CHARACTERIZATION OF THE PROPERTIES AND ROLES OF FE-S CENTERS IN

FERREDOXINS, AND RADICAL S-ADENOSYL METHIONINE

ENZYMES

by

SOWMYA SUBRAMANIAN

(Under the Direction of Michael K. Johnson)

ABSTRACT

The objectives of the research presented in this dissertation are to characterize the properties and to understand the role of Fe-S centers in [2Fe-2S]-ferredoxins, glutaredoxins and anaerobic sulfatase maturating enzymes (anSMEs), by using a combination of mutagenesis, analytical and spectroscopic studies. The spectroscopic techniques include UV-visible absorption, circular dichroism, variable temperature magnetic circular dichroism (VTMCD), EPR, Mössbauer, resonance Raman and MALDI-TOF MS. A detailed spectroscopic characterization of Cys-to-Ser variants of [2Fe-2S]-ferredoxin from Clostridium pasteurianum and Aquifex aeolicus were undertaken in order to characterize the electronic properties of + valence-delocalized [Fe2S2] center, which constitutes the building block of higher-nuclearity Fe-S clusters. VTMCD and magnetization studies enabled the first ever measurement of the double exchange parameter (B) for a valence delocalized [2Fe-2S]+ cluster with sulfide bridging ligands. Spectroscopic studies also revealed that the [2Fe-2S]+ cluster in the valence-delocalized S = 9/2 state at room temperature converts, either partially or fully to valence-localized S = 1/2 state upon freezing. The results are interpreted to be an outcome of changes in solvent packing and H-bonding interactions near the reducible iron site that occur upon freezing and shift the equilibrium in favor of the valence-localized form. Studies carried out to understand the nature and function of Fe-S centers in Arabidopsis thaliana GrxS16, a monothiol in chloroplast, revealed that recombinant GrxS16 is able to assemble a [2Fe-2S] cluster as-purified, and [2Fe-2S] and [4Fe-4S] clusters on in vitro reconstitution. The ability of GrxS16 to transfer both [2Fe-2S] and [4Fe-4S] cluster to plausible acceptor suggests a role for S16 in the maturation of Fe-S proteins in chloroplast. To elucidate the nature of Fe-S centers and the mechanism of action of anSMEs, analytical and spectroscopic studies were undertaken on the wild-type and variant anSMEs from Clostridium perfringens and Bacteriodes thetaiotamicron. The results reveal that anSMEs can bind three [4Fe-4S] clusters, and activate both Cys-type and Ser-type sulfatases via radical mechanism initiated by the reductive cleavage of S-adenosyl methionine bound to the radical SAM cluster. The two additional [4Fe-4S] clusters are proposed to play a role in the activation of substrate and/or a relay that shuttles electron during enzymatic turnover.

INDEX WORDS: Valence delocalization, ferredoxin, glutaredoxin, radical SAM enzymes, electron paramagnetic resonance, resonance Raman, magnetic circular dichroism, Mössbauer.

CHARACTERIZATION OF THE PROPERTIES AND ROLES OF FE-S CENTERS IN

FERREDOXINS, GLUTAREDOXINS AND RADICAL S-ADENOSYL METHIONINE

ENZYMES

by

SOWMYA SUBRAMANIAN

B.Sc., S.D.N.B. Vaishnav College, Madras, India, 1997

M.Sc., Anna University, Madras, India, 1999

M.S., University of Delaware, 2003

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2010

© 2010

Sowmya Subramanian

All Rights Reserved

CHARACTERIZATION OF THE PROPERTIES AND ROLES OF FE-S CENTERS IN

FERREDOXINS, GLUTAREDOXINS AND RADICAL S-ADENOSYL METHIONINE

ENZYMES

by

SOWMYA SUBRAMANIAN

Major Professor: Michael K. Johnson

Committee: Michael W. W. Adams Robert A. Scott

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia December 2010

iv

TABLE OF CONTENTS

Page

LIST OF TABLES...... vii

LIST OF FIGURES ...... viii

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ...... 1

Iron-sulfur proteins: Background...... 1

Brief summary of presented work...... 3

Structure and coordination of Fe-S clusters...... 4

Redox properties of Fe-S clusters...... 6

Electronic properties of Fe-S clusters...... 7

Valence delocalization in [2Fe-2S]+ clusters...... 9

Glutaredoxins: roles in Fe-S cluster biogenesis and iron homeostasis...... 13

Radical SAM enzymes...... 17

Abbreviations...... 21

References...... 22

2 SPECTROSCOPIC AND REDOX STUDIES OF VALENCE-

DELOCALIZED [2FE-2S]+ CENTERS IN THIOREDOXIN-LIKE

FERREDOXINS...... 54

Abbreviations...... 55

Abstract...... 56

v

Introduction...... 58

Materials and Methods...... 61

Results...... 62

Discussion...... 73

Acknowledgements...... 80

References...... 81

3 NATURE AND FUNCTION OF FE-S CLUSTERS ASSEMBLED ON

GRXS16, A CHLOROPLASTIC MONOTHIOL GLUTAREDOXIN...... 127

Abbreviations...... 128

Abstract...... 129

Introduction...... 130

Materials and Methods...... 133

Results...... 137

Discussion...... 143

Acknowledgements...... 147

References...... 148

4 ANAEROBIC SULFATASE-MATURATING ENZYMES, FIRST

DUAL SUBSTRATE RADICAL S-ADENOSYLMETHIONINE ENZYMES .....168

Abbreviations...... 169

Abstract...... 170

Introduction...... 171

Experimental Procedures ...... 173

Results...... 179 vi

Discussion...... 187

Acknowledgements...... 191

References...... 192

5 ANAEROBIC SULFATASE-MATURATING ENZYME: A MECHANISTIC LINK

WITH GLYCYL RADICAL ACTIVATING ENZYMES?...... 223

Abbreviations...... 224

Abstract...... 225

Introduction...... 226

Experimental Procedures ...... 227

Results...... 231

Discussion...... 240

Acknowledgements...... 244

References...... 245

6 CONCLUSIONS AND FUTURE WORK ...... 279

References...... 285

vii

LIST OF TABLES

Page

Table 2.1: Redox potential and pKa of the WT and variant forms of AaeFd4 and CpFd ...... 84

Table 3.1: Mössbauer parameters for the cluster bound forms of At GrxS16 ...... 152

Table 3.2: Spin Hamiltonian and magnetic hyperfine parameters used in fitting the Mössbauer

spectrum of the S = 5/2 linear [3Fe-4S]+ cluster component observed in fraction 1 of

At GrxS16 reconstituted in the presence of GSH...... 153

viii

LIST OF FIGURES

Page

Figure 1.1: Crystallographically defined structures for biological iron-sulfur clusters that are

involved in electron transfer ...... 33

Figure 1.2: Crystallographically defined structures of selected biological Fe-S centers involved

in substrate binding and activation ...... 35

Figure 1.3: Range of mid-point potentials (mV vs. NHE) for biological Fe-S centers ...... 37

Figure 1.4: Ground state spin (S) and valence-delocalization schemes for the fundamental types

of Fe–S centers...... 39

Figure 1.5: Illustration of the Robin-Day classification of mixed valence complexes...... 41

Figure 1.6: Schematic comparison of an antiferromagnetically coupled Fe(II)/Fe(III) pair and a

ferromagnetically coupled Fe(II)/Fe(III) pair (A); Energy level diagram for an

exchange coupled [2Fe-2S]+ cluster as a function of increasing spin-dependent

resonance delocalization (B)...... 43

Figure 1.7: A view of the superposition of the Anabaena thioredoxin-2 and monomer of

WT AaeFd4 (A); Electron density map of the oxidized [2Fe-2S] center in the

Cys59Ser variant of AaeFd4 crystal structure solved at a resolution of

1.05 Å (B) ...... 45

Figure 1.8: Domain organization in Class 1 through Class 6 Grxs ...... 47

Figure 1.9: Stereoview of the contrasted orientation of glutaredoxin monomers toward each

other in E. coli monothiol Grx4 (A) and in human dithiol Grx2 (B)...... 49

ix

Figure 1.10: A model illustrating possible roles for Grxs in Fe-S cluster assembly machineries

in plant plastid...... 51

Figure 1.11: General scheme of reactions involved in the generation of the S-adenosyl radical

in Radical SAM enzymes (A); Illustration of the chemical reaction leading to the

formation of S-adenosyl radical in Radical SAM enzymes (B)...... 53

Figure 2.1: UV-visible absorption spectra of oxidized and dithionite-reduced WT AaeFd4 at

pH 11.0 and pH 7.0...... 86

Figure 2.2: UV-visible absorption spectra of oxidized and dithionite-reduced C55S AaeFd4 and

C56S CpFd at pH 7.0...... 88

Figure 2.3: UV-visible absorption spectra of oxidized and dithionite-reduced C55S AaeFd4 and

C56S CpFd at pH 11.0...... 90

Figure 2.4: UV-visible absorption spectra of oxidized and dithionite-reduced C59S AaeFd4 and

C60S CpFd at pH 7.0...... 92

Figure 2.5: UV-visible absorption spectra of oxidized and dithionite-reduced C59S AaeFd4 and

C60S CpFd pH 11.0...... 94

Figure 2.6: Dependence of the mid-point potential of the WT, C56S and C60S variants of

CpFd on pH, determined by film voltammetry...... 96

Figure 2.7: CD spectra of dithionite-reduced C55S AaeFd4 measured as a function of pH ...... 98

Figure 2.8: CD spectra of dithionite-reduced C59S AaeFd4 measured as a function of pH ...... 100

Figure 2.9: Comparison of the X-band EPR spectra of the dithionite-reduced C55S and C59S

AaeFd4 and C56S and C60S CpFd in the S = 1/2 region at pH values above and

below the pKa values...... 102

Figure 2.10: X-band EPR spectra in the low-field region for dithionite-reduced C55S (A) and x

C59S (B) AaeFd4 at pH 11.0 ...... 104

Figure 2.11: VTMCD spectra of the dithionite-reduced C55S AaeFd4 at pH 7.0 and at

pH 11.0...... 106

Figure 2.12: VTMCD spectra of the dithionite-reduced C59S AaeFd4 at pH 7.0 and at

pH 12.0...... 108

Figure 2.13: UV-visible-NIR VTMCD spectra of dithionite-reduced C56S CpFd at pH 6.0 and

pH 11.0...... 110

Figure 2.14: UV-visible-NIR VTMCD spectra of dithionite-reduced C60S CpFd at pH 6.0 and

pH 11.0...... 112

Figure 2.15: VHVT MCD magnetization data at 712 nm (A) and 1070 nm (B) for dithionite-

reduced C56S CpFd at pH 11.0 ...... 114

Figure 2.16: VHVT MCD magnetization data at 705 nm (A) and 1070 nm (B) for dithionite-

reduced C60S CpFd at pH 11.0 ...... 116

Figure 2.17: Comparison of the RTMCD spectra of C55S AaeFd4 and C56S CpFd at pH 7.0 and

12.0...... 118

Figure 2.18: Comparison of the RTMCD spectra of C59S AaeFd4 and C06S CpFd at pH 7.0 and

12.0...... 120

Figure 2.19: Schematic summary of the proposed ligation, spin states and redox properties of

the [2Fe-2S]2+,+ centers in the Cys-to-Ser variants of CpFd and AaeFd4 as a

function of pH...... 122

Figure 2.20: Schematic MO diagram for the Fe-Fe interactions in a valence-delocalized S = 9/2

[2Fe-2S]+ cluster with polarizations and assignments for the predicted

electronic transitions ...... 124

xi

Figure 2.21: Comparison of the low temperature MCD spectra of the valence-delocalized S = 9/2

[2Fe-2S]+ center in high pH reduced C60S CpFd with those of higher nuclearity

Fe-S clusters believed to contain valence delocalized [2Fe-2S]+ fragments ...... 126

Figure 3.1: UV-visible absorption and CD spectra of recombinant At Grx S16, as purified

under anaerobic conditions, fraction 1 of a sample reconstituted in the presence of

GSH, and reconstituted in the presence of DTT...... 155

Figure 3.2: Comparison of the resonance Raman spectra of the anaerobically purified [2Fe-2S]

cluster-bound forms of At Grx S16 and At Grx S14 with 514-, 488- and 457-nm

laser excitation ...... 157

Figure 3.3: Comparison of the resonance Raman spectra of anaerobically purified At GrxS16

and fraction 1 of the At GrxS16 reconstituted in the presence of GSH with 514-,

488- and 457-nm laser excitation...... 159

Figure 3.4: Mössbauer spectra of cluster-bound forms of At GrxS16: anaerobically purified;

fractions 1 and 2 of a sample reconstituted in the presence of GSH; reconstituted

in the presence of DTT ...... 161

Figure 3.5: Analytical gel filtration elution profiles for the reconstituted fractions of At GrxS16,

using a SuperdexTM column ...... 163

Figure 3.6: Time course of [2Fe-2S] cluster transfer from At GrxS16 to apo Syn Fd monitored

by UV-visible CD spectroscopy ...... 165

Figure 3.7: Time course of [4Fe-4S] cluster transfer from At GrxS16 to apo At Nfu2 monitored

by UV-visible CD spectroscopy ...... 167

Figure 4.1: Sequence alignment of the three anSME putative clusters in AtsB, anSMEcpe and

anSMEbt ...... 196

xii

Figure 4.2: (A) Gel electrophoresis analysis of anSMEcpe (lane 1) and anSMEbt (lane2);

(B) UV-visible absorption spectra of reconstituted anSMEbt and anSMEcpe...... 198

Figure 4.3: UV-visible absorption spectra of as isolated and reconstituted anSMEcpe...... 200

Figure 4.4: Resonance Raman spectra of anSMEcpe as isolated (A), reconstituted (B), and

reconstituted, in the presence of a 20-fold excess of SAM (C) ...... 202

Figure 4.5: EPR spectra of anSMEcpe after anaerobic reduction with a 10-fold excess of

sodium dithionite ...... 204

Figure 4.6: AdoMet reductive cleavage assayed by reverse phase HPLC ...... 206

Figure 4.7: In vitro maturation of 23-mer peptides with reconstituted anSMEcpe analyzed by

MALDI-TOF MS with CHCA (A) or DNPH matrix (B)...... 208

Figure 4.8: In vitro maturation of 23-mer peptides with reconstituted anSMEcpe analyzed by

MALDI-TOF MS with CHCA matrix ...... 210

Figure 4.9: MALDI-TOF MS analysis of the serine (A) and FGly-containing peptide (B)

purified by HPLC...... 212

Figure 4.10: In vitro maturation of 23-mer peptides with reconstituted anSMEbt analyzed by

MALDI-TOF MS with CHCA (A) or DNPH matrix (B)...... 214

Figure 4.11: In vitro maturation of 23-mer peptides with reconstituted anSMEbt analyzed by

MALDI-TOF MS with CHCA matrix ...... 216

Figure 4.12: In vivo maturation of C. perfringens sulfatase expressed alone (1) or in the

presence of anSMEcpe (2) or anSMEbt (3)...... 218

Figure 4.13: In vivo maturation of the serine mutant of C. perfringens sulfatase expressed

alone (1) or in the presence of anSMEcpe (2) or anSMEbt (3) ...... 220

Figure 4.14: Schematic mechanism for the maturation reaction catalyzed by anSMEs...... 222

xiii

Figure 5.1: Sulfatase maturation scheme leading from a cysteinyl or seryl residue to a FGly in

sulfatase active site (A); MALDI-TOF MS analysis of maturation of peptide

17C (B) and 17S (C) incubated with anSMEcpe...... 251

Figure 5.2: MALDI-TOF MS analysis of 17-mer peptides, 17C (A), 17S (B) and 17A (C) after

incubation with anSMEcpe, using a CHCA matrix...... 253

Figure 5.3: MALDI-TOF MS analysis of 17-mer peptides, 17C (A) and 17S (B) after incubation

with anSMEcpe, using a DNPH matrix ...... 255

Figure 5.4: HPLC analysis of incubation reactions of anSMEcpe with peptide 17C (A) and

17S (B) and time-dependant formation of FGly-containing peptide (C) and

5′-deoxyadenosine (D)...... 257

Figure 5.5: Sequence alignment of the three anSMEs putative clusters in AtsB, anSMEcpe and

anSMEbt (A) and UV visible absorption spectra of reconstituted wild-type and

M1, M2 and M3 variants of anSMEbt (B)...... 259

Figure 5.6: Gel electrophoresis analysis of wild-type and the M1, M2 and M3 variants of

anSMEbt ...... 261

Figure 5.7: UV-visible absorption spectra of oxidized and dithionite-reduced, as purified and

reconstituted wild-type and M1 mutant anSMEbt...... 263

Figure 5.8: X-band EPR spectra of dithionite-reduced reconstituted samples of wild-type and

M1 mutant anSMEbt in the absence (A) and presence (B) of a 20-fold stoichiometric

excess of AdoMet ...... 265

Figure 5.9: Low field X-band EPR spectra of dithionite-reduced WT anSMEcpe in the presence

and absence of a 20-fold excess of AdoMet ...... 267

Figure 5.10: HPLC (A) and MALDI-TOF MS (B) analysis of the peptide maturation

xiv

catalyzed by WT and M1, M2 and M3 mutants of anSMEbt...... 269

Figure 5.11: MALDI-TOF MS analysis of 17C peptide after incubations with wild-type and

M1, M2 and M3 mutants of anSMEbt ...... 271

Figure 5.12: Sequence alignment of anSMEcpe, quinohemoprotein amine dehydrogenase, PqqE

and the ST ...... 273

Figure 5.13: Two possible mechanisms for anSMEs with Cys-type sulfatases substrate (A).....275

(B) ....276

Figure 5.14: MALDI-TOF mass spectrometry analysis of 17C peptide before (1) and after a

16 18 4 hour incubation with anSMEcpe in H2 O (2) or H2 O (3) buffer (A) and

Potential mechanism for isotope exchange via hydrolysis of the thioaldehyde

intermediate (B) ...... 278

1

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Iron-sulfur proteins: Background

Proteins containing iron sulfur (Fe-S) clusters or iron coordinated by at least one sulfur

ligand (iron sulfur proteins) as a prosthetic group constitute one of the most versatile class of

proteins, finding representation in all life forms, starting from the prokaryotes including archaea

and bacteria to higher eukaryotes, including plants, animals and humans (1;2). The plethora of

functions associated with these proteins manifests in many of the vital processes needed to

sustain life, the most prominent ones being photosynthesis, respiration, and carbon and nitrogen

fixation. While the primary role of biological Fe-S centers lies in mediating electron transfer,

extensive research over the past five decades has identified new roles and functions such as

coupling of electron and proton transfer (3-7), substrate binding and activation (3;8;9), DNA damage recognition and repair (10-12), regulation of expression (13-19), regulation of enzyme activity (20-22), disulfide reduction (23-25), iron, electron and cluster storage (26-28) and as sacrificial sulfur donors (29-31). Iron sulfur proteins are also structurally diverse, with mononuclear Fe, homometallic sulfide-bridged [2Fe-2S], [3Fe-4S], [4Fe-4S] and [8Fe-7S] clusters, and heterometallic sulfide-bridged clusters, coordinated by a wide range of cysteine motifs and protein folds and exhibiting redox potentials that span the entire physiological range,

−700 to +500 mV (32). In addition to cysteine, which is the most common ligand, Fe-S centers can have partial histidine, aspartate, serine, arginine, backbone amide nitrogen, solvent or substrate ligation (3). Moreover, Fe-S centers exhibit rich electronic ground and excited 2

state properties, which result from a combination of Heisenberg exchange exchange interactions

and spin-dependent resonance delocalization or double exchange interactions (2;3). The mechanism of in vivo assembly of Fe-S clusters and their incorporation into apo proteins is currently an area of rapid research growth with important medical and biotechnology

implications. While major progress has been made over the past decade (33-36), a molecular level understanding of iron sulfur cluster assembly and trafficking has yet to emerge.

Defects in Fe-S cluster biogenesis can lead to metabolic disorders for two main reasons.

First, Fe-S proteins are crucial to numerous metabolic processes, and their deletion or disruption can be lethal or result in impaired cell viability. Second, free iron and sulfide that results from defective cluster biogenesis or cluster degradation under oxidative stress conditions is cytotoxic.

Fe-S clusters can be spontaneously assembled from Fe2+/Fe3+ and S2- under reducing conditions.

However, free cellular iron is toxic due to the generation of hydroxyl radicals by Fenton

chemistry (37) and free sulfide is an inhibitor of cyclooxygenase and short-chain fatty acid oxidation (38). Hence, cellular iron and sulfide trafficking are tightly regulated and Fe homeostasis is closely linked to the synthesis of Fe-S clusters. An example of deleterious effects due to the disruption of an Fe-S cluster has been observed in DNA repair enzymes, Fanconi anemia group J protein (FancJ) and Xeroderma pigmentosum group D protein (XPD) which facilitate DNA damage recognition and repair. Mutations that disrupt the [4Fe-4S] cluster in

FancJ and XPD result in the lack of helicase activity leading to autosomal recessive genetic disorders called Fanconi anemia and trichothiodystrophy, respectively (39;40). Diseases linked to excess Fe accumulation caused by defects in the Fe-S cluster assembly machinery and Fe homeostasis include Friedrich’s ataxia (FA) (41) and X-linked with ataxia

(XLSA/A) (42). FA is a progressive neurodegenerative disease caused by a deficiency in 3

frataxin (putative iron donor for the Fe-S cluster biosynthetic machinery in eukaryotes) that

results in an accumulation of free iron in the mitochondria. XLSA/A on the other hand, results

due to defects in the activity of the delta-amino levulinic acid synthase enzyme (43), which

catalyzes the first step in heme biosynthesis, thus resulting in anemia, and the resultant

accumulation of iron leading to ataxia. XLSA/A has also been observed to result from mutations

in the gene coding for the ABCB7 transporter in mitochondria (44), implicated in the transport of

a Fe-S cluster precursor from the mitochondria to the cytosol, which affects further downstream

processes such as the biosynthesis of heme, resulting in anemia and free iron accumulation.

Recently, it was also shown that mutations in the human monothiol glutaredoxin, GLRX5 gene,

which plays a complex and poorly understood role in Fe-S cluster biosynthesis and Fe

homeostasis resulted in sideroblastic anemia (45). Thus, Fe-S cluster biosynthesis and Fe

homeostasis are strongly inter-related and any disruption or imbalance results in metabolic

disorders (46;47).

