding site around the three conserved cysteine residues. Additionally, candidates for the remaining ligand could be processed. Mutagenic studies could then be attempted to incorporate a fourth cysteine residue in

NsrR potentially allowing formation of a stable [2Fe-2S] cluster. Similar mutagenic studies on

FNR, the global transcriptional regulator in response to changes in O2 tension, generated a protein variant L28H more stable than the wildtype protein [69, 70]. Consequently, O2 impact

132

on FNR’s ability to acquire [Fe-S] cluster has suggested the isc operon plays a more significant role than the suf operon [69].

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APPENDIX: PERMISSION TO INCLUDE PUBLISHED WORK

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VITA

Jacob Philip Bitoun is the son of the Maurice Bitoun and Ann Woods. He was born in

Harvey, Louisiana. He attended high school at Louisiana School for Math, Science, and the Arts located in Natchitoches, Louisiana. He attended Louisiana State University in the fall of 2001 and graduated with a Bachelor of Science in biochemistry with a minor in chemistry in the winter of 2004. He began his doctoral research immediately thereafter under the supervision of

Dr. Huangen Ding. He will graduate with a Doctor of Philosophy in biochemistry in the summer of 2009.

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