Brief summary of presented work

The work presented in this dissertation spans a variety of projects, touching on many

aspects of the properties, chemistry, function and assembly of Fe-S proteins. Chapter 2 explores the origin of valence delocalization in [2Fe-2S]+ clusters in Cys-to-Ser variants of thioredoxin-

like ferredoxins and Fe-S clusters in general, as such units are components of almost all higher nuclearity Fe-S clusters and facilitate rapid electron transport by minimizing reorganization

energy associated with oxidation or reduction. Chapter 3 investigates the nature and type of Fe-S

clusters that can be assembled in vitro on recombinant Arabidopsis thaliana GrxS16, a plant

monothiol glutaredoxin, and addresses its role in Fe-S cluster assembly in plant chloroplasts.

Finally, chapters 4 and 5 address the nature and role of the Fe-S clusters in two radical, 4

S-adenosylmethionine (SAM) sulfatase maturating enzymes which catalyze post-translational

modification of a cysteine/serine residue to form an active site aldehyde moiety in bacterial

sulfatases, under anaerobic conditions.

Structure and coordination of Fe-S clusters

Fe-S clusters are structurally classified based on the number of irons used to build the

cluster, the most common being the dinuclear [2Fe-2S] cluster, linear or cubane [3Fe-4S] cluster

and cubane [4Fe-4S] clusters. Iron coordinated tetrahedrally to four cysteinyl ligands, [Fe(Cys)4]

as in rubredoxin, is also considered to be a part of the family of iron-sulfur centers due to

similarities in the function and coordination of the Fe site, and because they provide information

on the properties of isolated Fe(III)/(II) sites with tetrahedral S ligation

The fundamental building block of all Fe-S clusters is the planar Fe2(µ2-S)2 rhomb involving two Fe sites attached via two bridging sulfides. The higher nuclearity clusters, like the cubane [3Fe-4S] and [4Fe-4S] clusters can be considered as two [2Fe-2S] rhombs fused together as in Fe3(µ2-S)3(µ3-S)(SCys)3 and Fe4(µ3-S)4(SCys)4, shown in Figure 1.1. Most of the Fe-S

clusters have an all-cysteinyl ligation providing tetrahedral S ligation at each Fe site, but there

are several instances of histidine, aspartic acid and arginine providing one or two of the cluster

ligands. For example, in Rieske proteins, the [2Fe-2S] cluster is coordinated by two cysteines at

one Fe site and two histidines at the other Fe site (5). The structures shown in Figure 1.1A-E,

illustrate the general structural themes of the clusters found in Fe-S proteins, and their function is

usually that of a one-electron donor or acceptor in electron transport processes. The more

complex double cubane [8Fe-7S] P cluster, see Figure 1.1F, is found only in and

has the potential to mediate two-electron transfer processes. 5

The general structural theme discussed so far varies when the Fe-S proteins are involved

in catalytic roles. Fe-S clusters involved in catalysis generally have one non-cysteinyl-ligated

iron site that binds and activates the substrate. This is well illustrated by the [4Fe-4S] cluster active sites of the IspH isoprenoid biosynthesis enzyme and the radical SAM family of enzymes.

IspH is the terminal enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. The non-mevalonate pathway is specific to most pathogenic bacteria and plant plastids, but is absent in animals, making this enzyme pathway an important drug target. The recent crystal structure of

IspH (48) shows the active-site [4Fe-4S] cluster with the unique iron bound to the alcohol moiety of the substrate, 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) (Figure 1.2A).

Although the mechanistic aspects of the system are still not clear concerning a radical or anionic intermediate, recent crystallographic and ENDOR studies suggest that there is a definite interaction between the unique iron and the allylic group of the substrate (49). In radical SAM enzymes, a [4Fe-4S] cluster binds S-adenosylmethionine at the unique Fe site of the [4Fe-4S] cluster via the amido and carboxylate groups (see Figure 1.2B), and initiates a reductive cleavage to yield a 5'-deoxyadenosyl radical and methionine (50;51). Catalytic biological Fe-S centers can also be generated by inclusion of a heterometal or attachment of an additional metal center. For example, carbon monoxide dehydrogenase, an enzyme involved in CO2 fixation (52;53)

catalyzes the oxidation of CO to CO2 at the [Ni-4Fe-5S] cluster active site and can be visualized

as a [3Fe-4S] cluster connected to a [Ni-(µ2-S)-Fe] fragment via three (µ3-S) sulfides, with the

square planar Ni binding the substrate CO (Figure 1.2C). Another example of an enzyme with a

heterometallic Fe-S cluster is , which catalyzes the reduction of N2 to NH3 in an ATP

dependent reaction as well as the reduction of a variety of multiple bonded substrates such as

alkynes, nitriles, CO and azides (54). The Fe-S clusters contained in the nitrogenase enzyme 6

include a typical [4Fe-4S] cluster, the P cluster and the FeMo-cofactor (see Figures 1.1E and F

and 1.2 D). The P cluster is comprised of two fused [4Fe-4S] clusters that share a µ6-sulfide and

two µ2- bridging Cys-S at the interface of the two clusters (55). The FeMo-cofactor is a

heterometallic Mo-Fe-S cluster, [Mo-7Fe-9S-X] that is attached to the protein by a single

cysteinyl-S-Fe and histidyl-N-Mo linkage with homocitrate ligated by the 2-hydroxy and 2-

carboxy groups completing octahedral coordination at the Mo center (56). The Mo-Fe-S cluster

itself can be described as a cubane MoFe3S3 fragment attached to a Fe4S3 fragment by three μ2-

2- S and a single μ6-X atom where X =(C, N or O).

Redox potential of Fe-S clusters

Biological Fe-S clusters exhibit a wide range of redox potentials which are fine tuned by

several factors, such as the nature of ligation at the reducible Fe, protein-ligand secondary

interactions and the extent of solvent exposure at the site of reduction (3). The range of mid-

point potentials for redox cycling between the most common redox states for the different types

of clusters is summarized in Figure 1.3 (57). The potential of the [2Fe-2S]2+,+ redox couple ranges from +380 mV to −150 mV for Rieske-type clusters, which have two electropositive histidine ligands coordinated to the reducible Fe, and from +100 mV to −460 mV for [2Fe-2S]

clusters with complete cysteinyl ligation. Redox cycling between the [2Fe-2S]+,0 states is about

1.0 V lower than the [2Fe-2S]2+,+ couple, and so far has been observed only in the Rieske type

[2Fe-2S] clusters, where the [2Fe-2S] is stabilized by protonation of the µ2-sulfide (58). In the

case of the cubane [3Fe-4S] clusters, the redox potential of the [3Fe-4S]+,0 couple is quite

similar to that of the all-cysteinyl-ligated [2Fe-2S]2+,+ and lies between +90 to -460 mV. [4Fe-

4S] clusters exhibit a wider range of redox potential, +500 to -700 mV due in large part to the

ability to cycle between either the 3+/2+ or 2+/1+ core oxidation states. The [4Fe-4S]2+,+ centers 7

have redox potentials between +80 to -715 mV and the High Potential Iron sulfur protein (HiPIP)

type centers cycle between the [4Fe-4S]3+,2+ states and have higher potentials between +50 to

+500 mV.

Electronic properties of Fe-S clusters

The ground state electronic properties of Fe-S clusters are dictated by intracluster

exchange interactions and the extent of valence delocalization. Figure 1.4 gives a summary of the ground state spin (S) and valence delocalization schemes for the fundamental types of Fe-S

clusters, and their electronic properties are discussed below (57).

[2Fe-2S] clusters. Biological and synthetic [2Fe-2S]2+,+ clusters generally have two

accessible redox states. The oxidized form exhibits a S = 0 ground state as a result of antiferromagnetic exchange interaction between two high-spin (S = 5/2) Fe3+ ions. The mixed-

valence reduced form exhibits a S = 1/2 ground state with an axial or rhombic g-tensor, as a

result of antiferromagnetic exchange between localized-valence high-spin (S = 5/2) Fe3+ and high-spin (S = 2) Fe2+ ions, as shown in Figure 1.4. However, an exception to this general rule

was observed for the [2Fe-2S]+ clusters in two Cys-to-Ser mutants of a thioredoxin-like [2Fe-2S]

ferredoxin from Clostridium pasteurianum (59;60), which was found to exist in a S = 9/2 ground

state due to ferromagnetic interaction between the high-spin (S = 5/2) Fe3+ and high-spin (S = 2)

Fe2+ ions, as depicted in Figure 1.4. Ferromagnetic interaction facilitates valence delocalization within the [2Fe-2S]+ core resulting in formal Fe2.5+ oxidation states for both Fe sites, as a result

of an extra electron having anti-parallel spin that can visit both Fe sites without undergoing a

spin flip.

[3Fe-4S] clusters. Cubane [3Fe-4S] clusters have been observed in biology, as either an

active component of redox active site in enzymes, such as succinate dehydrogenase or as a result 8

of oxidative degradation of the fourth iron of [4Fe-4S] clusters, as in aconitase (61;62). The

cubane-type [3Fe-4S] clusters involved in mediating electron transport have two accessible

oxidation states in vivo. In the oxidized form, the [3Fe-4S]+ cluster has three S = 5/2 Fe3+ ions

which couple antiferromagnetically to give a S = 1/2 ground state, with an isotropic or a near

axial EPR signal (62). In the reduced state, the [3Fe-4S]0 exhibits a S = 2 ground state resulting

from antiferromagnetic interaction between a valence-delocalized Fe2+/Fe3+ pair (S = 9/2) and a

valence-trapped Fe3+ site (S = 5/2), as depicted in Figure 1.4.

[4Fe-4S] clusters. The cubane [4Fe-4S] clusters constitute one of the most prevalent cofactors involved in electron transfer. The cubane [4Fe-4S] clusters usually undergo one- electron redox cycling between either the [4Fe-4S]3+,2+ or [4Fe-4S]2+,+ couples under

physiological conditions. From EPR and Mössbauer studies, the [4Fe-4S]3+ form has been

rationalized to exist in a S = 1/2 ground state resulting from antiferromagnetic exchange

interaction between the Fe3+/Fe3+ pair (S = 5) and the valence delocalized Fe2+/Fe3+ pair (S =

9/2), with an axial EPR signal. On the other hand, the [4Fe-4S]2+ in S = 0 ground state is EPR

silent resulting from anitferromagnetic exchange interaction between two valence delocalized

Fe2+/Fe3+ pairs (S = 9/2). The [4Fe-4S]+ form exists in a S =1/2 ground state, resulting from

antiferromagentic exchange interaction between Fe2+/Fe2+ pair (S = 4) and the valence delocalized Fe2+/Fe3+ pair (S = 9/2), with a distinct rhombic EPR signal. In some instances,

depending on the structural, electronic and environmental factors, the reduced [4Fe-4S]+ form can exist as a mixture of species with S = 1/2 and 3/2 ground states (63-65). The all-reduced

[4Fe-4S]0 state has by far been documented only in the Fe protein of the nitrogenase enzyme

where four high-spin Fe2+ ions couple in a 3:1 alignment of up:down spin vectors to give a S = 4

ground state (see Figure 1.4) (66;67). 9

Valence delocalization in [2Fe-2S]+ clusters

Mössbauer studies have shown that valence-delocalized [2Fe-2S]+ units are intrinsic

components of almost all higher nuclearity biological Fe-S clusters and are required to

rationalize the ground-state magnetic and electronic properties. Valence delocalization in Fe-S

clusters serve to minimize the reorganization energy associated with electron transport and

ensures rapid electron transfer in biological systems (68). Thus, it is essential to understand the

origins of valence delocalization, since it is critical to the interpretation of kinetics and

thermodynamics associated with electron transfer reactions, in addition to the electronic and

magnetic properties of iron sulfur proteins.

Complexes involved in electron transfer, such as Fe-S clusters have the ability to exist in

mixed-valence states, where the metals exist in Mn+/M(n+1)+ oxidation state. Robin and Day (69) classified mixed valence complexes into three classes based on the ease of intramolecular electron transfer between the metal sites, and an illustration of the potential energy curves for the three classes is shown in Figure 1.5 (70). If the valences on two metal sites remain isolated or if the extra added electron remains localized or “trapped” on a metal site upon reduction, it belongs to class I and the resonance energy, β = 0. On the other hand, if the intra-electron transfer between the two metal sites can be induced photolytically or thermally to overcome the activation barrier, then the complex falls under class II when |β| < λ, where λ is the reorganization energy. Finally, if the intra-electron transfer between the two metal sites is spontaneous, i.e., if the extra added electron is delocalized over the two sites, when |β| > λ, it belongs to class III. Iron-sulfur clusters can display either localized (class I) or delocalized valence (class III) depending on the environment and oxidation state of the cluster and the coordination geometry, ligation, redox potentials and solvent exposure at individual Fe sites. 10

Valence localization in the reduced cluster is promoted by a large localization energy

(ΔE), which includes contributions from vibronic coupling and the magnitude of the difference in

site redox potentials, and is manifest by strong antiferromagnetic exchange interaction, which restricts delocalization of the extra electron by requiring a spin flip to visit both Fe sites. In contrast, valence delocalization is expected to require a significant Fe-Fe interaction, such that the resonance delocalization energy overwhelms the localization energy, and results in the switch to ferromagnetic exchange interaction and a S = 9/2 ground state, so that the extra electron can

visit both Fe sites without undergoing a spin flip. The above discussion indicates that valence

delocalization in [2Fe-2S]+ clusters equates to spin-dependent resonance delocalization and it is

parameterized by the double exchange parameter, B, where 10B = 2β, and β is the classical

resonance energy that is more familiar to chemists. Figure 1.6A illustrates the ferromagnetic and

antiferromagnetic spin alignment of a [2Fe-2S]+ cluster fragment and the dependence of its

ground state properties on the relative magnitude of Heisenberg exchange and spin-dependent

resonance delocalization. The energy level scheme is based on a Hamiltonian with both

Heisenberg-Dirac-vanVleck (J) and double exchange (B) terms that results in E = -JS(S + 1)±

B(S + 1/2) (71). This simple model neglects vibronic interactions and assumes that the valence-

localized species with the extra electron on the two iron sites, FeA and FeB are isoenergetic. As

the extent of resonance delocalization (B/J) increases, the ground state changes from S = 1/2 to

9/2 in integer steps, becoming S = 9/2 for │B/J│ > 9 (Figure 1.6B). Inclusion of the factors

responsible for valence localization, i.e. vibronic coupling and inequivalence in the energies of

the valence trapped species, decreases the B/J range in which the ground state has S ±3/2 or ±7/2.

This diminishes the likelihood of observing these intermediate-spin ground states and leads towards a situation in which the ground state changes directly from valence-localized S = 1/2 to 11

valence-delocalized S = 9/2 with increasing B/J. Hence the absolute value of B/J and the

energetic factors responsible for valence localization, determines both the ground state spin and

the extent of valence delocalization.

Although valence delocalization is not required to explain the properties of known

examples of isolated [2Fe-2S] clusters in wild-type proteins, Mössbauer studies have shown that

valence delocalized [2Fe-2S]+ units are intrinsic components of almost all higher nuclearity

biological Fe-S clusters and are required to rationalize the ground state magnetic properties. As

illustrated in Figure 1.4, it subsequently became apparent that valence-delocalized [2Fe-2S]+ units are a fundamental building block in [3Fe-4S]0,- and [4Fe-4S]3+,2+,+ and are required to

explain the ground state magnetic properties. While these results emphasized the importance of

valence delocalization for understanding the electronic and magnetic properties of Fe-S clusters and their efficacy in mediating rapid electron transport, the structural and electronic origin of valence delocalization in [2Fe-2S]+ units were poorly understood due to the lack of examples of

magnetically isolated, valence-delocalized [2Fe-2S]+ clusters.

This situation changed in 1995 with the discovery of S = 9/2 valence-delocalized [2Fe-

2S]+ clusters in variants of the thioredoxin-like Clostridium pasteurianum [2Fe-2S] ferredoxin

(Cp [2Fe-2S] Fd) in which either of the cysteines coordinating the reducible Fe site were

substituted with serine (C56S and C60S). Both EPR and MCD studies of dithionite-reduced

samples revealed a mixture of S = 1/2 and 9/2 [2Fe-2S]+ clusters, and the similarity in the

electronic transitions of the S = 9/2 component as revealed by MCD with those of clusters known

to contain valence-delocalized [2Fe-2S]+ units, strongly suggested a ferromagnetically coupled,

valence-delocalized [2Fe-2S]+ cluster (59;60). Definitive evidence for valence delocalization was subsequently provided by Mössbauer spectroscopy (72). Subsequently, spectroscopic and 12

redox studies revealed that midpoint potential of the [2Fe-2S]2+,+ couple is pH-dependent due to

protonation of the reduced cluster with a pKa ~ 9 and that S = 9/2 component is optimal at

alkaline pH. This was tentatively interpreted in terms of serinate coordination at high pH

resulting in valence delocalization and protonation of the coordinated serinate with a pKa of ~9 leading to serine or water coordination at neutral or acidic pH values that favors valence

localization. However, Mössbauer and saturation magnetization studies showed that the ratio of S

= 9/2 and 1/2 [2Fe-2S]+ clusters was maximally 1:1 even at pH 11, and surprisingly revealed that

the S = 1/2 component at alkaline pH is valence localized at low temperatures, but becomes

valence delocalized without a spin-state change at high temperatures (transition temperature ~

100 K) (73). The ability to observe and investigate S = 9/2 valence-delocalized [2Fe-2S]+ clusters in Cp [2Fe-2S] Fd represents a major advance in understanding valence delocalization in

Fe-S clusters. Nevertheless, the inability to obtain a stable form of the Fd with a homogeneous S

= 9/2 valence-delocalized [2Fe-2S]+ cluster, coupled with the absence of high resolution

structural data for this Fd, has impeded structural, electronic and vibrational characterization and

progress in understanding the origin of valence delocalization.

The impetus for the studies proposed herein was provided by the availability of a crystal

structure for the [2Fe-2S] ferredoxin from the hyperthermophilic bacterium, Aquifex aeolicus

ferredoxin-4 (AaeFd4), which is a homolog of Cp [2Fe-2S] Fd (74). The crystal structure of the

oxidized wild-type protein at 2.3 Å resolution revealed a unique fold, similar to thioredoxin,

which led to the recognition of a new class of ferredoxins, termed thioredoxin-like-ferredoxins.

A view of the superposition of Anabaena thioredoxin-2 (orange) and a monomer of AaeFd4

(Figure 1.7A) exemplifies the similarity of the fold, and also the positioning of the cysteine

residues of AaeFd4, Cys9 and Cys22 in the region between β-strand 1 and α-helix 1 of the 13

thioredoxin fold similar to the positioning of Cys32 and Cys35 residues that form the disulfide bond in thioredoxin. The crystal structure prompted the engineering of cysteine-to-serine variants analogous to those investigated in Cp [2Fe-2S] ferredoxin in an effort to understand valence delocalization in the new class of ferredoxins. The crystal structures of the oxidized C55S and

C59S variants of AaeFd4 at 1.25 Å and 1.05 Å resolution, respectively, and of wild-type oxidized AaeFd4 at 1.5 Å, have provided structural data at atomic resolution (Figure 1.7B) (75).

The crystallographic data confirm serinate ligation of the oxidized [2Fe-2S]2+ cluster and reveal

that serinate ligation is accompanied by a significant distortion in the [2Fe-2S] core and

shortening of the Fe-Fe distance by 0.04 Å. Hence, the objectives of the proposed spectroscopic

studies of AaeFd4 were to determine if valence-delocalized S = 9/2 [2Fe-2S]+ clusters can be

observed in wild-type or variant forms, define the conditions to optimize the formation of these

species, and proceed with detailed spectroscopic and crystallographic characterization in order to

assess structural, electronic, magnetic, and vibrational determinants of valence delocalization.

Glutaredoxins: roles in Fe-S cluster biogenesis and iron homeostasis

Glutaredoxins (Grxs) are small dithiol-disulfide which have traditionally been considered to play an essential metabolic role as intrinsic components of redox regulation or oxidative stress protective mechanisms (76;77). They share functions with thioredoxins (Trxs) in maintaining and regulating the cellular redox state. However, recent results have indicated roles for Grxs in iron homeostasis and Fe-S cluster biogenesis (78). The involvement of Grxs in

Fe-S cluster biogenesis, coupled with the ability of some Grxs to bind [2Fe-2S] clusters and transfer them to acceptor proteins (79) has led to putative roles for Grxs in the assembly, delivery, storage, or regulation of Fe-S clusters (78). Furthermore, the recent re-classification of

Grxs, based on genomic analyses (80), has identified more complexity in the sequence/structure 14

of these small proteins and has also redefined the various roles taken up by Grxs in cellular

metabolism.

Grxs have been broadly grouped into 6 classes, based on the sequence of the motif

containing the active site cysteine, glutathione binding motifs and the number of protein domains

(Figure 1.8) (80). Dithiol Grxs with active sites such as CGYC, CPFC, CSYC/S and CPYC/S

active site belong to class 1 and have been classified under various subgroups and are widely

represented across all three kingdoms of life. All dithiol Grxs investigated so far, have been able

to catalyze the reduction of disulfides by either a monothiol or dithiol mechanism, and a handful

have been shown to bind [2Fe-2S] clusters. Functional studies on the cluster-bound dithiol Grxs

investigated so far, suggest a role for these proteins in protection against oxidative stress (81;82).

Monothiol Grxs with CGFS active site fall under class 2, and are also widely distributed across all three kingdoms of life, although multi-domain monothiol Grxs with CGFS active site are specific to eukaryotic cells. The CGFS monothiol Grxs have been shown to assemble Fe-S clusters, both in vivo and in vitro and play an important role in Fe-S cluster assembly and iron

homeostasis (78). Class 3, with a CCxC/S active site is specific to terrestrial plants, and has been

implicated in the reproductive cycle of flowering plants (83;84). Grxs with a CxDC/S and CPxC

active sites are present in terrestrial plants and algae, respectively, and constitute class 4 of Grxs.

They are also characterized by the presence of two domains of unknown function, in addition to

the Grx domain, and their cellular role is currently unknown. Class 5 and Class 6 have CPWG

and CPWC/S active sites, respectively. They are present exclusively in cyanobacteria and their

functions are currently unknown.

Most of the early evidence concerning the role of Grxs in Fe-S cluster biogenesis was

obtained from studies of the monothiol CGFS Grx, Grx5 in Saccharomyces cerevisiae. Deletion 15

of the grx5 gene resulted in impaired respiratory growth and increased sensitivity to oxidative stress due to deficient cluster assembly of Fe-S proteins in the mitochondria and accumulation of free iron in the cell (85;86). Further, 55Fe immunoprecipitation studies indicated that Grx5 plays

a role in mediating transfer of clusters preassembled on the Iscu1p scaffold protein to acceptor

proteins (87). Interestingly, the Fe-S cluster bound Grx5 has also been shown to be essential for

vertebrate heme synthesis and regulation of bone apoptosis (45;88).

The potential role for monothiol CGFS Grxs in Fe-S cluster assembly or trafficking was

further demonstrated by the recent crystal structure of the [2Fe-2S] cluster-bound form of E. coli

Grx4 (89). In accord with spectroscopic and analytical studies (79;82;90), the crystal structure

revealed a homodimer with one subunit bridging [2Fe-2S] cluster ligated by the cysteines of the

active site CGFS motifs (Cys30), with the cysteines of two trans glutathiones completing

tetrahedral coordination at each Fe site. The crystal structure revealed contrasting orientations of

the protomers in monothiol versus dithiol Grxs, suggesting a plausible mechanism for the

transfer of the [2Fe-2S] cluster to acceptor proteins by cluster-bound monothiol Grxs (Figure

1.9). The conformational changes in the loop region, β1-α2 presents a contrasted position of two

critical residues, Lys22 and Cys30 which facilitates binding of the [2Fe-2S] cluster, in the dimer

versus the apo monomer. Also, Ser33, specific to the monothiol active site is involved in an

interaction with Lys22 backbone amine, stabilizing the position of the β1-α2 loop in the

homodimer, versus the apo monomer. Thus, the residues involved in coordinating and stabilizing

the cluster are poised to bind the bridging [2Fe-2S] cluster in the homodimeric form versus the

monomeric form, suggesting interactions with acceptor and/or chaperones to trigger

conformational changes that would release the cluster, facilitating an intact cluster transfer. On

the other hand, the dithiol Grxs reflect no major conformational changes on going from a 16

monomeric to dimeric form, and perfectly superimpose on each other, giving evidence for a

structural change that could trigger cluster release.

Recent studies have identified two [2Fe-2S] cluster-bound homodimeric cystosolic

monothiol Grxs with CGFS active site in Saccharomyces cerevisiae, Grx3 and Grx4, that form

[2Fe-2S] cluster-bound heterodimeric complexes with Fra2, a eukaryotic homolog of the

ubiquitous prokaryotic BolA proteins (91). Moreover, the cluster-bound heterodimeric Grx3/4-

Fra2 complex has been shown to play a central role in regulation of the Fe regulon in yeast (91-

93). The role of Fe-S cluster-bound Grx3/4 as a sensor for the intracellular iron status and as an

intracellular iron trafficking protein has been further suggested by the recent work of Mühlenhoff and co-workers (94). In this work, it was demonstrated that the depletion of Grx3/4 affected mitochondrial and cytosolic Fe-S cluster biogenesis, and hence the activity of mitochondrial and

cytosolic Fe-S proteins and other iron-dependent enzymes, like heme and di-iron centers,

eventually leading to cell death. Further, mutation of the active site cysteine to alanine in Grx3/4

was shown to abolish mitochondrial enzymatic activity under iron replete conditions, suggesting

a role for Grx3/4 in intracellular iron trafficking.

In plants, monothiol Grxs with CGFS active sites are represented by four proteins:

GrxS14 and S16, localized in the chloroplast; GrxS15, localized in the mitochondria; and

GrxS17, which has been predicted to be cytosolic (79). Of the four Grxs, GrxS14, S16 and S17

were able to rescue the mutant grx5 defects in respiratory growth and sensitivity to oxidants in

yeast, but not GrxS15. It was also demonstrated that GrxS14 was able to assemble and transfer

an intact [2Fe-2S] cluster to apo ferredoxin at a rapid rate, indicating possible roles for monothiol

Grxs in the maturation of Fe-S proteins in chloroplast. Among the chloroplastic proteins, SufE1,

an essential component of the SUF (Sulfur Mobilization) assembly machinery contains an N- 17 terminal SufE domain fused to a BolA domain (95). The presence of two monothiol Grxs in the chloroplast, GrxS14 and GrxS16 which have been shown to assemble a [2Fe-2S] cluster then raises the possibility that these cluster bound Grxs could interact with the BolA domain of SufE and serve to regulate the SUF Fe-S cluster assembly machinery. Based on the current information available on monothiol Grxs, a summary of the proposed roles for monothiol Grxs in the chloroplast is presented in Figure 1.10. In the light of the new and emerging roles for monothiol CGFS Grxs, Chapter 3 presents the results of our biochemical and spectroscopic studies of cluster-bound forms of the chloroplastic monothiol GrxS16 from Arabdiopsis thaliana.

Radical SAM enzymes

Radical SAM enzymes are a superfamily of Fe-S enzymes that utilize a catalytically active [4Fe-4S]2+,+ cluster to bind and reductively cleave SAM to generate a highly reactive, 5'- deoxyadenosyl radical (Ado•), which initiates a diverse range of radical reactions by substrate or protein-based H abstraction (9;51). Though the first radical SAM enzyme was discovered in the

1970s, details regarding their diverse roles have proliferated over the past decade. In 2001, bioinformatics studies indicated that the radical SAM superfamily, characterized by CX3CX2C

[4Fe-4S] cluster-binding motif and a conserved SAM binding motif, contains over 600 members from 126 species spread across all the three domains of life (96). A recent search for “radical

SAM” in the InterPro database lists a taxonomic coverage of about 25,000 proteins in bacteria,

2000 in archeae, 1000 in cyanobacteria and about 300 in plants, indicative of the wide proliferation of these enzymes. In recent years, proteins with slightly different motifs, CX4CX2C and CX9CX2C in ThiC and Elp3, respectively were also found to coordinate a [4Fe-4S] cluster and reductively cleave SAM, generating the Ado• radical, adding more members to this already expanding class of enzymes (97;98). 18

A schematic of the events leading to the production of the Ado• radical by radical SAM

enzymes is shown in Figure 1.11A (99). An electron donor, usually flavodoxin reduces the [4Fe-

4S]2+ cluster to the [4Fe-4S]+, which transfers an electron to SAM causing a one electron reductive cleavage of the S-C bond to yield methionine and the Ado• radical. The Ado• radical abstracts a hydrogen atom from an appropriately placed substrate or protein residue creating a substrate or protein-based radical, thereby initiating the catalytic cycle. In radical SAM enzymes, the three cysteines from the radical SAM motif provide the ligands to the [4Fe-4S] cluster, leaving the fourth iron free to bind SAM. The SAM molecule coordinates to the unique iron site of the [4Fe-4S] cluster via the methionine amido and carboxylate group so that carbon-sulfonium bond can be cleaved by inner-sphere electron transfer from the reduced [4Fe-4S]+ cluster as

shown in Figure 1.11B.

The functions of radical SAM enzymes are diverse, and may be broadly classified in to

two groups; those which use SAM catalytically and those which use SAM as a substrate that is

consumed as a part of the reaction. The basic mechanism involved in the generation of the Ado•

radical is however the same in both cases. Two well characterized examples of radical SAM

enzymes that use SAM catalytically are lysine 2,3-aminomutase (LAM) and spore photoproduct lyase. LAM was the first radical SAM enzyme to be discovered and catalyzes the conversion of

α-lysine to β-Lysine. The product, β-lysine is used in the biosynthesis of antibiotics and in the metabolism of lysine, by anaerobes. LAM is a pyridoxal phosphate (PLP)-dependent enzyme, and the first step involves the reaction of PLP and lysine to form an aldimine. The Ado• radical then abstracts a β hydrogen, followed by 1,2-nitrogen migration via a cyclic azacyclopropylcarbinyl radical intermediate. The radical intermediates involved in the reaction mechanism were observed by EPR using isotopically labeled substrates and substrate analogs of 19

lysine and SAM (100-102). In spore photoproduct lyase, the Ado• radical serves to repair

bacterial DNA lesion by resolving the thymine dimers caused by exposure to UV light. The

reaction involves the abstraction of the C-6 hydrogen from the thymine dimer by Ado• radical,

followed by fragmentation of the dimer to yield thymine and thymine radical. The thymine

radical then abstracts a hydrogen back from 5'-dA to form thymine, thereby regenerating SAM

for next catalytic cycle. Experimental evidence for the above mechanism was obtained by using

a tritium labeled analog of the thymine dimer at the C-6 position, and 5'-3H SAM, which lead to

an exchange of tritium label in to SAM and thymine, respectively (103).

In comparison to the reactions that use SAM catalytically, a larger fraction of radical

SAM enzymes use SAM irreversibly and these include activases, sulfur-inserting enzymes and

methylating enzymes, to name a few. A subclass of this group of enzymes also contains

additional Fe-S clusters, which have been suggested to serve as sacrificial sulfur donors, to

provide a site for binding and/or activating the substrate, or to provide an electron transfer

pathway for delivering electrons to the radical-SAM [4Fe-4S] cluster. The pyruvate-formate

lyase-activating enzyme (PFL-AE) is one of the first members to be identified in the class of

activases. PFL-AE activates PFL, which converts pyruvate to acetyl-coA during the fermentation

of glucose. The first step involves the generation of a glycyl radical in PFL by the stereospecific

abstraction of HS-atom from the active site glycine by the Ado• radical generated by PFL-AE

(104). The glycyl radical in turn generates a thiyl radical by abstracting a hydrogen atom from

the active site cysteine in PFL, which forms an addition product with pyruvate. The cysteine-

pyruvate adduct undergoes fragmentation to yield formate, and subsequently acetylation of CoA results in acetyl CoA (105;106). Other radical SAM activases appear to function by a similar mechanism, and three of them, i.e. benzylsuccinate synthase (107), glycerol dehydratase 20

(108;109) and 4-hydroxyphenyl-acetate decarboxylase (110) activases contain two additional cysteine clusters that appear to ligate additional [4Fe-4S] clusters. Although the role of these additional clusters has still to be established, preliminary mutagenesis studies for a hydroxyphenylacetate decarboxylase activating enzyme indicated a role in mediating electron transfer to the radical-SAM [4Fe-4S] cluster (110).

The sulfur-inserting radical SAM enzymes, in addition to using SAM as a substrate, also possess additional Fe-S clusters which are believed to be used as sacrificial S donors. Well- characterized examples of sulfur-inserting radical SAM enzymes include the biotin synthase

(BioB) and lipoyl synthase (LipA) that cleave unreactive C-H bonds and insert sulfur atom(s) during the biosynthesis of biotin and lipoic acid, respectively. The mechanism of sulfur insertion is not known, but in both enzymes, the Ado• radical abstracts a hydrogen from the highly unreactive alkyl bonds generating an organic radical which subsequently reacts with sulfur to form the C-S bonds. The source of sulfur in both enzymes is believed to be the auxiliary Fe-S cluster present, which is a [2Fe-2S] cluster in BioB and a [4Fe-4S] cluster in LipA

(29;31;111;112). However, this dictates the need to rebuild the sacrificial Fe-S cluster in order to effect catalytic turnover and as yet it has not been possible to accomplish more than one turnover in vitro for any member of this subclass of radical SAM enzymes.

Another group of radical SAM enzymes that consume SAM as a part of their catalytic cycle, and possess additional Fe-S clusters are the anaerobic sulfatase maturating enzymes

(anSMEs) which are responsible for the post-translational modification of sulfatases under anaerobic conditions. Sulfatases are found in both prokaryotes and eukaryotes and catalyze the hydrolytic desulfonation of sulfate esters and sulfamates to form the corresponding alcohol and sulfate (113). They are broadly classified in to Cys-type and Ser-type sulfatases depending on 21

whether the active site cysteine or serine of the signature sequence motif, C/SXP/AXR is

modified to 3-oxoalanine (formylglycine, FGly) (114). While the Ser-type sulfatases are found exclusively in prokaryotes, Cys-type sulfatases are present in both prokaryotes and eukaryotes.

The oxidation of the active-site cysteine/serine to FGly is believed to be catalyzed by two different enzymatic systems, namely the formylglycine generating enzyme system, which is an oxygen–dependent specific to Cys-type sulfatases that is found only in eukaryotes and an oxygen-independent, anaerobic radical SAM enzyme catalyzed system thought to be specific for Ser-type sulfatases found only in prokaryotes. The anSMEs have three conserved clusters, one of which is the CX3CX2C motif that ligates the SAM-binding [4Fe-4S]

cluster in radical SAM enzymes (115). In order to investigate the presence and role of additional

Fe-S clusters and address the mechanism of anSMEs, spectroscopic and mechanistic studies of

wild-type and site-directed variants of the enzymes purified from Clostridium perfringens and

Bacteroides thetaiotaomicron are presented in Chapters 4 and 5.

Abbreviations

Grxs, Glutaredoxins; SUF, Sulfur Mobilization; SAM, S-adenosyl methionine; Ado•, 5'- deoxyadenosyl radical; anSME, anaerobic sulfatase maturating enzyme; FGly, Formyl glycine;

Cp, Clostridium pasteurinaum; AaeFd4, Aquifex aeolicus ferredoxin-4.

22

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32

Figure 1.1 Crystallographically defined structures for biological iron-sulfur clusters that are

involved in electron transfer. Structures are taken from the coordinates deposited in the Protein

Data Bank: A. Fe Rd, PDB ID# 8RXN, rubredoxin from Desulfovibrio vulgaris; B. [2Fe-2S],

PDB ID# 1FRD, Anabaena pcc7120 Fd; C. Rieske [2Fe-2S] center in Sulfolobus acidocaldarius,

PDB ID# 1JM1; D. [3Fe-4S], PDB ID# 6FDR, Azotobacter vinelandii FdI; E. [4Fe-4S], PDB

ID# 6FDR, A. vinelandii FDI; F. [8Fe-7S] PDB ID# 1MIN dithionite reduced A. vinelandii

nitrogenase MoFe protein. Color code: brown, Fe; yellow, S; blue, N. Adapted from reference 3.

33

AB

Fe Rd [2Fe-2S]

C D

[2Fe-2S]R [3Fe-4S]

E F

[4Fe-4S] [8Fe-7S]

34

Figure 1.2 Crystallographically defined structures of selected biological Fe-S centers involved in substrate binding and activation. A.HMBPP-bound [4Fe-4S] center in IspH from Escherichia coli, PDB ID# 3KE8 (48); B. S-Adenosyl Methionine bound [4Fe-4S] center in Escherichia coli

HemN, PDB ID# 1OLT; C. [Ni-4Fe-5S] center in reduced CO dehydrogenase II from

Carboxydothermus hydrogenoformans, PDB ID# 1SU8; D. [Mo-7Fe-9S-X] FeMo cofactor in

Azotobacter vinelandii nitrogenase, PDB ID# 1MIN. Color code: brown, Fe; yellow, S; gray, C; red, O; orange, P; green, Ni; pink, Mo; black, unknown low Z atom (O/C/N). Adapted from reference 3.

35

AC

HMBPP

IspH [4Fe-4S] + HMBPP [Ni-4Fe5S]

BD SAM

Homocitrate

[4Fe-4S] + SAM Mo-7Fe-9S-X

36

Figure 1.3 Ranges of mid-point potentials (mV vs. NHE) for biological Fe-S centers.

2+,+ [2Fe-2S] R, Rieske-type Fe-S center. Adapted from reference 57.

37

38

Figure 1.4 Ground state spin (S) and valence-delocalization schemes for the fundamental types of Fe–S centers. Color code: red, Fe3+; blue, Fe2+; green, Fe2.5+; yellow, S; black, O. Adapted from reference 57.

39

40

Figure 1.5 Illustration of the Robin-Day classification of mixed valence complexes. EA and EB represent the potential energy curves, when there is no orbital interaction between sites, A and B.

E- and E+ represent the potential energy curve of ground state and excited states, respectively. β,

is the resonance energy, λ, the reorganization energy, Eθ, the thermal activation barrier, and Eop is the energy needed for the optical transition. Reproduced with permission from reference 70.

41

Class I

Class II

Class III

42

Figure 1.6 (A) Schematic comparison of an antiferromagnetically coupled Fe(II)/Fe(III) pair

and a ferromagnetically coupled Fe(II)/Fe(III) pair (From [Beinert, H., Holm, R. H., and Münck,

E. (1997) Iron-sulfur clusters; Nature's modular, multipurpose structures, Science 277, 653-659].

Reprinted with permission from AAAS). (B) Energy level diagram for an exchange coupled

[2Fe-2S]+ cluster as a function of increasing spin-dependent resonance delocalization.

43

(A)

(B)

44

Figure 1.7 (A) A view of the superposition of the Anabaena thioredoxin-2 (orange) and monomer of WT AaeFd4 (green) (Reprinted from J. Mol. Biol. 300, Yeh, A. P., Chatelet, C.,

Soltis, S. M., Kuhn, P., Meyer, J., and Rees, D. C., Structure of a thioredoxin-like [2Fe-2S] ferredoxin from Aquifex aeolicus, 587-595, Copyright (2000), with permission from Elsevier).

(B) Electron density map of the oxidized [2Fe-2S] center in the Cys59Ser variant of AaeFd4 crystal structure solved at a resolution of 1.05 Å.

45

(A)

Trx Fd

(B)

46

Figure 1.8 Domain organization in Class 1 through Class 6 Grxs. Adapted from reference 78.

47

Class 1

GrxC1 to C5, S12, HsGrx1 & 2, CxxC/S EcGRX1 & 3, ScGRX1, 2, 6, 7

Class 2 GrxS14/S15, ScGRX5, HsGRX5, CGFS EcGrx4

GrxS16 CGFS (specific to plants)

GrxS17 Trx CGFS CGFS CGFS (higher plants)

GrxS17 (lower plants, Trx CGFS CGFS algae), HsGRX3

ScGrx3p, ScGrx4p Trx CGFS

Fusion proteins (xanthomonadales- CGFS HesB Rhod myxococales)

Desulfotalea psychrophila LSc54 CGFS FTRc

Class 3 (specific to higher plants) CCxC/S

Class 4 CxDC/S (specific to plants)

(specific to algae) CPxC

Class 5 (specific to cyanobacteria) CPWG

Class 6 CPWC/S (specific to cyanobacteria)

100 AA

48

Figure 1.9 Stereoview of the contrasted orientation of glutaredoxin monomers toward each

other in E. coli monothiol Grx4 (A) and in human dithiol Grx2 (B). Glutathione molecules are represented as yellow sticks in Grx4, and blue sticks in Grx2, and the cluster atoms, Fe and S are depicted in orange and yellow, respectively (Reproduced with permission from reference 89.

Copyright 2009 American Chemical Society).

49

50

Figure 1.10 A model illustrating possible roles for Grxs in Fe-S cluster assembly machineries in plant plastid. Adapted from reference 78.

51

52

Figure 1.11 (A) General scheme of reactions involved in the generation of the S-adenosyl radical

in Radical SAM enzymes (Reprinted from Curr. Opin. Chem. Biol., 8, Layer, G., Heinz, D. W.,

Jahn, D., and Schubert, W.-D., Structure and function of radical SAM enzymes, 468-476,

Copyright (2004), with permission from Elsevier). (B) Illustration of the chemical reaction

leading to the formation of S-adenosyl radical in Radical SAM enzymes (Marsh, E. N. G.,

Patterson, D. P., and Li, L.: Adenosyl radical: Reagent and catalyst in enzyme reactions.

Chembiochem. 2010. 11. 604-621. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.

Reproduced with permission).

53

(A)

(B)

Ado•

SAM 54

CHAPTER 2

SPECTROSCOPIC AND REDOX STUDIES OF VALENCE-DELOCALIZED [2FE-2S]+

CENTERS IN THIOREDOXIN-LIKE FERRREDOXINS1

1Sowmya Subramanian†, Evert C Duin‡, Jacques Meyer§, Michael K. Johnson†, to be submitted to Journal of the American Chemical Society, † Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA, ‡ Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, § Département Réponse et Dynamique Cellularies, CEA-Grenoble, Grenoble, France. 55

1Abbreviations. CpFd, Clostridium pasteurianum [2Fe-2S] ferredoxin; AaeFd4, Aquifex aeolicus ferredoxin-4; VTMCD, variable temperature magnetic circular dichroism; VHVT, variable-field and variable-temperature; RTMCD, room-temperature magnetic circular dichroism; NIR, near-infrared; EPR, electron paramagnetic resonance; WT, wild-type

56

Abstract

Aquifex aeolicus Ferredoxin 4 (AaeFd4) is the best structurally characterized member of the

class of the thioredoxin-like [2Fe-2S] ferredoxins. The reduced forms of the C56S and C60S

variants of the highly homologous Clostridium pasteurianum [2Fe-2S] ferredoxin (CpFd) currently provide the only known examples of valence-delocalized [2Fe-2S]+ clusters. Such

cluster fragments constitute a fundamental building block of all higher nuclearity Fe-S clusters

and understanding the origin of valence delocalization is central to the understanding of

biophysical properties of Fe-S clusters. In this work, we have revisited earlier work on the CpFd

variants and carried out detailed redox and spectroscopic studies on the [2Fe-2S]2+,+ center in

WT and the equivalent Cys-to-Ser variants of AaeFd4 using UV-visible absorption, EPR, and variable temperature MCD spectroscopies to assess if these variants also contain valence- delocalized S = 9/2 [2Fe-2S]+ clusters. The [2Fe-2S]+ centers in the equivalent AaeFd4 and CpFd

variants were found to reversibly interconvert between similar valence-localized S = 1/2 and

valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3-9.0.

However, on freezing the high pH samples for spectroscopic studies, the [2Fe-2S]+ centers are

partially or fully converted from valence-delocalized S = 9/2 to valence-localized S = 1/2 [2Fe-

2S]+ clusters. Analysis of variable-field and variable-temperature MCD saturation magnetization

data for valence-delocalized S = 9/2 [2Fe-2S]+ centers facilitated assessment of transition polarizations and thereby assignments of low energy MCD bands in terms of transitions associated with the Fe−Fe interaction. The results provide the first experimental assessment of

the double exchange parameter, B = 930 cm-1, and resonance energy β = 4650 cm-1, for [2Fe-

2S]+ centers and demonstrate that variable-temperature MCD spectroscopy provides a means of

detecting and investigating the properties of valence-delocalized S = 9/2 [2Fe-2S]+ fragments in 57 higher nuclearity Fe-S clusters. The origin of valence delocalization in thioredoxin-like ferredoxin Cys-to-Ser variants and Fe-S clusters in general is discussed in light of these new results.

58

Introduction

Valence delocalization is an intrinsic property of numerous high-nuclearity biological

Fe-S clusters, e.g. [3Fe-4S]0, [4Fe-4S]3+,2+,+, [8Fe-7S]4+,3+ clusters, and is important for understanding ground and excited state electronic properties and facilitating rapid electron transport by minimizing reorganization energy associated with oxidation/reduction (1;2). It is hence important to understand the origins of valence delocalization in order to interpret the electronic properties of Fe-S clusters and to rationalize the thermodynamics and kinetics of intercluster electron transfer. Based on Fe-S cluster biogenesis studies, Fe2(μ2-S)2 units ([2Fe-

2S]) constitute the basic building blocks of all Fe-S clusters (3), and spectroscopic studies have demonstrated that valence-delocalized [2Fe-2S]+ fragments with ferromagnetically coupled S =

9/2 ground states are intrinsic components of all homometallic and heterometallic high nuclearity

Fe-S clusters in at least one oxidation state (4;5). However, understanding the origin and

properties of valence-delocalized [2Fe-2S]+ units has been impeded by the fact that all known

synthetic and naturally occurring biological [2Fe-2S]+ centers are valence localized and exhibit S

= 1/2 ground states as a result of antiferromagnetic coupling (6).

Valence localization in the reduced cluster is promoted by a large localization energy

(ΔE), which includes contributions from vibronic coupling and the magnitude of the difference in

site redox potentials, and is manifest by strong antiferromagnetic exchange interaction, which restricts delocalization of the extra electron by requiring a spin flip to visit both Fe sites. In contrast, valence delocalization is expected to require a significant Fe-Fe interaction, such that the resonance delocalization energy overwhelms the localization energy, and results in the switch to ferromagnetic exchange interaction and a S = 9/2 ground state, so that the extra electron can

visit both Fe sites without undergoing a spin flip. Hence valence delocalization in [2Fe-2S]+ 59

clusters requires spin-dependent resonance delocalization and it is parameterized by the double

exchange parameter, B, where 10B = 2β, and β is the classical resonance energy that is more

familiar to chemists. The ground state properties of a [2Fe-2S]+ cluster fragment depends on the

relative magnitudes of Heisenberg-Dirac-vanVleck (J) exchange and double exchange (B) terms and results in energies E = −JS(S + 1) ± B(S + 1/2) (7). This simple model neglects vibronic interactions and assumes that the valence-localized species with the extra electron on the two iron sites, FeA and FeB, are isoenergetic. As the extent of resonance delocalization (B/J) increases, the ground state changes from S = 1/2 to 9/2 in integer steps, becoming S = 9/2 for

│B/J│ > 9. Inclusion of the factors responsible for valence localization, i.e. vibronic coupling

and inequivalence in the energies of the valence trapped species, decreases the B/J range in

which the ground state has S = ±3/2 or ±7/2. This diminishes the likelihood of observing these

intermediate-spin ground states and leads towards a situation in which the ground state changes

directly from valence-localized S = 1/2 to valence-delocalized S = 9/2 with increasing B/J.

Hence the absolute value of B/J and the energetic factors responsible for valence localization,

determine both the ground state spin and the extent of valence delocalization.

As indicated above, the structural and electronic origins of valence delocalization in

[2Fe-2S]+ units are not well understood, due to the lack of examples of magnetically isolated,

valence-delocalized [2Fe-2S]+ clusters. Hence, the observation of S = 9/2 valence-delocalized

[2Fe-2S]+ clusters in variants of Clostridium pasteurianum [2Fe-2S] ferredoxin (CpFd) in which

either of the cysteines coordinating the reducible Fe site were substituted with serine (C56S and

C60S) provided the opportunity to characterize the properties of a valence-delocalized [2Fe-2S]+ cluster and assess the determinants of valence delocalization in these variants. The initial discovery of valence-delocalized [2Fe-2S]+ clusters in these variants came from EPR and 60

variable-temperature magnetic circular dichroism (VTMCD) studies of dithionite-reduced

samples at alkaline pH which revealed a mixture of S = 1/2 and 9/2 [2Fe-2S]+ clusters and the

similarity in the electronic transitions of the S = 9/2 component as revealed by VTMCD with

those of clusters known to contain valence-delocalized [2Fe-2S]+ fragments (5;8). Definitive evidence for valence delocalization was subsequently provided by Mössbauer spectroscopy (9).

In addition, Mössbauer and saturation magnetization studies indicated that the ratio of S = 9/2

and 1/2 [2Fe-2S]+ clusters was maximally 1:1 even at pH 11, and surprisingly indicated that the

S = 1/2 component at alkaline pH is valence localized at low temperatures, but becomes valence

delocalized without a spin-state change at high temperatures (transition temperature ~ 100 K)

(10).

Structural data are not available for Clostridium pasteurianum [2Fe-2S] ferredoxin.

However, high resolution crystal structures are now available for the oxidized form of a close

homolog of the CpFd, the wild-type (WT) [2Fe-2S] ferredoxin-4 from the hyperthermophilic

bacterium Aquifex aeolicus (AaeFd4) (solved at 1.5 Å resolution) and the corresponding oxidized

Cys-to-Ser variants, C55S and C59S (solved at 1.25 Å and 1.05 Å resolution, respectively)

(11;12). The crystallographic data indicated a thioredoxin-like fold and confirmed serinate

ligation to the oxidized [2Fe-2S]2+ cluster in both variants. Moreover serinate ligation was

accompanied by a significant distortion in the [2Fe-2S] core and shortening of the Fe-Fe distance by 0.04 Å which was proposed to be an important determinant for valence delocalization in the reduced samples. However, there is currently no evidence in support of a valence-delocalized

[2Fe-2S]+ cluster in reduced samples of the C55S and C59S variants of AaeFd4. To remedy this situation, we report here a detailed comparison of the spectroscopic and redox properties of the oxidized and reduced forms of WT and the corresponding Cys-to-Ser variants of CpFd and 61

AaeFd4 using the combination of EPR and UV-visible absorption, CD and VTMCD spectroscopies. The implications of the results for the properties and origin of valence- delocalized S = 9/2 [2Fe-2S]+ clusters in the reduced Cys-to-Ser variants of these ferredoxins are discussed.

Materials and Methods

Sample preparation and redox measurements. WT and variant forms of CpFd and

AaeFd4 were provided by Dr. Jacques Meyer, Grenoble, France. All sample preparations were carried out under anaearobic conditions in a Vacuum Atmosphere’s glove box under an argon atmosphere (<5 ppm O2). Samples of as prepared and dithionite-reduced WT and variant forms of AaeFd4 variants were prepared at pH 7.0-8.0 and at pH 11.0-12.0 and 50% (v/v) ethylene glycol was used as the glassing agent for VTMCD studies. Low pH samples were prepared in

200 mM Tris-HCl buffer with 0.1 M NaCl, and high pH samples were prepared in 200 mM

CAPS buffer with 0.1 M NaCl. Samples of as prepared and dithionite-reduced WT and variant forms of CpFd were prepared in 200 mM MES buffer with 0.2 M NaCl at pH 6.0, 200 mM Tris-

HCl buffer with 0.1 M NaCl at pH 7.0, or in 200 mM CAPS buffer with 0.1 M NaCl at pH 11.0.

Samples used for VTMCD spectroscopy contained 55% (v/v) glycerol as the glassing agent.

Samples of the AaeFd4 and CpFd variants for RTMCD studies were prepared in a mixed buffer system (50 mM Tris, 50 mM CHES, 50 mM CAPS and 50 mM phosphate) with 0.1 M NaCl and were adjusted to pH 7 or 12. Redox potentials were determined by direct cyclic voltammetry of protein samples adsorbed on modified electrodes in the laboratory of Dr. Fraser Armstrong,

University of Oxford, UK. The dependence of the redox potential on pH were determined by applying 100 μM solution of the protein as a film on the electrode with 200 μg mL-1 of 62

polymyxin as co-adsorbate, in 10 mM mixed buffer and 0.1 M NaCl, and the voltammograms were recorded at 0 °C with a scan rate of < 20 mV s-1.

Spectroscopic methods. UV-visible absorption and CD spectra were recorded in anaerobic 1-mm cuvettes using a Shimadzu 3101 PC spectrophotometer and a Jasco J-715 spectropolarimeter, respectively. UV-visible-NIR VTMCD spectra were recorded on samples

containing 50% (v/v) ethylene glycol or glycerol using Jasco J-715 and J-730

spectropolarimeters interfaced to an Oxford Instruments Spectromag 4000 (0-7 T) split coil

superconducting magnet (sample temperatures: 1.5 – 300 K) using the published protocols

(13;14). Variable-field and variable-temperature (VHVT) MCD saturation magnetization data

were collected at sweep rates of 0.78 T/min from 0-6 T at fixed temperatures and analyzed

according to the published procedures (15) using software supplied by Prof. Edward I. Solomon

(Stanford University). Room-temperature magnetic circular dichroism (RTMCD) spectra in the

UV-visible-NIR region were recorded using a Jasco J-730 spectropolarimeter interfaced to a

Jasco MCD-1B electromagnet (0-1.5 T). X-band EPR spectra were recorded on a Bruker

Instruments ESP 300E spectrometer equipped with an ER-4116 dual mode cavity and an Oxford

Instruments ESR 900 flow cryostat (4.2–300 K). Spectra were quantified under non-saturating

conditions by double integration against a 1 mM Cu-EDTA standard. Spin Hamiltonian

parameters for S = 9/2 resonances were determined using the Rhombo program provided by Dr.

Wilfred Hagen, Delft University, the Netherlands.

Results

UV-visible absorption. The UV-visible absorption spectra of the oxidized and dithionite-

reduced WT AaeFd4 at pH 7.0 (Figure 2.1) are very similar to those reported for WT CpFd, with

prominent bands at 335, 425, 464 and 545 nm for the oxidized [2Fe-2S]2+ center and a band 63

around 550 nm characteristic of a valence-localized, dithionite-reduced [2Fe-2S]+ center (16;17).

Neither oxidized nor dithionite-reduced WT AaeFd4 exhibited any change in absorption

properties as a function of pH in the range pH 6-11. The electronic spectra of the oxidized Cys-

to-Ser variants, C55S and C59S AaeFd4 were very similar to the analogous C56S and C60S

CpFd variants, with prominent bands around 320, 415, 440 and 530 nm, and were independent of

pH over the range pH 6-11 (Figures 2.2-2.5). The observed spectra are, therefore characteristic of

a [2Fe-2S] ferredoxin and the blue shifts in the absorption spectra of the variants are consistent

with serinate ligation to the oxidized [2Fe-2S]2+ cluster (17). In contrast, the absorption spectra

of the dithionite-reduced variants at high pH (11-12) are quite different from those observed at

neutral pH. The band at 550 nm that is characteristic of a S = 1/2 [2Fe-2S]+ valence-localized

centers in the dithionite-reduced variants at neutral pH, is replaced by broad absorption bands in

400-500 nm and 600–750 nm regions (Figures 2.2-2.5) at high pH. The drastic change in the

absorption properties of the reduced AaeFd4 variants as a function of pH is similar to that

observed for the equivalent CpFd variants and suggests the presence of different [2Fe-2S]+ centers with distinct spectral properties at low and high pH in both sets of ferredoxin Cys-to-Ser variants.

Redox properties and CD spectra. The redox properties of protein-bound [2Fe-2S] clusters depend on a number of factors such as the nature of ligating residue, solvent exposure at the reducible iron site, secondary hydrogen bonding interactions and cluster environment (6). A

Cys-to-Ser mutation at the reducible iron site, while other factors remain the same is expected to cause a negative shift in midpoint potential (ΔEm) as a consequence of the more electronegative

serinate stabilizing the Fe(III) oxidation state. For example, in E. coli fumarate reductase, the

midpoint potentials of the [2Fe-2S]2+,+ centers in the C57S and C62S variants are shifted to 64

-103 and -243 mV, respectively, versus NHE at pH 7.0, compared to a midpoint potential of -79

mV for WT (18). The results of extensive site-directed mutagenesis and spectroscopic studies of

Cp [2Fe-2S] Fd have established that Cys56 and Cys60 serve as ligands to the reducible iron and

hence the redox potential of the C56S and C60S variants were determined at pH 7.0 (17). As

expected, the mutation resulted in a 100-102 mV decrease in midpoint potential for the variants

compared to the WT (Em = -280 mV versus NHE at pH 7.0) and the analogous variants from

AaeFd4 were also found to exhibit a similar trend (Table 2.1). To probe the dependence of

midpoint potential on pH, the redox potential of WT and the Cys-to-Ser variants of CpFd were

monitored as a function of pH by film voltammetry at 0 °C. While the WT showed no significant

change in midpoint potential in the pH range 5-10, the C56S and the C60S variants showed a

steady decrease in the midpoint potential with increasing pH, reaching a constant value of ~ -460

mV at pH > 10 (Figure 2.6). A non-linear regression fit of the data from Figure 2.6 was

performed using Equation 2.1, where E0′ is the reduction potential measured as a function of pH,

0 E ′alk is the reduction potential in the limit of high pH, n is the number of electrons transferred

and y is the number of protons transferred with a dissociation constant, Ka.

+ y 00 RT ⎡ H )]([ ⎤ EE alk ⎢1ln'' ++= ⎥ Equation 2.1 nF ⎣ K a ⎦

Results of the fit indicate that the midpoint potentials of C56S and C60S variant show a strong

pH dependence with a drop of about -50 mV/pH unit in the pH range 6-8 and that the reduced

clusters are associated with a single deprotonation event that occurs with a pKa of 8.7 and 8.9 for the C56S and C60S variants, respectively. Since serine is good candidate for deprotonation at pKa ~ 9.0 and the negative shift in redox potential relative to WT is characteristic of serinate

coordination at the reducible Fe site of the [2Fe-2S]2+,+ center, the dramatic pH-induced changes 65

in the excited state electronic properties of the reduced cluster are interpreted in terms of

protonation of the serinate ligand.

CD spectroscopy rather than film voltammetry was used to determine the pKa of the reduced clusters in the C55S and C59S variants of AaeFd4. The CD spectra of the reduced

AaeFd4 variants at pH ~ 6.5 are characterized by the presence of two intense positive bands between 350-400 nm, a broad less-intense positive band around 670 nm and an intense negative band at ~ 470 nm (Figures 2.7 and 2.8). As the pH is increased, these bands shift and become less intense with the observation of one set of isosbestic points that indicates direct

interconversion between low and high pH species. Plots of the CD intensity around 385 nm with

increasing pH were used to determine the pKa values and number of protons involved in the

conversion between the low and high pH forms of both variants (insets in Figures 2.7 and 2.8).

The data were fit using Equation 2.2, where Aobs is the observed CD intensity at a particular

wavelength, A1 and An are the CD intensities at this wavelength at low and high pH,

respectively.

− AA )( 10− pH A = 1 n + A Equation 2.2 obs ( − pH +1010 − pKa ) n

The results indicate a single deprotonation event with pKa ~ 9.0 and 8.3 for the C55S and C59S

variant, respectively. The significantly lower pKa for the C59S variant provides rationalization

for the significantly lower midpoint potential that is observed at pH 7 for the C59S variant (-420

mV) compared to the C55S variant (-380 mV) (Table 1). Hence redox properties of the WT and

variant forms of AaeFd4 and CpFd are clearly very similar except that the AaeFd4 potentials are

~ 20 mV more negative and that the pKa values for the two variants in AaeFd4 are significantly

different (Table 2.1). 66

Since high resolution crystallographic studies have provided definitive evidence for

serinate cluster ligation in the C55S and C59S variants of oxidized AaeFd4 (12) and film

voltammetry of the C56S and C60S variants of CpFd indicates well defined and symmetrical

oxidation and reduction waves at pH > 10 (data not shown), we conclude that serinate cluster

ligation is retained at pH > 10 on reduction of [2Fe-2S]2+,+ centers in both the AaeFd4 and CpFd

variants. The deprotonation event with pKa values between 8.3 and 9.0 is therefore attributed to

protonation of the serine ligand. As protonated serine is expected to be a weak cluster ligand and

the reducible Fe site of the cluster is solvent exposed, it seems likely that serine is replaced as a

ligand by water when samples are reduced at pH values below the pKa. Support for this

hypothesis comes from the film voltammetry results for the CpFd variants at pH < 8 which

reveal unsymmetrical oxidative and reductive waves (data not shown) that are indicative of a

conformational change accompanying the coupled proton/electron redox event.

EPR studies. The ground state properties of the [2Fe-2S]+ clusters in the AaeFd4 and

CpFd variants at low and high pH were investigated by EPR and VHVT MCD saturation magnetization studies. WT samples of dithionite-reduced AaeFd4 and CpFd exhibit almost

identical rhombic S = 1/2 EPR signals (g ≈ 2.00, 1.95, 1.92; gav ≈ 1.96) accounting for 1.0 ±0.1

spins/cluster that are invariant to pH over the pH range 6-11 (19). Comparisons of the X-band

EPR spectra of anaerobically prepared, dithionite-reduced samples of the Cys-to-Ser variants

AaeFd4 and CpFd in the S = 1/2 region at pH values significantly above and below the observed

pKa values are shown in Figure 2.9. Compared to WT, the g-value anisotropy increases and the

average g-value decreases (gav = 1.925-1.938) for each of the Cys-to-Ser variants, as expected for

replacing a cysteinate ligand with an oxygenic ligand at the reducible Fe site (18;20;21). In addition, different S = 1/2 EPR signals are observed at low and high pH values, with the high pH 67

samples invariably exhibiting greater g-value anisotropy, suggesting distinct types of oxygenic

ligands at low and high pH. Spin quantitations indicate that essentially all the [2Fe-2S]+ clusters have S = 1/2 ground states for the low pH samples of the CpFd and AaeFd4 variants. However, spin quantitations at pH 11.0 indicate that the S = 1/2 resonances accounted for ~ 0.9 spins/cluster for the AaeFd4 variants and ~ 0.5 spins/cluster for the CpFd variants. As discussed below, the sub-stoichiometric spin quantitations are a consequence of some of the [2Fe-2S]+ clusters having S = 9/2 ground states.

Previous EPR studies of dithionite-reduced C56S and C60S CpFd at pH 11.0 at temperatures between 4.2 and 60 K reveled low field resonances from [2Fe-2S]+ clusters with S

= 9/2 ground states (E/D = 0.12, D < 0, g0 = 2.00 for C56S and E/D = 0.16, D < 0, g0 = 2.04 for

C60S) (5;8). These resonances were attributed to valence-delocalized [2Fe-2S]+ clusters; an

interpretation that was subsequently confirmed by Mössbauer spectroscopy (9). Very similar

resonances with analogous temperature-dependent behavior, albeit with much lower intensity,

were also observed in the low field region of the dithionite-reduced C55S and C59S AaeFd4

samples at pH 11.0, see Figure 2.10. As shown in Figure 2.10, the effective g-values and the

observed temperature dependence for the low-field components are readily interpreted in terms

of a conventional S = 9/2 spin Hamiltonian with E/D = 0.12, D < 0, and g0 = 2.00 for the C55S

variant and E/D = 0.175, D < 0, and g0 = 2.04 for the C59S variant. The observation of S = 9/2

resonances at pH > pKa in the AaeFd4 and CpFd variants by EPR is therefore interpreted to result

from serinate cluster ligation and the consequent changes in cluster redox properties. It also

seems likely that the changes in the excited state electronic properties of the reduced clusters in

the Cys-to-Ser variants as a function of pH at room temperature reflects the change in spin state

of the reduced cluster with increasing pH. Since VTMCD spectroscopy provides a more 68

discriminating method for elucidating electronic excited state properties of paramagnetic chromophores, as well as means of assessing ground state properties via the analysis of VHVT

MCD saturation magnetization data at discrete wavelengths, MCD studies were employed to

further investigate the change in the spin-state and electronic excited state properties as a

function of pH.

VTMCD studies. The VTMCD spectra of the C55S and C59S variants of AaeFd4

(Figure 2.11 and 2.12) surprisingly showed features characteristic of pure valence-localized S =

1/2 [2Fe-2S]+ clusters (22;23) at both low and high pH. The MCD bands in the 350-525 nm region, assigned primarily to (Cys)S → Fe(III) charge transfer bands, the intense positive band at

2- ~ 540 nm, assigned primarily to the μ2-S → Fe(III) charge transfer transition and weak positive bands in the 650-750 nm region, assigned to d-d transitions, are all characteristic of valence- localized [2Fe-2S]+ cluster. The subtle shifts in the position of the VTMCD bands at pH 11.0

indicate slightly perturbed excited state electronic properties for the valence-localized S = 1/2

[2Fe-2S]+ cluster, which correlate with the slightly altered electronic ground state properties as

evidenced by distinct EPR g-values for the S = 1/2 resonances at high and low pH, see Figure

2.9. Moreover, VHVT MCD saturation magnetization data at discrete wavelengths showed that all the observed MCD bands originate from [2Fe-2S]+ clusters with S = 1/2 ground states (data

not shown) and provided no evidence for contributions from [2Fe-2S]+ clusters with S = 9/2

ground states that were observed by EPR. The origin of this discrepancy appears to be related to

the addition of 50% (v/v) ethylene glycol, as parallel EPR studies of the high-pH dithionite-

reduced C55S and C59S AaeFd4 samples used for VTMCD studies did not show low-field resonances indicative of S = 9/2 [2Fe-2S]+ clusters. Hence medium effects in the vicinity of the

solvent exposed reducible Fe site of the [2Fe-2S] cluster appear to play a crucial role in 69

determining whether or not the high-pH reduced [2Fe-2S]+ clusters can exist in the valence-

delocalized S = 9/2 form in frozen solution.

Unlike the AaeFd4 variants, the VTMCD spectra of the dithionite-reduced CpFd C56S

and C60S variants are quite different at low and high pH (Figure 2.13 and 2.14). At low pH, the

MCD bands are characteristic of valence-localized S =1/2 [2Fe-2S]+ clusters, and the VTMCD

spectra are very similar to those of the corresponding AaeFd4 variants. However, the VTMCD

spectra of the dithionite-reduced C56S and C60S samples are both markedly different at high pH

compared to low pH, and both exhibit very different temperature dependence behavior for

individual bands suggesting a mixture of S = 1/2 and 9/2 [2Fe-2S]+ clusters in accord with parallel EPR studies of the VTMCD samples. In contrast to the reduced AaeFd4 variants, EPR studies of the CpFd variants indicated that the approximate 50:50 mixture of S = 1/2 and 9/2

[2Fe-2S]+ centers was not significantly perturbed by the addition of 50% (v/v) ethylene glycol.

Since the S = 9/2 [2Fe-2S]+ centers approach magnetic saturation much more rapidly than the S =

1/2 [2Fe-2S]+ centers with decreasing temperature at 6 T, the VTMCD spectra of the S = 9/2 centers dominate at ~50 K. Hence the S = 9/2 [2Fe-2S]+ centers in the C56S and C60S variants

have very similar VTMCD spectra (intense positive bands centered near 470 and 710 nm, weak

positive bands centered near 870 and 1070 nm, and strong negative bands centered near 330 and

600 nm) that are quite distinct from the VTMCD spectra of the S = 1/2 [2Fe-2S]+ centers, indicating very different excited state electronic properties for the S = 1/2 and 9/2 [2Fe-2S]+ centers.

VHVT MCD magnetization studies were used to investigate the ground state properties and transition polarizations of the S = 9/2 [2Fe-2S]+ centers in high pH dithionite-reduced C56S

and C60S CpFd, see Figures 2.15 and 2.16, respectively. The data obtained by varying the 70

magnetic field (0-6 T) at three fixed temperatures (1.64, 4.22 and 10.0 K) were fit based on the

EPR-determined spin Hamiltonian parameters (D < 0 and E/D = 0.12 for C56S; and D < 0 and

E/D = 0.16 for C60S) and using D and the transition dipole moments in the xy, xz, and yz planes

(Mxy, Mxz, and Myz) as variable parameters using the methodology developed by Neese and

Solomon (15). Satisfactory theoretical fits for the VHVT MCD magnetization data collected at

~710 nm required a small contribution from S = 1/2 [2Fe-2S]+ centers (15% for C56S and 10%

for C60S). For both variants, the dominant S = 9/2 components were found to exhibit almost

exclusively xy-polarized transitions at ~710 nm (Mxy:Mxz:Myz = +1.00:+0.05:+0.08 for C56S at

712 nm; Mxy:Mxz:Myz = +1.00:+0.07:+0.05 for C60S at 705 nm) and best fits were obtained with

D = −1.5 ±0.2 cm-1. Very different VHVT MCD magnetization data were observed for the weak

bands in the NIR region at ~1070 nm. For both variants, only the S = 9/2 components were found

to contribute at ~1070 nm and these transitions were shown to be almost exclusively z-polarized

(Mxy:Mxz:Myz = +0.15:−1.00:+0.69 for C56S at 1070 nm which corresponds to 93% z-polarized;

Mxy:Mxz:Myz = +0.15:-1.00:+0.55 for C60S at 1070 nm which corresponds to 91% z-polarized)

and best fits were again obtained with D = −1.5 ±0.2 cm-1. Uniaxial VTMCD transitions are

expected to be weak in intensity as MCD C-terms require two non-zero perpendicular transition

moments. The observation of a low energy z-polarized transition for a valence-delocalized S =

9/2 [2Fe-2S]+ center is particularly important as it is an excellent candidate for the σ → σ*

transition associated with the Fe-Fe interaction.

Medium effects on the spin-state heterogeneity: The results of EPR and VTMCD studies

on the Cys-to-Ser variants in AaeFd4 and CpFd summarized thus far, indicate that the [2Fe-2S]+ centers in these variants can exist in two different spin states at high pH in frozen solutions: valence-localized S =1/2 and valence-delocalized S = 9/2. EPR spin quantitations indicate that 71

maximally 10% and 50% of the reduced clusters are present in the S = 9/2 state in reduced high-

pH AaeFd4 and CpFd variants, respectively. Since one of the objectives of this work was to find conditions conducive to obtaining samples of reduced AaeFd4 variants with homogeneous

valence-delocalized S = 9/2 [2Fe-2S]+ centers in order to facilitate structural characterization of

the determinants of spin-dependent resonance delocalization, we investigated the effects of the

medium and freezing rate on the spin-state heterogeneity using EPR spectroscopy. To

investigate the possibility that the rate of freezing affects the extent of valence delocalization,

EPR samples of the reduced C55S AaeFd4 at pH 11.0/12.0 were prepared by rapid freezing in an

isopentane bath using an UpdateTM freeze quench apparatus and slow freezing in liquid nitrogen.

The rate of freezing however did not affect the extent of valence-localization as EPR spin quantitations revealed that 90% of the clusters remained in the valence localized S = 1/2 form.

To investigate buffer effects and the possibility of apparent pH changes upon freezing, samples at high pH were also prepared in different buffers; phosphate and in mixed-buffers. However, the samples revealed no difference in the spin-state heterogeneity as determined from the spin quantitative EPR studies. Samples were also prepared in mixed-buffers at different ionic strengths by varying the salt concentration (0.025-0.5 M NaCl), but again no significant change in spin-state heterogeneity was apparent.

Since the addition of ethylene glycol to the AaeFd4 variants at high pH for VTMCD

studies resulted in the observation of ~ 100 % valence-localized S = 1/2 [2Fe-2S]+ clusters, different glassing agents were used to perturb the solvent medium around the solvent exposed Fe site, and promote the observation of valence delocalized S = 9/2 state. However, neither the addition 50 % glycerol or polyethylene glycol as the glassing agent were successful in bringing forth any change in the spin state of the [2Fe-2S]+ center, which remained in the valence 72

localized S = 1/2 state. Moreover, variable temperature Mössbauer studies (10) reported a

temperature-dependent transition of the valence-localized S = 1/2 population to valence-

delocalized S = 1/2 state in the CpFd variants with a transition temperature of about 100 K. To investigate if such a transition is observable for the AaeFd4 variants, the VTMCD spectra of the

C55S AaeFd4 variant at pH 11.0 was recorded at 2, 4, 10, 50, 100 and 230 K. The data revealed that the MCD bands observed were characteristic of a valence-localized S = 1/2 [2Fe-2S]+ center

and the relative intensity of the bands remained unchanged through 230 K, indicating that

valences remain localized with increasing temperature (data not shown).

RTMCD Studies. The observation that the dramatic changes in the room-temperature UV-

visible absorption spectra for the low and high pH forms of the CpFd and AaeFd4 variants

appear to mirror the changes in the electronic transitions for the S = 1/2 valence-localized and S

= 9/2 valence-delocalized [2Fe-2S]+ centers as judged by VTMCD spectroscopy, raised the

possibility that the high pH samples are fully valence delocalized at room temperature and only

become valence localized on freezing samples for EPR, VTMCD and Mössbauer studies. To

investigate this hypothesis, the RTMCD of the high and low pH samples of the reduced AaeFd4

and CpFd variants were measured and compared with each other and with the low temperature

MCD spectra of the S = 1/2 valence-localized and S = 9/2 valence-delocalized [2Fe-2S]+ centers in frozen solution. The RTMCD spectra of the reduced AaeFd4 and the CpFd variants at low pH

(Figure 2.17 and 2.18), are all very similar to those of the valence-localized S = 1/2 [2Fe-2S]+

cluster in frozen solution as determined by VTMCD, with positive bands centered around 400,

540 and 690 nm and a negative band around 460 nm. However, at high pH, unlike the VTMCD

data for the AaeFd4 variants, the spectra are characteristic of a valence-delocalized S = 9/2 [2Fe-

2S]+ cluster (Figure 2.17 and 2.18). The complete absence of the positive band centered around 73

540 nm, and the presence of positive bands centered at 480, 720 and ~1000 nm and a negative

band centered at 600 nm, unambiguously identifies the excited state electronic structure of a

valence delocalized S = 9/2 [2Fe-2S]+ clusters in the high pH samples at room temperature.

Hence we conclude that valence localization of the high pH variants only occurs on freezing

samples for spectroscopic studies and that high pH samples of both the AaeFd4 and CpFd

variants are predominantly or exclusively valence-delocalized with an S = 9/2 ground state at

room temperature.

Discussion

The thioredoxin-class of [2Fe-2S] ferredoxins, typified by CpFd, is unique in providing

the only known examples of biological or synthetic valence-delocalized [2Fe-2S]+ centers. The

objectives of this work were to compare the detailed spectroscopic and redox properties of the

[2Fe-2S] centers in the highly homologous CpFd and AaeFd4 proteins with a view towards

characterizing valence-delocalized [2Fe-2S]+ centers in the structurally characterized Cys-to-Ser

variants of AaeFd4 (12) and thereby addressing the origins of valence delocalization in Fe-S clusters. Valence-delocalized S = 9/2 [2Fe-2S]+ fragments are integral constituents of all

biological and synthetic Fe-S clusters containing 3 or more Fe atoms (4;6). However, valence-

delocalized S = 9/2 [2Fe-2S]+ clusters have thus far only been observed in C56S and C60S

variants of CpFd, where the reduced clusters were found to be present as mixtures of S = 9/2 valence-delocalized and S = 1/2 valence-localized forms in frozen solution (5;8-10). The spectroscopic results presented in this work demonstrate that the [2Fe-2S]+ clusters in the

reduced forms of C56S and C60S CpFd and C55S and C59S AaeFd4 are predominantly or

exclusively present as S = 9/2 valence delocalized centers at pH ≥ 10 in aqueous solution at

room temperature. However, in all four variants the reduced clusters revert completely or 74

partially to S = 1/2 valence-localized centers on freezing samples for spectroscopic studies.

Nevertheless, the equivalent variants of CpFd and AaeFd4 are shown to have almost identical S =

9/2 valence delocalized [2Fe-2S]+ centers and the significant concentrations of S = 9/2 valence delocalized [2Fe-2S]+ centers that are present in the frozen samples of reduced C56S and C60S

CpFd have facilitated detailed characterization of the ground and excited state electronic

properties of these novel species using VTMCD and EPR spectroscopies.

A schematic in Figure 2.19 summarizes the ligation, spin states and redox properties of

the [2Fe-2S]2+,+ centers in the Cys-to-Ser variants of CpFd and AaeFd4 as a function of pH,

based on the high resolution structural studies of oxidized C55S and C59S AaeFd4 (12) and the

spectroscopic and redox studies of C56S and C60S CpFd and C55S and C59S AaeFd4 reported

in this work. Crystallographic data provide direct evidence for serinate ligation at the solvent

exposed reducible Fe site of the S = 0 antiferromagnetically coupled [2Fe-2S]2+ centers in

oxidized C55S and C59S AaeFd4 (12). Moreover, the absence of any changes in the UV-visible

absorption and CD spectra of the oxidized proteins as a function of pH in the range pH 6-11

indicate that serinate remains a ligand to the oxidized cluster at high and low pH. In contrast, the reduced clusters of all four variant proteins undergo protonation equilibria, with pKa values between 8.3 and 9.0, which appear to involve the serinate ligand as protonation induces marked changes in the UV-visible absorption and CD properties of the reduced clusters in the Cys-to-Ser variant ferredoxin but not in the WT ferredoxin. At pH > pKa, serinate remains as a ligand to the

reduced cluster, as evidenced by sharp and symmetrical oxidation and reduction waves in cyclic

voltammograms. The resultant high-pH reduced [2Fe-2S]+ cluster is proposed to be valence delocalized with a S = 9/2 ground state at room temperature, based on comparison of the

RTMCD with the low temperature MCD of the S = 9/2 valence-delocalized [2Fe-2S]+ centers in 75

high pH samples of reduced C56S and C60S CpFd in frozen solution. However, the combination of EPR and VTMCD studies demonstrate that the [2Fe-2S]+ centers are completely or partially

converted to a serinate ligated, valence-localized S = 1/2 form on freezing high pH samples for

spectroscopic investigation. For each variant, lowering the pH of reduced samples well below the

pKa or reducing oxidized samples at pH values well below the pKa of the reduced samples,

results in a valence-localized S = 1/2 [2Fe-2S]+ center with EPR and VTMCD spectra that are

distinct from the corresponding high-pH valence-localized S = 1/2 [2Fe-2S]+ center. The solvent-

exposed nature of the reducible Fe site, coupled with the observation of unsymmetrical oxidative

and reductive waves in the cyclic voltammograms at low pH, therefore suggest that the serinate

ligand is protonated and replaced by a water ligand in the low-pH valence-localized S = 1/2 [2Fe-

2S]+ centers.

The spectroscopic and redox results for CpFd and AaeFd4 clearly indicate that serinate

ligation at the reducible Fe site is required to observe valence-delocalized S = 9/2 [2Fe-2S]+

centers in the thioredoxin-class of [2Fe-2S] Fds. However, detailed characterization of the

ground and excited state properties of the valence-delocalized S = 9/2 [2Fe-2S]+ centers at high

pH in the reduced AaeFd4 Cys-to-Ser variants was not possible due to near quantitative

conversion to valence-localized S = 1/2 [2Fe-2S]+ centers on freezing samples for EPR and

VTMCD. Nevertheless, the electronic properties are expected to be very similar to those of the equivalent high pH CpFd variants based on the close similarity in the observed EPR signals (E/D

= 0.12 and D < 0 for both C56S CpFd and C55S AaeFd4; E/D = 0.16 and D < 0 for C60S CpFd

and E/D = 0.175 and D < 0 for C59S AaeFd4). Although S = 9/2 valence-delocalized and S =

1/2 valence-localized [2Fe-2S]+ centers are present in an approximately 50:50 mixture in frozen

samples of reduced C56S and C60S CpFd at high pH, contributions from each to the VTMCD 76

spectra can be deconvoluted based on dramatic differences in the temperature dependence of

transitions originating from the S = 9/2 and 1/2 ground states. Moreover, using the EPR-

determined ground state-spin Hamiltonian parameters, VHVT MCD saturation magnetization

data can be used to assess the magnitude of the axial zero-field splitting parameter, D, and transition polarizations. The latter are particularly important for assigning low energy transitions in the near-IR region that originate from the Fe-Fe orbital overlap which is responsible for spin- dependent resonance delocalization. A schematic MO diagram for the Fe-Fe interactions of a S =

9/2 [2Fe-2S]+ cluster is shown in Figure 2.20. The tetrahedral crystal field splitting at each Fe2.5+ site, ~ 5000 cm-1 is based on the detailed electronic studies of tetrahedral Fe2+ and Fe3+

2 2 complexes with thiolate ligands (24;25). The overlap of dz -dz orbitals along the Fe-Fe axis is

crucial for the delocalization of the electron between the two sites and is the dominant interaction

responsible for the observation of S = 9/2 ground state, with progressively smaller π interactions,

2 2 between pairs of dxy and dyz orbitals, and δ interactions, between pairs of dxy and dx -y orbitals.

The σ → σ* transition is electric dipole allowed and z-polarized which dictates weak VTMCD intensity since MCD intensities require two perpendicular non-zero transition dipole moments.

Hence the weak z-polarized band that is observed at ~1070 nm (~9300 cm-1) in both variants is

assigned to the σ → σ* transition. The σ → π and σ → π* transitions are both predicted to be predominantly xy polarized if the dxz and dyz orbitals are close in energy and both should

therefore exhibit broad derivative shaped temperature dependent MCD bands (pseudo A terms).

The former is electric-dipole forbidden and hence weak in MCD, but is clearly evident

particularly in the C56S variant with the positive component at ~900 nm with the negative

component buried under the intense band at 710 nm suggesting an energy around 12000 cm-1.

Analysis of VHVT MCD saturation magnetization data for the C56S variant at 905 nm 77

confirmed xy-polarization for this transition (data not shown). The σ → π* transition is electric-

dipole allowed and hence is assigned to the intense xy-polarized pseudo A-term centered at 660

nm (15,000 cm-1) (positive band at ~710 nm and negative band at ~600 nm) that dominates the

NIR region of the VTMCD and absorption spectra of the S = 9/2 valence delocalized [2Fe-2S]+ center.

The importance of assigning σ → σ* transition is that the energy of this transition corresponds to 10B = 2β, and thus provides the first direct measurement of the double exchange

parameter (B) and thereby the resonance energy (β) associated with the spin-dependent resonance delocalization. Theoretical estimates of the value of B in [2Fe-2S]+ centers or fragments span a wide range from 30-970 cm-1 (7;19;26-30). The values of B = 930 ± 30 cm-1 and β = 4650 ± 150 cm-1 for the valence delocalized S = 9/2 [2Fe-2S]+ clusters C56S and C60S

CpFd are at the high end of this range. Moreover, these values are in excellent agreement with

those estimated for [2Fe-2S]+ cluster (B = 965 cm-1 and β = 4825 cm-1), based on extrapolation

2+ from the Fe−Fe electronic coupling in a complex with a [Fe2(μ2-OH)3] core that is currently the

best characterized example of a valence delocalized S = 9/2 diiron complex (31;32).

In addition, assignment of the transitions associated with the Fe-Fe interactions in the

VTMCD spectra of valence-delocalized S = 9/2 [2Fe-2S]+ clusters provides a means of

identifying and assigning analogous transitions associated with valence-delocalized [2Fe-2S]+ fragments in higher nuclearity clusters. This is illustrated in Figure 2.21 which compares the low temperature MCD spectrum of the valence-delocalized S = 9/2 [2Fe-2S]+ center in C60S CpFd

with those of higher nuclearity that are known to or have the potential to contain valence-

delocalized [2Fe-2S]+ fragments, i.e. S = 5/2 [Zn3Fe-4S]+ (contains a cubane-type S = 5/2 [3Fe-

4S]− cluster that results from antiferromagnetic coupling of S = 2 Fe2+ with S = 9/2 [2Fe-2S]+), 78

cubane-type S = 2 [3Fe-4S]0 (S = 5/2 Fe3+ antiferromagnetically coupled with S = 9/2 [2Fe-2S]+),

S = 1/2 [4Fe-4S]+ (S = 4 [2Fe-2S]0 antiferromagnetically coupled with S = 9/2 [2Fe-2S]+), S = 3

or 4 [8Fe-7S]4+ double cubane oxidized nitrogenase P cluster (has the potential to contain a S =

9/2 [2Fe-2S]+ fragment in each cubane cluster). In each case a set of low energy bands

corresponding to the σ → σ*, σ → π and σ → π* transitions associated with Fe−Fe interactions

of a S = 9/2 valence-delocalized [2Fe-2S]+ fragment is apparent. The poor resolution of the bands

in the [8Fe-7Fe]4+ P-cluster is tentatively attributed to the overlap of transitions from two slightly

different S = 9/2 valence-delocalized [2Fe-2S]+ fragments. The results demonstrate the potential

of VTMCD spectroscopy to identify the presence of valence-delocalized [2Fe-2S]+ fragments in

higher nuclearity Fe-S clusters and to assess the values of the double exchange parameter (B) and

thereby the resonance delocalization energies (β) for these fragments via the energies of the σ →

σ* transitions. Clearly the identification and characterization of S = 9/2 valence-delocalized

[2Fe-2S]+ clusters in Cys-to-Ser variants of thioredoxin-like Fds constitutes a major step in

understanding the ground and excited state properties of Fe-S clusters.

Finally we address what the results presented in this work tell us about the origin of

valence delocalization in the Cys-to-Ser variants of thioredoxin-like Fd [2Fe-2S]+ centers and

why valence-delocalized [2Fe-2S]+ clusters are so common in high nuclearity Fe-S clusters, but

have never been observed thus far in naturally occurring or synthetic [2Fe-2S]+ clusters. Valence

delocalization resulting in an S = 9/2 ground state requires *B/J* > 9 in an idealized system in

which the two valence-localized configurations have the same energy (7). This can be

accomplished by increasing B, decreasing J, or a combination of both. Asymmetric Fe

2− coordination as a result of serinate coordination or exchange coupling mediated μ3-S rather

2− than μ2-S as in higher nuclearity clusters, would both be expected to decrease J and this is 79

probably an important contributing factor for observing valence delocalization in the Cys-to-Ser

[2Fe-2S] Fd variants and in the [2Fe-2S] fragments of higher nuclearity clusters. Whether or not

B increases in the serinate-coordinated valence-delocalized reduced [2Fe-2S]+ cluster forms

remains to be determined, but it is interesting to note that B is expected to correlate with the

Fe−Fe distance (31), which has been shown to decrease by 0.04 Å for the oxidized [2Fe-2S]2+ centers in the C55S and C59S variant of AaeFd4 compared to WT (12). High resolution structural data for reduced WT and the reduced low-pH valence-localized and high-pH valence- delocalized forms of these variants will be required to address the extent to which a decrease in J or an increase in B is responsible for valence delocalization. Room temperature crystallographic studies are planned to address this issue.

The degree of valence delocalization for a given spin state is determined by the ratio of B to the localization energy, ΔE, which contains the energetic terms reflecting both the vibronic and static preference for valence localization. On the basis of the simple resonance Hamiltonian model, valence trapping will generally occur unless 2*B(S + 1/2)* > ΔE (7). Although it is difficult to separate the vibronic and static contributions to ΔE, the static component, which corresponds to the energy difference when the extra electron is on FeA or FeB, is likely to be minimized in the Cys-to-Ser Fd variants. By decreasing the potential at the reducible Fe site by approximately 200 mV, we hypothesize that serinate coordination makes the two Fe sites almost isopotential thereby minimizing the static localization energy. Hence we propose that the difference in site potentials for the Fe centers in [2Fe-2S] cluster coupled with the value of B/J are both a major determinants of the degree of valence (de)localization in [2Fe-2S]+ centers.

Since both of these determinants would be perturbed by changes in H-bonding interactions involving the solvent-exposed serinate ligand, we propose that changes in the H-bonding 80

interactions associated with organization of water molecules at the solvent exposed Fe site in

frozen solution is responsible for the valence-delocalized to valence-localized transition on

freezing samples for spectroscopic studies.

On the basis of this discussion, it seems probable that further examples of valence

delocalized S = 9/2 [2Fe-2S]+ clusters will come through mutagenesis studies of proteins rather

than from chemical synthesis. In order to decrease the Heisenberg exchange coupling,

asymmetric Fe coordination is probably required, but it is difficult to see how this can be

accomplished in a synthetic cluster while maintaining isopotential Fe sites. In contrast, the nature

of the Fe ligands in a protein-bound cluster is only one of the factors that determine the redox

potentials at the Fe sites, making it possible to obtain isopotential Fe sites with asymmetric Fe coordination via site-directed mutagenesis.

Acknowledgements

This work was supported by grants from the National Institutes of Health (GM51962 and

GM62524 to MKJ).

81

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18. Werth, M. T., Cecchini, G., Manodori, A., Ackrell, B. A. C., Schröder, I., Gunsalus, R. P., and Johnson, M. K. (1990) Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: Modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster, Proc. Natl. Acad. Sci. USA 87, 8965- 8969.

19. Orio, M. and Mouesca, J. M. (2008) Variation of Average g Values and Effective Exchange Coupling Constants among [2Fe−2S] Clusters: A Density Functional Theory Study of the Impact of Localization (Trapping Forces) versus Delocalization (Double- Exchange) as Competing Factors, Inorganic Chemistry 47, 5394-5416.

20. Bertrand, P. and Gayda, J.-P. (1979) A theoretical interpretation of the variations of some physical parameters within the [2Fe-2S] ferredoxin group, Biochim. Biophys. Acta 579, 107-121.

21. Bertrand, P., Guigliarelli, B., Gayda, J.-P., Beardwood, P., and Gibson, J. F. (1985) A ligand-field model to describe a new class of 2Fe-2S clusters in proteins and their synthetic analogs, Biochim. Biophys. Acta 831, 261-266.

22. Fu, W., Drozdzewski, P. M., Davies, M. D., Sligar, S. G., and Johnson, M. K. (1992) Resonance Raman and magnetic circular dichroism studies of reduced [2Fe-2S] proteins, J. Biol. Chem. 267, 15502-15510.

23. Thomson, A. J., Cammack, R., Hall, D. O., Rao, K. K., Briat, B., Rivoal, J. C., and Badoz, J. (1977) The low temperature magnetic circular dichroism spectra of iron-sulfur proteins. II Two-iron ferredoxins, Biochim. Biophys. Acta 493, 132-141. 83

24. Gebhard, M. S., Deaton, J. C., Koch, S. A., Millar, M. M., and Solomon, E. I. (1990) - Single-crystal spectra studies of Fe(SR)4 [R = 2,3,5,6-(Me)4C6H]: The electronic structure of the ferric tetrathiolate active site, J. Am. Chem. Soc. 112, 2217-2231.

25. Gebhard, M. S., Koch, S. A., Millar, M. M., Devlin, F. J., Stephens, P. J., and Solomon, 2- E. I. (1991) Single-crystal spectroscopic studies of Fe(SR)4 (R = 2-(Ph)C6H4): Electronic structure of the ferrous site in rubredoxin, J. Am. Chem. Soc. 113, 1640-1649.

26. Kröckel, M., Grodzicki, M., Papaefthymiou, V., Trautwein, A. X., and Kostikas, A. (1996) Tuning of electron delocalization in polynuclear mixed-valence clusters by super- exchange and double exchange, J. Biol. Inorg. Chem. 1, 173-176.

27. Blondin, G. and Girerd, J.-J. (1996) Value of the β transfer integral in Fe-S clusters, J. Biol. Inorg. Chem. 1, 170-172.

28. Noodleman, L., Case, D. A., and Mouesca, J.-M. (1996) Valence electron delocalization in polynuclear iron-sulfur clusters, J. Biol. Inorg. Chem. 1, 177-182.

29. Bertini, I. and Luchinat, C. (1996) Experimental data and calculated parameters in Fe-S polymetallic centers in proteins, J. Biol. Inorg. Chem. 1, 183-185.

30. Solomon, E. I., Xie, X., and Dey, A. (2008) Mixed valent sites in biological electron transfer, Chem. Soc. Rev. 37, 623-638.

31. Gamelin, D. R., Bominaar, E. L., Kirk, M. L., Wieghardt, K., and Solomon, E. I. (1996) Excited-state contributions to ground-state properties of mixed-valence dimers: Spectral 2+ + and electronic-structural studies of the [Fe2(OH)3(tmtacn)2] related to the [Fe2S2] active site of plant-type ferredoxins, J. Am. Chem. Soc. 118, 8085-8097.

32. Gamelin, D. R., Bominaar, E. L., Mathonière, C., Kirk, M. L., Wieghardt, K., Girerd, J.- J., and Solomon, E. I. (1996) Excited-state distortions and electron delocalization in mixed-valence dimers: Vibronic analysis of the near-IR absorption and resonance Raman 2+ profiles of [Fe2(OH)3(tmtacn)2] , Inorg. Chem. 35, 4323-4335.

84

a b Table 2.1 Redox potential and pKa of the WT and variant forms of AaeFd4 and CpFd.

Redox potential (mV) pKa AaeFd4 -280±10 - AaeFd4 C55S variant -380±10 9.0 AaeFd4 C59S variant -420±10 8.3 CpFd -262±10 - CpFd C56S variant -364±10 8.7 CpFd C60S variant -362±10 8.9 aThe redox potential of the AaeFd4 and CpFd WT and variant forms were determined by film b voltammetry at pH 7.0 versus SHE at 0 °C. The pKa of AaeFd4 variants were determined by monitoring the pH dependence via CD spectrophotometric titrations (Figure 2.7 and 2.8). The pKa of CpFd variants were determined by monitoring the pH dependence via film voltammetry (Figure 2.6).

85

Figure 2.1 UV-visible absorption spectra of oxidized and dithionite-reduced WT AaeFd4 (0.12 mM) in 200 mM CAPS buffer with 0.1 M NaCl at pH 11.0, and in 200 mM Tris-HCl buffer with

0.1 M NaCl at pH 7.0, recorded in 0.1 cm cuvettes.

86

WT AaeFd4 0.2 200 mM CAPS, pH 11.0

bance OX r 0.1 bso A RED

0

WT AaeFd4 0.2 200 mM Tris-HCl, pH 7.0 bance

r OX 0.1 bso A

RED

0 300 400 500 600 700 800 Wavelength (nm)

87

Figure 2.2 UV-visible absorption spectra of oxidized and dithionite-reduced C55S AaeFd4

(0.34 mM) and C56S CpFd (0.46 mM) in 200 mM Tris-HCl buffer with 0.1 M NaCl at pH 7.0, recorded in 0.1 cm cuvettes.

88

0.6 C55S AaeFd4 200 mM Tris-HCl, pH 7.0

0.4

OX bance r bso A 0.2 RED

0.0

0.8 C56S CpFd 200 mM Tris-HCl, pH 7.0

0.6 OX bance r 0.4 RED bso A

0.2

0.0 300 400 500 600 700 800

Wavelength (nm)

89

Figure 2.3 UV-visible absorption spectra of oxidized and dithionite-reduced C55S AaeFd4 (0.43 mM) and C56S CpFd (0.42 mM) in 200 mM CAPS buffer with 0.1 M NaCl at pH 11.0, recorded in 0.1 cm cuvettes.

90

0.8 C55S AaeFd4 200 mM CAPS, pH 11.0 0.6

bance OX r 0.4 bso RED A x5 0.2

0.0

C56S CpFd 200 mM CAPS, pH 11.0 0.6

bance OX r 0.4 bso A 0.2 RED x5

0.0 300 400 500 600 700 800 Wavelength (nm)

91

Figure 2.4 UV-visible absorption spectra of oxidized and dithionite-reduced C59S AaeFd4

(0.22 mM) and C60S CpFd (0.33 mM) in 200 mM Tris-HCl buffer with 0.1 M NaCl at pH 7.0, recorded in 0.1 cm cuvettes.

92

0.4 C59S AaeFd4 200 mM Tris-HCl, pH 7.0

bance OX r 0.2 bso A RED

0.0

C60S CpFd 200 mM Tris-HCl, pH 7.0 0.4

OX bance r bso

A 0.2 RED

0.0 300 400 500 600 700 800 Wavelength (nm)

93

Figure 2.5 UV-visible absorption spectra of oxidized and dithionite-reduced C59S AaeFd4

(0.36 mM) and C60S CpFd (0.27 mM) in 200 mM CAPS buffer with 0.1 M NaCl at pH 11.0, recorded in 0.1 cm cuvettes.

94

0.6 C59S AaeFd4 200 mM CAPS, pH 11.0

0.4 OX bance r bso A 0.2 RED x5

0.0

C60S CpFd 0.4 200 mM CAPS, pH 11.0

OX bance r

bso 0.2 A x7 RED

0.0 300 400 500 600 700 800 Wavelength (nm)

95

Figure 2.6 Dependence of the midpoint potential of the WT (▲) and the C56S (■) and C60S (●)

variants of CpFd on pH, determined by film voltammetry. The solid line shows a non-linear regression fit to equation 2.2.

96

97

Figure 2.7 CD spectra of dithionite-reduced C55S AaeFd4 measured as a function of pH. The titration was carried out under anaerobic conditions in a 1.0 cm cuvette, using 0.09 mM dithionite-reduced C55S AaeFd4 in a mixed buffer system (50 mM Tris, 50 mM CHES, 50 mM

CAPS, 50 mM phosphate and 0.1 M NaCl), by adding aliquots of NaOH. Arrows indicate the direction of change in CD intensity at selected wavelengths with increasing pH. The inset is a plot of the ellipticity at 387 nm versus pH, and the solid line represents the fit to a single deprotonation event with a pKa = 9.0.

98

40 ↓

20 ↓

0

40

35 Ellipticity (mdeg) -20 30

25

20 Ellipticity at 387 nm (mdeg) nm 387 at Ellipticity ↓ 15 -40 678910111213 pH

400 500 600 700 800

Wavelength (nm)

99

Figure 2.8 CD spectra of dithionite-reduced C59S AaeFd4 measured as a function of pH. The titration was carried out under anaerobic conditions in a 1.0 cm cuvette, using 0.11 mM dithionite-reduced C59S AaeFd4 in a mixed buffer system (50 mM Tris, 50 mM CHES, 50 mM

CAPS, 50 mM phosphate and 0.1 M NaCl), by adding aliquots of NaOH. Arrows indicate the direction of change in CD intensity at selected wavelengths with increasing pH. The inset is a plot of the ellipticity at 384 nm versus pH, and the solid line represents the fit to a single deprotonation event with a pKa = 8.3.

100

60 ↓ 40

20 ↓ ↓

0

50

Ellipticity (mdeg) -20 40

30

-40 20 Ellipticity nm (mdeg) at 384 ↓ 10 6 7 8 9 10 11 12 pH -60 400 500 600 700 800

Wavelength (nm)

101

Figure 2.9 Comparison of the X-band EPR spectra of the dithionite-reduced C55S and C59S

AaeFd4 and C56S and C60S CpFd in the S = 1/2 region at pH values above and below the pKa values. The spectra were recorded at 35 K with a microwave frequency of 9.58 GHz, modulation amplitude of 0.64 mT and a microwave power of 0.5 mW.

102

g = 2.008 pH 11.0 g = 2.020 g = 2.007 pH 11.0 g = 1.994 pH 8.0 pH 7.0

g = 1.920 g = 1.928

g = 1.925 g = 1.937

g = 1.866 g = 1.860 g = 1.850 C55S AaeFd4 g = 1.885 C59S AaeFd4 g = 2.008 g = 2.007 pH 11.0 pH 11.0 g = 2.012 pH 6.0 g = 2.015 pH 6.0

g = 1.920 g = 1.922

g = 1.920 g = 1.935

C56S CpFd g = 1.885 g = 1.878 1.862 C60S CpFd g = 1.885

325 345 365 385 325 345 365 385 Magnetic Field (mT) Magnetic Field (mT)

103

Figure 2.10 X-band EPR spectra in the low-field region for dithionite-reduced C55S (A) and

C59S (B) AaeFd4 at pH 11.0. The spectra were recorded at a microwave frequency of 9.58 GHz and modulation amplitude of 0.64 mT with a microwave power of 50 mW.

104

g = 6.83 A g = 8.53 g = 9.58

g = 4.31 Effective g-values for zero-field 20 K doublets of a S = 9/2 ground state with E/D = 0.12, D < 0, and g0 = 2.00 MS> gx gy gZ ±1/2> 0.63 16.86 1.36 5x g = 4.33 ±3/2> 4.23 8.51 6.62 ±5/2> 9.52 1.72 1.94 7 K ±7/2> 13.90 0.07 0.08 ±9/2> 17.98 0.00 0.00

5x

4.2 K

0 100 200 300 Magnetic Field (mT)

g = 10.5 g = 9.1 B

g = 5.6

g = 3.7 g = 3.3 Effective g-values for zero-field doublets of a S = 9/2 ground state 20 K with E/D = 0.175, D < 0, and g0 = 2.04 MS> gx gy gZ ±1/2> 0.32 17.72 0.58 ±3/2> 3.43 10.51 5.62 ±5/2> 9.05 3.21 3.73 10 K ±7/2> 14.05 0.22 0.26 2x ±9/2> 18.31 0.00 0.00

8 K g = 4.3 2x

6 K

0 100 200 300 Magnetic Field (mT) 105

Figure 2.11 VTMCD spectra of the dithionite-reduced C55S AaeFd4 (0.3 mM) at pH 7.0 in

200 mM Tris-HCl and at pH 11.0 in 200 mM CAPS buffer. Both samples contained 50% (v/v) ethylene glycol and the spectra were recorded at a magnetic field of 6.0 T.

106

2.20 K pH 11.0 200 4.22 K 10.2 K ) -1 100 cm -1 M Δε ( 0

1.98 K 4.22 K pH 7.0 200 10.0 K 25.0 K

) -1

cm 100 -1 M Δε (

0

400 500 600 700 800 Wavelength (nm)

107

Figure 2.12 VTMCD spectra of the dithionite-reduced C59S AaeFd4 (0.6 mM) at pH 7.0 in

200 mM Tris-HCl and at pH 12.0 in 200 mM CAPS buffer. Both samples contained 50% (v/v) ethylene glycol and the spectra were recorded at a magnetic field of 6.0 T.

108

2.1 K pH 11.0 4.2 K 200 9.9 K -1

cm 100 -1 M Δε 0

-100

2.2 K 4.2 K pH 7.0 10.0 K

-1 cm

-1 100 M Δε

0

-100 400 500 600 700 800 Wavelength (nm)

109

Figure 2.13 UV-visible-NIR VTMCD spectra of dithionite-reduced C56S CpFd at pH 6.0

(0.72 mM in 100 mM MES buffer) and pH 11.0 (0.21 mM in 100 mM CAPS buffer). The samples contained 50% (v/v) ethylene glycol and the spectra were recorded in 0.1-cm cuvettes at a magnetic field of 6.0 T at the temperature indicated on the spectra. The intensities of all MCD bands increase in intensity with decreasing temperature.

110

1.64 K 4.22 K pH 11.0 10.0 K 80 19.9 K 48.8 K 600 60 40 450 20 )

-1 300 0 cm -1

M 150 Δε ( 0

-150

1.70 K pH 6.0 4.22 K 400 10.0 K ) -1 200 cm -1 M Δε ( 0

-200 400 600 800 1000 1200 1400 Wavelength (nm)

111

Figure 2.14 UV-visible-NIR VTMCD spectra of dithionite-reduced C60S CpFd at pH 6.0

(0.82 mM in 100 mM MES buffer) and pH 11.0 (0.20 mM in 100 mM CAPS buffer). The samples contained 50% (v/v) ethylene glycol and the spectra were recorded in 0.1-cm cuvettes at a magnetic field of 6.0 T at the temperature indicated on the spectra. The intensities of all MCD bands increase in intensity with decreasing temperature.

112

pH 11.0 1.64 K 4.22 K 80 10.0 K 20.0 K 60 50.3 K 40

400 20

0

) 200 -1

cm 0 -1 -200 (mM

Δε -400

-600

600 1.64 K pH 6.0 4.22 K 10.0 K 400 ) -1

cm 200 -1

(mM 0 Δε -200

-400

200 600 1000 1400 Wavelength (nm)

113

Figure 2.15 VHVT MCD magnetization data at 712 nm (A) and 1070 nm (B) for dithionite- reduced C56S CpFd at pH 11.0. Sample is as described in Fig. 2.13. MCD magnetization data were collected at temperatures of 1.64 K (○). 4.22 K, (□), and 10.0 K (Δ) at magnetic fields in

the range 0-6 T. (A) Solid lines correspond to theoretical VHVT MCD magnetization data for

85% of a predominantly xy-polarized transition (Mxy:Mxz:Myz = +1.00:+0.05:+0.08) from a

S = 9/2 ground state with D = −1.5 cm-1 and E/D = 0.12 and 15% of an S = 1/2 ground state with

g = 2.01, 1.92, and 1.88. (B) Solid lines correspond to theoretical VHVT MCD magnetization

data for a predominantly (93%) z-polarized transition (Mxy:Mxz:Myz = +0.15:−1.00:+0.69) from a

S = 9/2 ground state with D = −1.5 cm-1 and E/D = 0.12.

114

115

Figure 2.16 VHVT MCD magnetization data at 705 nm (A) and 1070 nm (B) for dithionite- reduced C60S CpFd at pH 11.0. Sample is as described in Fig. 2.14. MCD magnetization data were collected at temperatures of 1.64 K (○). 4.22 K, (□), and 10.0 K (Δ) at magnetic fields in

the range 0-6 T. (A) Solid lines correspond to theoretical VHVT MCD magnetization data for

90% of a predominantly xy-polarized transition (Mxy:Mxz:Myz = +1.00:+0.07:+0.05) from a

S = 9/2 ground state with D = −1.4 cm-1 and E/D = 0.17 and 10% of an S = 1/2 ground state with

g = 2.02, 1.93, and 1.86. (B) Solid lines correspond to theoretical VHVT MCD magnetization

data for a predominantly (91%) z-polarized transition (Mxy:Mxz:Myz = +0.15:−1.00:+0.55) from a

S = 9/2 ground state with D = −1.4 cm-1 and E/D = 0.17.

116

117

Figure 2.17 Comparison of the RTMCD spectra of C55S AaeFd4 and C56S CpFd at pH 7.0 and

12.0 using an applied magnetic field of 1.5 T. The samples (0.5 -1.0 mM in [2Fe-2S] clusters) were prepared in 200 mM mixed buffer with 0.1 M NaCl, and the pH was adjusted to 7.0 or 12.0.

118

6.0 C55S AaeFd4 C56S CpFd ) -1 3.0 cm -1

(M pH 12.0

Δε 0.0

1.2 ) -1 0.6 cm -1 pH 7.0 (M 0 Δε

-0.6 400 600 800 1000 Wavelength (nm)

119

Figure 2.18 Comparison of the RTMCD spectra of C59S AaeFd4 and C06S CpFd at pH 7.0 and

12.0 using an applied magnetic field of 1.5 T. The samples (0.5 -1.0 mM in [2Fe-2S] clusters) were prepared in 200 mM mixed buffer with 0.1 M NaCl, and the pH was adjusted to 7.0 or 12.0.

.

120

C59S AaeFd4 C60S CpFd )

-1 3.0 cm -1 pH 12.0 (M

Δε 0.0

1.8 )

-1 0.9 cm

-1 pH 7.0 (M 0 Δε

-0.9 400 600 800 1000 Wavelength (nm)

121

Figure 2.19 Schematic summary of the proposed ligation, spin states and redox properties of the

[2Fe-2S]2+,+ centers in the Cys-to-Ser variants of CpFd and AaeFd4 as a function of pH.

122

123

Figure 2.20 Schematic MO diagram for the Fe-Fe interactions in a valence-delocalized S = 9/2

[2Fe-2S]+ cluster with polarizations and assignments for the predicted electronic transitions. The z-axis is along the Fe-Fe coordinate.

124

125

Figure 2.21 Comparison of the low temperature MCD spectra of the valence-delocalized S = 9/2

[2Fe-2S]+ center in high pH reduced C60S CpFd with those of higher nuclearity Fe-S clusters believed to contain valence delocalized [2Fe-2S]+ fragments. The 6-T and 50-K spectrum is shown for C60S CpFd in order to emphasize the transitions from the S = 9/2 [2Fe-2S]+ component. All other spectra were recorded at 6 T and ~1.6 K.

126

127

CHAPTER 3

NATURE AND FUNCTION OF FE-S CLUSTERS ASSEMBLED ON GRXS16,

A CHLOROPLASTIC MONOTHIOL GLUTAREDOXIN1

1 † ‡ ‡ § § Sowmya Subramanian , Sunil G Naik ,, Boi Hanh Huynh ,, Jean Pierre Jacquot , Nicolas Rouhier , Michael K. Johnson†, to be submitted to Biochemistry, † Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA, ‡ Department of Physics, Emory University, Atlanta, Georgia 30322, § Unité Mixte de Recherches 1136 INRA Nancy University, Interactions Arbres Microorganismes, IFR 110 EFABA, Faculté des Sciences, BP 239 54506, Vandoeuvre Cedex, France. 128

1Abbreviations. Grx, glutaredoxin; At, Arabidiopsis thaliana; CD, circular dichroism; GSH,

glutathione; DTT, dithiothreitol; IPTG, isopropyl-β-D-thiogalactopyranoside; IscS, cysteine desulfurase; Syn Fd , Synechocystis ferredoxin; SUF, Sulfur Mobilization.

129

Abstract

Glutaredoxins (Grxs) with CGFS active site have recently been discovered to play a

critical role in the biogenesis of iron-sulfur clusters and cellular Fe homeostasis. In S. cerevisiae,

the deletion of grx5, which encodes for a mitochondrial monothiol Grx with CGFS active site,

renders the organism more susceptible to oxidative stress and results in the accumulation of free

iron and impaired Fe-S cluster biogenesis. Two monothiol Grxs from plant chloroplast, GrxS14

and GrxS16 were shown to rescue the yeast grx5 deletion strain, however, their exact role in

plant chloroplasts is not known. In this work, we report the results of our spectroscopic and

biochemical studies on recombinant GrxS16 from A. thaliana. Resonance Raman and Mössbauer

studies provide definitive evidence for the presence of a [2Fe-2S]2+ cluster in vivo, and either

[2Fe-2S]2+ or [4Fe-4S]2+ clusters in vitro. The results of analytical gel filtration studies indicate

that the [2Fe-2S] cluster-bound form of GrxS16 is likely to exist as a dimer, with the cluster

bound at the dimer interface, similar to the structurally characterized E. coli Grx4, and the [4Fe-

4S] cluster-bound form is likely to exist as a tetramer, with four S16 molecules providing an all-

cysteinyl ligation through the active site cysteine. In vitro kinetic studies monitored by CD

spectroscopy reveal that GrxS16 is able to transfer the [4Fe-4S] cluster to apo Nfu2 from plant

chloroplast effectively, in contrast to the slow and incomplete transfer of the [2Fe-2S] cluster to

apo chloroplast ferredoxin, indicating that GrxS16 is likely to be involved in the maturation of

[4Fe-4S] cluster-containing proteins in plant chloroplast.

130

Introduction

Glutaredoxins (Grxs) are small thiol-disulfide oxidoreductases which have traditionally

been considered to play an essential metabolic role as intrinsic components of cellular

regulatory/protective mechanisms (1;2). However, in recent years, evidence that has

accumulated in support of a role for Grxs in iron homeostasis and iron-sulfur (Fe-S) cluster biogenesis has added a new dimension to the biological functions of these ubiquitous proteins

(3). Fe-S proteins are central to metabolic processes across all kingdoms of life, as they are involved in two critical processes essential to life, photosynthesis and respiration, in addition to a plethora of other metabolic processes (4). The involvement of Grxs with Fe-S cluster biogenesis has opened new avenues of research concerning how these ubiquitous and versatile cofactors are assembled and delivered to acceptor proteins.

Grxs have been broadly grouped under 6 classes, based on the sequence of the motif containing the active site cysteine, glutathione binding motifs and the number of protein domains

(5). Of these classes, some class 1 dithiol Grxs with CGYC or CSYC/S active sites and all class

2 monothiol Grxs with CGFS active sites have been shown to assemble Fe-S clusters (6-9). Most of the evidence concerning the role of Grxs in Fe-S cluster biogenesis has come from studies on the monothiol CGFS Grx, Grx5, from Saccharomyces cerevisiae. In yeast, deletion of the grx5 gene resulted in impaired respiratory growth and increased sensitivity to oxidative stress due to deficient cluster assembly of Fe-S proteins in the mitochondria and accumulation of free iron in the cell (10;11). Furthermore, 55Fe immunoprecipitation studies in yeast suggest that Grx5 plays

a role in mediating transfer of clusters preassembled on the Iscu1p scaffold protein to acceptor

proteins (12). Interestingly, Fe-S cluster bound Grx5 was also shown to be essential for

vertebrate heme synthesis and regulation of bone apoptosis (13;14). 131

Recent crystallographic studies of the as purified recombinant monothiol CGFS Grx from

E. coli, Grx4 (15), confirmed the presence of a subunit bridging [2Fe-2S] cluster ligated by the

cysteines of the CGFS active site and two trans glutathiones as originally deduced by

biochemical, analytical and spectroscopic studies of this and other monothiol Grxs (6;8).

Moreover, compared to the analogously ligated [2Fe-2S] clusters in the dithiol human Grx2 and plant GrxC1 (9;16), which cannot be transferred to acceptor proteins, the [2Fe-2S] cluster-bound form of monothiol Grxs shows interesting structural differences (15) which provide insight into the mechanism of facile cluster transfer to acceptor proteins that is the hallmark of monothiol

Grxs (6). In the monothiol [2Fe-2S] Grx crystal structure, the homodimer is centered on the

[2Fe-2S] cluster with tetrahedral coordination at each iron atom completed by the active-site cysteine (Cys30) of a Grx monomer and the cysteine moiety of a glutathione (GSH). Lys22 that was previously shown by mutagenesis studies to be critical, in addition to Cys30, for Fe−S

cluster assembly (8), makes contact with both the GSH and the adjacent Grx. The GSH

molecules interact with the cluster in the same way as in dithiol human Grx2 and forms hydrogen

bonds with the same residues. However, whereas the orientation of the monomers with respect to

each other is similar in both crystallographically defined dithiol Grxs (8;9), E. coli Grx4 shows a

different orientation whereby one monomer is rotated 90° relative to the other. As a result, Grx4

monomers have a direct interaction with each other, which is not the case in dithiol Grxs.

Comparing the apo-monomeric and dimeric [2Fe-2S] cluster-bound structures of E. coli

Grx4 reveals another important difference. Super-positioning of the monomer and homodimer

structures shows that in the monomeric apo structure, the position of the critical residues for

cluster assembly (Cys30 and Lys22) are moved by 7.0 and 6.4 Å, respectively, from their

positions in the cluster-bound homodimer, hence making it difficult to establish the interactions 132

required for cluster binding. In contrast, comparison of the apo monomeric, GSH bound monomeric and cluster-bound dimeric structures of the dithiol human Grx2 reveals that the three structures superimpose almost perfectly with only minor variations. Based on these differences, it was proposed that the monothiol Grx dimers would need to undergo major conformational changes as a result of interactions with acceptor proteins and/or molecular chaperones in order to release the intact [2Fe-2S] cluster, consistent with roles in storage, transport, and delivery of clusters to apo acceptor proteins. In contrast, the cluster in dithiol Grxs is proposed to serve as a sensor, which is degraded under oxidizing conditions to yield a functional apo monomeric dithiol

Grx without any conformational change in the structure of the Grx.

In plants, monothiol CGFS Grxs are represented by four proteins: GrxS14 and S16 that are localized in the chloroplast; GrxS15 that is localized in the mitochondria; GrxS17 that is predicted to be cytosolic. Of the four Grxs, GrxS14, S16 and S17 were able to rescue the mutant grx5 defects in respiratory growth and sensitivity to oxidants, but not GrxS15 (6). It was

recently demonstrated that recombinant GrxS14 is expressed both in monomeric apo form and

[2Fe-2S] cluster-bound dimeric form, and that an identical cluster can be reconsitituted on apo-

GrxS14 in a cysteine desulfurase (IscS) mediated reaction, and transferred intact to apo ferredoxin at a rapid rate, suggesting a role for monothiol Grxs in the maturation of chloroplastic

Fe-S proteins (6). Unlike GrxS14, which comprises only the monothiol Grx domain, GrxS16 is a modular protein with a C-terminal monothiol Grx domain and a ~180 amino acid N-terminal domain of unknown function that is unique among monothiol Grxs. While preliminary studies of recombinant GrxS16 demonstrated that it is heterologously expressed in E. coli in a [2Fe-2S]

cluster-bound form (6), the specific roles of GrxS14 and S16 in chloroplastic Fe-S cluster

biogenesis remain to be addressed. In this work, we demonstrate that GrxS16 can accommodate 133

[2Fe-2S]2+, linear [3Fe-4S]+, and [4Fe-4S]2+ clusters and we assess the ability of [2Fe-2S] and

[4Fe-4S] cluster-bound forms of GrxS16 to transfer these clusters to physiologically relevant

acceptor proteins.

Materials and Methods

Materials. GSH and dithiothreitol (DTT) were obtained from Acros Organics and Fisher,

respectively. The antibiotics, ampicillin and kanamycin were purchased from Sigma-Aldrich and

Boehringer Mannheim, respectively. IPTG (isopropyl-β-D-thiogalactopyranoside) was obtained

from Molecula and the gel filtration molecular weight markers (MW-GF-70) were obtained from

Sigma-Aldrich.

Analytical and spectroscopic methods. Protein concentrations were determined by the DC

protein assay (Bio-Rad), using bovine serum albumin as the standard. Iron concentrations were

determined colorimetrically using bathophenanthroline under reducing conditions, after digestion of the protein in 0.8% KMnO4/0.2 M HCl (17). Sample concentrations and extinction

coefficients are based on protein concentrations unless otherwise stated. Analytical gel filtration

analyses were performed using a 25 mL SuperdexTM 10/300 column (Pharmacia Biotech) with

50mM Tris-HCl buffer, 100mM KCl at pH 7.6 as the elution buffer at a flow rate of 0.4 ml/min

using the published procedure (18;19). The molecular weight standards used were blue dextran

(Mr 2,000,000), alcohol dehydrogensae (Mr 150,000), albumin (Mr 66,000), carbonic anhydrase

(Mr 29,000) and cytochrome c (Mr 12,400).

Samples for spectroscopic studies were prepared under Ar in a Vacuum Atmospheres

glove box at oxygen levels < 2 ppm. UV-visible absorption and CD spectra were recorded at

room temperature using a Shimadzu UV-3101PC spectrophotometer and Jasco J-715

spectropolarimeter, respectively. Resonance Raman spectra were recorded as previously 134

described (20), using an Instruments SA Ramanor U1000 spectrometer coupled with a Coherent

Sabre argon ion laser, with 20 μL frozen droplets of 2-3 mM sample mounted on the cold finger

of an Air Products Displex Model CSA-202E closed cycle refrigerator. Mössbauer spectra were

recorded as described previously (21) . The zero velocity of the spectra refers to the centroid of

room temperature spectrum of a metallic Fe foil. Analysis of the Mössbauer data was performed

with the WMOSS program (Web Research).

Heterologous expression and purification of At GrxS16 in E. coli. The E. coli expression

strain BL21(DE3), containing the helper plasmid pSBET, was transformed with the pET3d

vector containing the At grxS16 gene encoding for GrxS16 with the 62 residue plastid targeting

sequence removed, as previously described (6). Generally, a 12 L LB media was innoculated

with an overnight culture resulting from the transformation of the recombinant plasmid, while

shaking at 37 ºC under aerobic conditions. The cell culture was induced in the exponential phase

(O.D600 0.6-0.8) with 0.1 mM IPTG and the cells were allowed to grow further for 4 hours at 32

ºC. The cells were harvested by centrifugation for 5 minutes at 9000 g and stored at -80 ºC until

further use. 57Fe-labeled samples of the as-isolated protein for Mössbauer studies were obtained

by growing the E. coli recombinant strain in LB media as described above and supplemented

with 57Fe during the cell culture. The protein was purified anaerobically under Ar in a Vacuum

Atmospheres glove box at oxygen levels < 2 ppm to isolate the as-purified form of the protein

for spectroscopic analyses. Aerobic purification of the protein was used to obtain apo protein for

Fe-S cluster reconstitution experiments.

Protein purification involved resuspension of the harvested cells in 100 mM Tris-HCl, pH

8.0, 1 mM GSH (buffer A) and removal of the cell debris by sonication, followed by centrifugation. The reddish-brown cell-free extract containing holo Grx S16 thus obtained was 135

subjected to a 30-60 % ammonium sulfate cut followed by centrifugation, when the protein

fractionated as a brown pellet. The protein pellet was resolubilized in buffer B (buffer A plus 1

M (NH4)2SO4 and loaded onto a 10 mL Hitrap Phenyl Sepharose (PS) column (GE Healthcare)

pre-equilibrated with buffer B. The protein was then eluted with a 1-0 M (NH4)2SO4 gradient, and the purest fractions, as judged by SDS-PAGE analysis were pooled and (NH4)2SO4 was removed by ultrafiltration dialysis using YM10 membrane and buffer A.

IscS mediated reconstitution of At GrxS16. Reconstitution of Fe-S cluster on apo GrxS16 was carried out under anaerobic conditions, in the presence of GSH or DTT. Reconstitution in the presence of GSH was carried out by incubating apo GrxS16 in 100 mM Tris-HCl, pH 8.0 buffer, with 5 mM GSH, 12-fold excess of ferrous ammonium sulfate, 12-fold excess of L- cysteine and catalytic amounts of IscS for 2 hours in an ice bath. Reagents in excess were removed anaerobically by loading the reconstitution mixture on to a 10 mL Hitrap Q-Sepharose column (GE Healthcare), and the protein was eluted with an increasing salt gradient, 0-1 M

NaCl. The cluster-bound protein eluted as two different fractions, and was collected separately

and concentrated using Amicon ultrafiltration with an YM10 membrane. Reconstitution in the

presence of DTT was carried out using the same protocol, replacing GSH with DTT in the

reconstitution mixtures and elution buffers. Only a single fraction containing cluster bound

protein eluted under increasing NaCl gradient in the presence of DTT.

Apo GrxS16 was also prepared from cluster-bound anaerobically purified samples using

the following procedure. Fractions containing >60% GrxS16, as judged by SDS-PAGE after

purification using the PS column, were pooled and concentrated using Amicon ultrafiltration

with a YM10 membrane. 10 mM EDTA and a 10-fold excess of potassium ferricyanide were

added to the concentrated protein and the resulting mixture was loaded on to a 300 mL 136

SuperdexTM 75 gel filtration column (GE Healthcare) pre-equilibrated with 100 mM Tris-HCl,

pH 8.0 buffer. The purest fractions, as judged by SDS-PAGE were pooled and concentrated, as described before.

Fe-S Cluster transfer experiments

Cluster transfer from holo [2Fe-2S] At GrxS16 to apo Synechocystis ferredoxin.

Synechocystis [2Fe-2S] ferredoxin (Syn Fd) used for cluster transfer experiments was

heterologously expressed in E. coli and purified according to published procedures (22). Apo Fd

was prepared by treating the holo protein with 10 mM EDTA and a 10-fold excess of potassium

ferricyanide inside the glove box, and removing the excess reagents by ultrafiltration dialysis

using an YM10 membrane and buffer A. The time course of the cluster transfer from [2Fe-2S]

GrxS16 to apo Fd was monitored at room temperature, under anaerobic conditions in small

volume 1-cm cuvettes using UV-visible CD spectroscopy. The reaction was carried out in buffer

A with 5mM DTT and apo Syn Fd was added 30 min prior to the initiation of cluster transfer

reaction by the addition of [2Fe-2S] GrxS16.

Cluster transfer from holo [4Fe-4S] At GrxS16 to apo At Nfu2. At Nfu2 was

heterologously expressed in E. coli. The pET3d vector with ampicillin resistance and At Nfu2

insertion was transformed into BL21[DE3] pSBET competent cells and an overnight culture of

the transformed cells was used to innoculate the LB growth media. When OD600 ~ 0.8, At Nfu2

was induced by adding 200 μg/mL IPTG and the incubation was continued at 34 °C for an

additional 4 hr period. The cells were then harvested by centrifugation at 9000 g at 4 °C for 10 min, and stored at -80 °C for later use. Protein purification involved resuspension of the harvested cells in 100 mM Tris-HCl, pH 7.5, with 1 mM DTT (buffer C), and the cells were lysed either by sonication or by incubation with lysozyme. The homogenate was centrifuged at 137

39700 g at 4 °C for 1 hr, followed by a 40% and an 80% ammonium sulfate cut. The pellet

obtained after the second ammonium sulfate cut was resuspended in buffer C supplemented with

1 M (NH4)2SO4 and purified using a Hi-trap phenyl sepharose FF column followed by a Hi-trap

Q sepharose HP column. Fractions containing At Nfu2, identified by SDS-PAGE, were pooled

and concentrated using ultrafiltration. Any residual cluster that was present was removed by

treatment with 10 mM EDTA and a 10-fold excess of potassium ferrocyanide followed by desalting to afford the apo protein for cluster transfer experiments.

The time course of the cluster transfer from [4Fe-4S] GrxS16 (reconstituted in the

presence of DTT) to apo At Nfu2 was monitored at room temperature, under anaerobic

conditions in 1 cm cuvettes using UV-visible CD spectroscopy. The reaction was carried out in

100 mM Tris buffer, pH 8.0 with 5mM DTT and apo At Nfu2 was added 30 min prior to the

initiation of cluster transfer reaction by the addition [4Fe-4S] At GrxS16.

Results

Nature and properties of Fe-S clusters assembled on At GrxS16. The nature of Fe-S

clusters assembled on At GrxS16 was determined by carrying out spectroscopic and analytical

studies on as-purified and reconstituted samples. Aerobic purification of At GrxS16 in the

presence of GSH resulted in a brown colored fraction, which had absorption characteristics

typical of a [2Fe-2S]2+ center. However, due to low Fe content (typically < 0.5 Fe per

homodimer), samples for spectroscopic characterization were prepared by purifying the protein

under anaerobic conditions. Anaerobic purification of At GrxS16 in the presence of GSH

afforded a [2Fe-2S]2+ cluster, as evident from very similar absorption and CD spectra (thin line,

Figure 3.1) compared to other [2Fe-2S]2+ cluster bound monothiol Grxs (6). On the basis of the

-1 -1 theoretical and experimental ε280 values for the apo protein (28 mM cm ), the ε280 and ε410 138

2+ -1 -1 values for the [2Fe-2S] center are estimated to be 5.5 and 4.9 mM cm , respectively, and the

A410/A280 was found to be 0.15 ± 0.01. The cluster extinction coefficients are in good agreement

with structurally and spectroscopically characterized monothiol Grxs containing one [2Fe-2S]

cluster per homodimer and this is confirmed by quantitative Fe and protein analyzes which

indicated 1.1 ± 0.1 Fe/ GrxS16 monomer.

In vitro IscS-mediated reconstitution of apo GrxS16 under anaerobic conditions, using

ferrous ammonium sulfate and L-cysteine, yielded different results depending on the presence of

GSH or DTT. In the presence of GSH, two brown colored fractions were obtained upon

purification of the reconstitution mixture, fraction 1 and 2, while a single brown colored fraction

was obtained in the presence of DTT. Fraction 2 of the sample reconstituted in the presence of

GSH and the sample reconstituted in the presence of DTT (thick line in Figure 3.1) exhibited

very similar absorption and CD properties. The absorption spectra comprise a broad absorption

centered at 400 nm and both samples exhibit weak CD spectra, properties that are characteristic

of a [4Fe-4S]2+ cluster rather than a [2Fe-2S]2+ cluster. Both samples contained 1.02 ± 0.2

Fe/protein monomer suggesting one [4Fe-4S]2+ center per tetramer. Moreover, this conclusion is

-1 -1 in accord with the ε400 value of 4.5 ± 0.5 mM cm based on GrxS16 monomer which translates to 18 ± 2 mM-1cm-1 per tetramer and is the range established for [4Fe-4S]2+ clusters, 14-18

mM-1cm-1 (23). Fraction 1 of the sample reconstituted in the presence of GSH exhibit absorption

and CD spectra (broken line in Figure 3.1) more similar to those of the [2Fe-2S]2+ center in anaerobically purified GrxS16 (thin line in Figure 3.1). However, there are some significant differences, notably increased absorption intensity and more pronounced absorption bands at 520 and 570 nm, coupled with the appearance of a positive CD band at 560 nm and decreased negative CD intensity at 400 nm, that are characteristic of the presence of a linear [3Fe-4S]+ 139 component (24;25) and (S. Bandyopadhyay, B. Zhang, and M. K. Johnson, unpublished results).

Iron and protein analyses indicated 1.3 ± 0.2 Fe/monomer suggesting the sample is a mixture of forms containing one [2Fe-2S]2+ and one [3Fe-4S]+ cluster per homodimer. As discussed below the cluster composition of as purified and reconstituted At GrxS16 suggested by analytical and

UV-visible absorption and CD studies are supported by resonance Raman and Mössbauer data.

Resonance Raman studies of the anaerobically purified At GrxS16 and fraction 1 of the

At GrxS16 reconstituted in the presence of GSH were carried out to assess cluster content to gain an insight in to the vibrational properties and nature of cluster ligation. Figure 3.2 shows a comparison of the resonance Raman spectra of the two anaerobically purified plant chloroplast monothiol Grxs, At GrxS16 and GrxS14 at three different excitation wavelengths. The spectra are indicative of a [2Fe-2S] as the sole type of Fe-S center and the close correspondence between the spectra at all three excitation wavelengths indicates analogous [2Fe-2S] cluster ligation and environment for both Grxs. The Fe-S stretching frequencies are similar to those of structurally characterized [2Fe-2S] ferredoxins with complete cysteinyl cluster and are readily assigned to

b t b 2− t vibrational modes of the Fe2S 2S 4 unit (S , bridging S ; S = terminal or cysteinyl S) by direct analogy with published data (26;27). Figure 3.3 shows the comparison of the resonance Raman spectra of anaerobically purified At GrxS16 (thick line) and fraction 1 of the At GrxS16 reconstituted in the presence of GSH (thin line) at three different excitation wavelengths. In addition to the Fe-S stretching modes associated with the [2Fe-2S]2+ center, additional bands are observed at ~286 and 388 cm-1 for fraction 1 of the At GrxS16 reconstituted in the presence of

GSH. Since the dominant resonance Raman bands of the linear [3Fe-4S]+ center in reconstituted

S. cerevisiae Grx5 are observed at ~280 and 388 cm-1 (S. Bandyopadhyay, B. Zhang, and M. K. 140

Johnson, unpublished results), these bands are attributed to a linear [3Fe-4S]+ cluster component in accord with the UV-visible absorption and CD results.

Mössbauer studies of cluster-bound forms of GrxS16 were undertaken to address the nature and stoichiometry of clusters in the anaerobically purified and reconstituted samples. The results are shown in Figure 3.4 and the parameters for the Fe components used in simulating the spectra are given in Tables 3.1 and 3.2. The anaerobically purified spectrum is well simulated as

[2Fe-2S]2+ cluster bound form (94%), with a minor contribution from an adventitious Fe2+ species (6%). The spectra of cluster-bound fractions obtained by reconstitution in the presence of

GSH can only be simulated as a mixture of different Fe-S cluster species. Fraction 1 is simulated as a mixture of 57% [2Fe-2S]2+ and 27% linear [3Fe-4S]+ clusters with minor contribution from

a [4Fe-4S]2+ cluster (4%) and an adventititous Fe2+ species (12%). Fraction 2 is simulated as a

mixture of 21% [2Fe-2S]2+ and 70% [4Fe-4S]2+ clusters with minor adventitious Fe2+ species

(9%). In contrast, the Mössbauer spectra of the At GrxS16 reconstitution in the presence of DTT

is simulated as [4Fe-4S]2+ cluster-bound form (92%) with a minor contribution from an

adventitious Fe2+ species (8%). The S = 0 [2Fe-2S]2+ clusters in different samples of At GrxS16

are simulated with identical parameters as a sum of symmetric, quadrupole doublets from each

Fe site. The isomer shift (δ) and the quadrupole splitting (ΔEQ) values, see Table 3.1, are

characteristic of a [2Fe-2S]2+ clusters with tetrahedral S ligation at each site, suggesting all

cysteinyl ligation, similar to that reported for At GrxS14 (6). Likewise, the S = 0 [4Fe-4S]2+ clusters in different samples of At GrxS16 are simulated with identical parameters as a sum of symmetric, quadrupole doublets from two valence delocalized [2Fe-2S]+ pairs. The isomer shift

2+ (δ) and the quadrupole splitting (ΔEQ) values, see Table 3.1, are characteristic of a [4Fe-4S] clusters with tetrahedral S ligation at each site, suggesting all cysteinyl ligation (28). The S = 5/2 141

linear [3Fe-4S]+ cluster that is observed in fraction 1 of samples reconstituted in the presence of

GSH exhibits magnetic hyperfine and is simulated with spin Hamiltonian and magnetic hyperfine parameters very similar to those used for the linear [3Fe-4S]+ cluster that forms in

partially unfolded aconitase at alkaline pH values (24), see Table 3.2. The Mössbauer results are

clearly in excellent agreement with the UV-visible absorption/CD, resonance Raman and

analytical studies and provide quantitative assessment of the cluster composition in cluster-

bound forms of At GrxS16 prepared under different conditions.

Oligomeric state of Fe-S cluster-bound forms of At GrxS16. The oligomeric states of

anaerobically purified and reconstituted samples of At GrxS16 were assessed by analytical gel

filtration chromatograpy using a SuperdexTM 75 column and monitoring the absorbance at 280

nm. Apo and anaerobically purified At GrxS16 eluted as single bands with apparent MWs of

~33 kDa and ~64 kDa, respectively, suggesting a monomeric apo form and a dimeric [2Fe-2S] cluster-bound anaerobically purified form, based on the actual MW of 25428 Da. The elution profiles of the cluster-bound reconstituted samples investigated in this work are shown in Figure

3.5. Both fractions 1 and 2 of the samples reconstituted in the presence of GSH, eluted as three different peaks at elution volumes of 7.6, 9.2 and 10.7 mL which correspond to apparent MWs of

~135 kDa, ~65 kDa and ~35 kDa indicating tetrameric, dimeric and monomeric components. In contrast, the samples reconstituted in the presence of DTT showed only bands at elution volumes of 7.6 and 10.7 mL indicating only tetrameric and monomeric forms. Based on UV-visible absorption and CD studies of the eluted fractions, the monomeric form is apo, whereas the

tetrameric and dimeric forms contain [4Fe-4S]2+ and [2Fe-2S]2+/linear [3Fe-4S]+ clusters,

respectively. Moreover, the intensities of the different cluster-bound forms in the elution profiles

correlate to a good approximation with the Mössbauer results. Taken together, the analytical gel 142

filtration and the spectroscopic/analytical results indicate that the [4Fe-4S] cluster-bound form is a tetramer with one cluster per tetramer and that the [2Fe-2S] and linear [3Fe-4S] cluster-bound

forms are dimers with one cluster per dimer.

Cluster transfer studies involving [2Fe-2S] and [4Fe-4S] cluster-bound At GrxS16. The

above results demonstrate that GrxS16 can assemble a [2Fe-2S]2+ cluster, both in vivo and in vitro, and [4Fe-4S]2+ and linear [3Fe-4S]+ clusters, in vitro. However, the role of the cluster

bound forms of GrxS16 remains unknown. The ability to assemble [2Fe-2S] and [4Fe-4S]

clusters suggests that GrxS16 may function in the assembly, transfer or storage of biological Fe-

S clusters. In order to assess the role of GrxS16 in cluster trafficking, cluster transfer

experiments were carried out to the apo forms of ferredoxin and Nfu2, which constitute

potentially relevant chloroplastic [2Fe-2S] and [4Fe-4S] acceptor proteins, respectively.

The marked difference in the CD spectra of the [2Fe-2S]2+ centers of holo Fd and GrxS16

offers a convenient way of monitoring the process of cluster transfer, as a function of time. By

following the increase in the CD intensity of the acceptor, and the simultaneous decrease in the

CD intensity of the donor, the extent of cluster transfer can be quantitatively assessed.

Comparison of the time course of the CD changes in a reaction mixture containing a 1:1

stoichiometry of GrxS16 [2Fe-2S] clusters and apo Fd (Figure 3.6) with that of the simulated

data for a 0-100% cluster transfer indicates that the cluster transfer comes to an equilibrium with

~50% of the clusters on the donor and acceptor after about 90 min. The data thus indicate that

though GrxS16 is able to transfer its cluster to apo Fd, the transfer occurs slowly and does not go

to completion. The data therefore demonstrate that GrxS16 is competent for [2Fe-2S] cluster

transfer, but that apo Fd is unlikely to be a physiologically relevant acceptor protein in vivo.

Analogous experiments, using [2Fe-2S] GrxS14 as the cluster donor, showed rapid and 143

complete cluster transfer with a second order rate constant of 20,000 M-1min-1 (6) indicating that

GrxS14 is a much better candidate for the cluster donor to Fd in plant chloroplasts.

At Nfu2 was chosen as a candidate for a chloroplastic [4Fe-4S]-cluster acceptor protein

because the corresponding Nfu protein in cyanaobacteria (NfuA) has been shown to bind one

[4Fe-4S]2+ cluster per homodimer that can be used for maturation of the apo form of PsaC of

Photosystem 1 (29). At GrxS16 with one [4Fe-4S]2+ per tetramer and At Nfu2 with one [4Fe-

4S]2+ cluster per dimer exhibit very similar UV-visible absorption spectra but very different UV- visible CD spectra, see Figure 3.7. Hence CD was used to monitor the time course of [4Fe-4S]2+ cluster transfer, although the experiments are more difficult than for [2Fe-2S]2+ cluster transfer as the CD intensity is an order of magnitude weaker for [4Fe-4S] centers, cf Figures 3.6 and 3.7.

The CD-monitored time course of a cluster transfer reaction involving a 1:2 stoichiometry of

GrxS16 [4Fe-4S] clusters and monomeric apo Nfu2 is shown in Figure 3.7. The cluster transfer is 60-70% complete after 20 mins and almost 100% complete after 180 mins and the data at discrete wavelengths 360 and 450 nm can be fit to a good approximation by second order kinetics with a rate constant of 3000 ± 600 M-1min-1. This rate of [4Fe-4S] cluster transfer is

comparable with those reported in the literature for other [4Fe-4S] cluster transfers from donor to

acceptor proteins (30;31). Taken together with the observation that the reverse cluster transfer

from [4Fe-4S] Nfu2 to apo GrxS16 does not occur, this result demonstrates that GrxS16 has the

potential to be involved with [4Fe-4S] trafficking in plant chloroplasts.

Discussion

Plant chloroplasts are replete with proteins containing [2Fe-2S] and [4Fe-4S] clusters, as

a part of the photosynthetic electron transport chain and metabolic processes, such as nitrogen

and sulfur assimilation, but little is currently known concerning the mechanism of maturation of 144

chloroplastic Fe-S proteins. Most of the current understanding of Fe-S cluster biogenesis in plant

chloroplasts has come from the study of the components of SUF (Sulfur Mobilization)

machinery and Nfu proteins (32). The recent discovery that monothiol Grxs can assemble and

transfer [2Fe-2S] clusters (6;8;15), coupled with the presence of two monothiol Grxs, GrxS14

and GrxS16 in chloroplasts, have suggested possible roles for Grxs in the maturation of Fe-S

proteins either in de novo Fe-S cluster biosynthesis or as Fe-S cluster carrier proteins that receive

clusters assembled by the SUF machinery and deliver them to specific acceptor proteins or other

cluster scaffold or carrier proteins such as the Nfu proteins. Although very little is known about

the physiological roles played by monothiol glutaredoxins including GrxS16, recent

bioinformatic studies indicate that the genes encoding for monothiol CGFS Grxs site are often

found clustered along with various Fe-S cluster assembly genes, and genes that code for Fe-S

proteins, including ferredoxins in microbial genomes (3).

In this study, the nature and properties of the Fe-S clusters assembled on anaerobically

purified and in vitro reconstituted At GrxS16 have been investigated. Using a range of

spectroscopic and analytical techniques, the results indicate that GrxS16 is able to assemble an

all-cysteinyl ligated [2Fe-2S]2+, linear [3Fe-4S]+ and [4Fe-4S]2+ clusters. Analytical gel filtration

data indicate that both the [2Fe-2S] and linear [3Fe-4S] cluster-bound forms are likely to be

homodimers containing one [2Fe-2S] or linear [3Fe-4S] cluster per dimer, whereas the [4Fe-4S]

cluster-bound form is a homotetramer containing one [4Fe-4S] per tetramer. Moreover, GSH

appears to be required for the assembly of [2Fe-2S] and linear [3Fe-4S] clusters, but not for the

[4Fe-4S] cluster since it is the only cluster that can be assembled in the absence of GSH. Based

on the close correspondence of the spectroscopic properties compared to GrxS14 and the structurally characterized E. coli Grx4 (15), the [2Fe-2S] cluster-bound form of homodimeric 145

GrxS16 is almost certainly coordinated by the active-site cysteines of the two CGFS Grxs and

two trans GSHs. An analogous set of coordinating cysteines, including two Grxs and two GSHs,

is also likely for the linear [3Fe-4S] cluster in the linear [3Fe-4S] cluster-bound form of

homodimeric GrxS16.

The specific cysteine residues that ligate the [4Fe-4S] cluster in the [4Fe-4S] cluster-

bound form of tetrameric GrxS16 have yet to be determined. Since GrxS16 has an active site

(CGFS) cysteine and a second partially conserved cysteine, Cys221, which is conserved in 60% of known CGFS Grxs, one possibility is that the cluster is ligated at the dimer interface by the

active-site CGFS cysteines and the two partially conserved cysteines with two apo GrxS16

molecules stabilizing a tetrameric complex. If this is correct, the available in vivo evidence

argues against the [4Fe-4S] cluster-bound form being physiologically relevant since

complementation studies indicate that the partially conserved cysteine in yeast Grx5 is not

required to rescue the Fe-S cluster biogenesis defects in mutant grx5 strains. However, the

observation that At GrxS14, which also has the partially conserved cysteine, is unable to

reconstitute any type of Fe-S cluster in the absence of GSH using the same procedure used for

reconstituting a [4Fe-4S] cluster on GrxS16, argues against involvement of the partially

conserved cysteine in ligating the [4Fe-4S] cluster in GrxS16. Hence we favor the alternative

hypothesis, namely that the [4Fe-4S] cluster in GrxS16 is coordinated at the tetramer interface

using the active site CGFS cysteines of all four Grxs. Crystallographic studies of [4Fe-4S]-

GrxS16 and cysteine mutagenesis experiments are planned to test this hypothesis.

The ability of At GrxS16 to bind [2Fe-2S]2+, linear [3Fe-4S]+, and [4Fe-4S]2+ clusters is

in stark contrast to the other plant chloroplast monothiol Grx, GrxS14, which has only been found to bind a [2Fe-2S] cluster in the presence of GSH despite numerous reconstitution 146

attempts under a wide variety of different conditions (S. Bandyopadhyay and M.K. Johnson,

unpublished results). This suggests that the different roles for GrxS14 and GrxS16 in chloroplast

Fe-S cluster biogenesis are likely to be related to the ~180 amino acid N-terminal domain, that is

only present in GrxS16. Evidence for different roles for GrxS14 and GrxS16 comes from a

comparison of their ability to transfer [2Fe-2S] clusters to the same plant-type apo Fd under

analogous conditions. For [2Fe-2S]-GrxS16, the transfer occurs slowly and comes to equilibrium at about 50% completion after ~ 90 mins, whereas [2Fe-2S] GrxS14 is a much more effective cluster donor, capable of rapid and complete cluster transfer with a second order rate constant of

20,000 M1min1 (6). However, GrxS16 was able to serve as an effective in vitro [4Fe-4S] cluster

donor to Nfu2 which has been implicated to play a role in the maturation of [4Fe-4S] centers in

Photosystem I based on both in vivo and in vitro studies (29;33). Hence it is tempting to speculate that GrxS14 plays a role in the maturation of [2Fe-2S] cluster-containing proteins and that GrxS16 is primarily involved with the maturation of [4Fe-4S] cluster-containing proteins in plant chloroplasts. Additional in vitro cluster transfer studies from GrxS14 and GrxS16 to a wider range of chloroplastic [2Fe-2S] and [4Fe-4S]-containing proteins, coupled with in vivo gene knock out studies in A. thaliana, are planned to address the specific roles of the two monothiol Grxs in chloroplastic Fe-S cluster biogenesis.

The linear [3Fe-4S]+ clusters observed when GrxS16 is reconstituted in the presence of

GSH could be a physiologically relevant species or an artifact of the reconstitution conditions.

Recombinant forms of several as-purified and reconstituted monothiol Grxs, including yeast

Grx5, have been found to contain linear [3Fe-4S]+ clusters (S. Bandyopadhyay, B. Zhang, and

M. K. Johnson, unpublished results). In addition many of the published absorption spectra of

cluster-containing monothiol Grxs, see for example Ref. (8), have contributions from linear 147

[3Fe-4S]+ clusters even though they are interpreted as exclusively containing [2Fe-2S]2+ clusters.

To date, there is no evidence that protein-bound linear [3Fe-4S]+ clusters are physiologically

relevant, although they have been observed under oxidative denaturing conditions for many Fe-S

proteins, e.g. bovine heart [4Fe-4S] aconitase (24), [4Fe-4S] dihydroxy acid dehydratase

(DHAD) (34), [3Fe-4S]/[4Fe-4S] Fds (35;36), [3Fe-4S] and [2Fe-2S] Fds (37;38). Only one

protein, human iron regulatory protein 1 (IRP1), has been purified in a form containing a linear

[3Fe-4S]+ cluster (39). However, synthetic and biological linear [3Fe-4S]+ clusters can be used for the synthesis of [2Fe-2S]2+ clusters under oxidizing conditions with loss of Fe3+ ions and

[4Fe-4S]2+ under reducing conditions in the presence of Fe2+ ions (24;40). Hence the ability to

+ accommodate linear [Fe3S4] clusters may make monothiol Grxs unusually versatile scaffold or

carrier proteins able to service proteins requiring either [2Fe-2S]2+ and [4Fe-4S]2+ clusters or

may be related to a role in recycling clusters released as a result of protein unfolding under

aerobic or oxidative stress conditions.

Acknowledgements This work was supported by grants from the National Institutes of Health (GM62524 to

MKJ and GM47295 to BHH).

148

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25. Richards, A. J. M., Thomson, A. J., Holm, R. H., and Hagen, K. S. (1990) The magnetic 3- circular dichroism spectra of the linear trinuclear clusters [Fe3S4(SR)4] in purple aconitase and in a synthetic model, Spectrochim. Acta 46A, 987-993.

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27. Han, S., Czernuszewicz, R. S., and Spiro, T. G. (1989) Vibrational spectra and normal mode analysis for [2Fe-2S] protein analogues using 34S, 54Fe, and 2H substitution: Coupling of Fe-S stretching and S-C-C bending modes, J. Am. Chem. Soc. 111, 3496- 3504.

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31. Unciuleac, M.-C., Chandramouli, K., Naik, S., Mayer, S., Huynh, B. H., Johnson, M. K., and Dean, D. R. (2007) In vitro activation of apo-aconitase using a [4Fe-4S] cluster- loaded form of the IscU [Fe-S] cluster scaffolding protein, Biochemistry 46, 6812-6821.

32. Balk, J. and Lobréaux, S. (2005) Biogenesis of iron-sulfur proteins in plants, Trends Plant Sci. 10, 324-331.

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34. Flint, D. H., Emptage, M. H., Finnegan, M. G., Fu, W., and Johnson, M. K. (1993) The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase, J. Biol. Chem. 268, 14732-14742. 151

35. Griffin, S., Higgins, C. L., Soulimane, T., and Wittung-Stafshede, P. (2003) High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin. Linear clusters form at high pH on polypeptide unfolding, Eur. J. Biochem. 270, 4736-4743.

36. Jones, K., Gomes, C. M., Huber, H., Teixeira, M., and Wittung-Stafshede, P. (2002) Formation of a linear [3Fe-4S] cluster in a seven-iron ferredoxin triggered by polypeptide unfolding, J. Biol. Inorg. Chem. 7, 357-362.

37. Higgins, C. L. and Wittung-Stafshede, P. (2004) Formation of linear three-iron clusters in Aquilex aeolicus two-iron ferredoxins: effect of protein unfoding speed, Arch. Biochem. Biophys. 427, 154-163.

38. Pereira, M. M., Jones, K. L., Compos, M. G., Melo, A. M. P., Saraiva, L. M., Louro, R. O., Wittung-Stafshede, P., and Teixeira, M. (2002) A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus, Biochim. Biophys. Acta-Proteins and Proteomics 1601, 1-8.

39. Gailer, J., George, G. N., Pickering, I. J., Prince, R. C., Kohlhepp, P., Zhang, D., Walker, F. A., and Winzerling, J. J. (2001) Human cytosolic iron regulatory protein 1 contains a linear iron-sulfur cluster, J. Am. Chem. Soc. 123, 10121-10122.

40. Hagen, K. S., Watson, A. D., and Holm, R. H. (1983) Synthetic routes to Fe2S2, Fe3S4, 2- Fe4S4, and Fe6S9 clusters from the common precursor [Fe(SC2H5)4] : Structures and 3- 4- properties of [Fe3S4(SR)4] and [Fe6S9(SC2H5)2] , examples of the newest types of Fe-S- SR clusters, J. Am. Chem. Soc. 105, 3905-3913.

152

Table 3.1: Mössbauer parameters for the cluster bound forms of At GrxS16

Sample Cluster type δ (mm/s) ΔEQ (mm/s) Γ (mm/s) As-purified [2Fe-2S]2+ site 1 0.28 0.69 0.28 site 2 0.28 0.44 0.28

Fe2+ site 1 0.76 3.41 0.57 & 0.52 site 2 1.30 3.77 0.53 & 0.59

At GrxS16 reconstituted [2Fe-2S]2+ in the presence of GSH; site 1 0.27 0.70 0.28 Fraction 1 site 2 0.27 0.44 0.28

linear [3Fe-4S]+ see Table 3.2 see Table 3.2 see Table 3.2

[4Fe-4S]2+ pair 1 0.45 1.00 0.31 pair 2 0.46 1.26 0.31

Fe2+ 1.30 3.00 0.65

Fraction 2 [2Fe-2S]2+ site 1 0.27 0.70 0.28 site 2 0.27 0.44 0.28

[4Fe-4S]2+ pair 1 0.45 1.00 0.31 pair 2 0.46 1.26 0.31

Fe2+ 1.30 3.00 0.65

At GrxS16 reconstituted [4Fe-4S]2+ in the presence of DTT pair 1 0.45 1.00 0.31 pair 2 0.46 1.26 0.31

Fe2+ 1.30 3.00 0.65

153

Table 3.2: Spin Hamiltonian and magnetic hyperfine parameters used in fitting the Mössbauer spectrum of the S = 5/2 linear [3Fe-4S]+ cluster component observed in fraction 1 of At GrxS16 reconstituted in the presence of GSH.

Site 1 Site 2 Site 3 D (cm-1) 2.0 2.0 2.0 E/D 0.31 0.31 0.31

gx 2.0 2.0 2.0

gy 2.0 2.0 2.0

gz 2.0 2.0 2.0

Ax/gnβn (T) -13.0 -12.0 10.0

Ay/gnβn (T) -13.7 -13.2 9.9

Az/gnβn (T) -13.7 -10.0 10.5

ΔEQ (mm/s) -0.64 -0.73 0.57 δ (mm/s) 0.28 0.28 0.38 Γ (mm/s) 0.35 0.35 0.35 η -1.2 -1.34 0.45

154

Figure 3.1 UV-visible absorption and CD spectra of recombinant At Grx S16, as purified under anaerobic conditions (thin line), fraction 1 of a sample reconstituted in the presence of GSH

(broken line), and reconstituted in the presence of DTT (thick line).

155

8 Absorption

6 ) -1 cm

-1 4 (mM ε 2

0 CD

4 ) -1 cm

-1 0 (M Δε -4

-8 300 400 500 600 700

Wavelength (nm)

156

Figure 3.2 Comparison of the resonance Raman spectra of the anaerobically purified [2Fe-2S]

cluster-bound forms of At Grx S16 (thick line) and At Grx S14 (thin line) with 514-, 488- and

457-nm laser excitation. Samples were ~2 mM in [2Fe-2S] cluster and were in the form of a frozen droplet at 21 K. Each spectrum is the sum of 100 scans, with each scan involving counting photons for 1 s each 0.5 cm-1 with 7 cm-1 spectral resolution. Lattice modes of ice have

been subtracted.

157

400 402 292 348 325 424 288 347 402 292 514 nm 349 288 329 424 402 Raman Intensity 325 292 346 488 nm 402 288 327 346 400

457 nm

250 300 350 400 450

Raman Shift (cm-1) 158

Figure 3.3 Comparison of the resonance Raman spectra of anaerobically purified At GrxS16

(thick line) and fraction 1 of the At GrxS16 reconstituted in the presence of GSH (thin line) with

514-, 488- and 457-nm laser excitation. Samples were ~2 mM in [2Fe-2S] cluster and were in the form of a frozen droplet at 21 K. Each spectrum is the sum of 100 scans, with each scan involving counting photons for 1 s each 0.5 cm-1 with 7 cm-1 spectral resolution. Lattice modes of ice have been subtracted.

159 388 400 286 349 292 329 348 325

388 514 nm 292 402 349 325 290 Raman Intensity 325 286

347 488 nm 326 400 292 388 346 327

457 nm

250 300 350 400 450

Raman Shift, cm-1

160

Figure 3.4 Mössbauer spectra of cluster-bound forms of At GrxS16: anaerobically purified;

fractions 1 and 2 of a sample reconstituted in the presence of GSH; reconstituted in the presence

of DTT. The anaerobically purified Mössbauer sample was prepared by growing cells on 57Fe-

enriched media and the reconstituted samples were prepared by IscS-mediated reconstitution of

apo At GrxS16 using 57Fe(II) and cysteine. The Mössbauer spectra were recorded at 4.2 K with a

magnetic field of 50 mT applied parallel to the γ-beam. The solid black lines are theoretical

simulations, and the red, orange, blue and green lines correspond to the constituent [2Fe-2S]2+, linear [3Fe-4S]+, [4Fe-4S]2+ and high-spin Fe(II) components for each theoretical simulation, respectively. The parameters used in simulating the Fe species are listed in Tables 3.1 and 3.2.

161

[2Fe-2S]2+ 94 % Anaerobically purified Fe2+ 6%

[2Fe-2S]2+ 57% Reconstituted with GSH Linear [3Fe-4S]+ 27% Fraction 1 [4Fe-4S]2+ 4% Fe2+ 12%

[2Fe-2S]2+ 21% Reconstituted with GSH [4Fe-4S]2+ 70% Fraction 2 Fe2+ 9%

Reconstituted with DTT [4Fe-4S]2+ 92% Fraction 1 Fe2+ 8% 162

Figure 3.5 Analytical gel filtration elution profiles for the reconstituted fractions of At GrxS16, using a SuperdexTM column.

163

Dimer

At GrxS16 reconstituted Tetramer in presence of GSH; Monomer Fraction 1 nm (arb. units) nm (arb.

Fraction 2 Absorption at 280

At GrxS16 reconstituted in presence of DTT

0 5 10 15 20 25 Elution Volume (mL) 164

Figure 3.6 Time course of [2Fe-2S] cluster transfer from At GrxS16 to apo Syn Fd monitored by

UV-visible CD spectroscopy at 25°C in a small volume 1-cm cuvette. CD spectra were recorded

at 5 min intervals for a period of 60 min, and at 90 min for a reaction mixture that was initially

160 μM in GrxS16 [2Fe-2S] clusters and 160 μM apo Fd. The spectrum at zero time (thick line)

corresponds to [2Fe-2S] GrxS16 in the same reaction mixture in the absence of apo Fd. The

broken line corresponds to the spectrum of holo Syn Fd. The arrows indicate the direction of

intensity change with time at selected wavelengths. The Δε values are expressed per [2Fe-2S]2+ cluster.

165

20 ) 1 ↑ 10 cm- -1 (M Δε

0 ↓

-10 300 400 500 600 700 800

Wavelength (nm)

166

Figure 3.7 Time course of [4Fe-4S] cluster transfer from At GrxS16 to apo At Nfu2 monitored

by UV-visible CD spectroscopy at 25°C in a small-volume 1-cm cuvette. CD spectra were

recorded at 20 min intervals for 100 min and then at 180 min, for a reaction mixture that was

initially 80 μM in GrxS16 [4Fe-4S] clusters and 160 μM in monomeric apo At Nfu2. The thick

line corresponds to [4Fe-4S] GrxS16 in the same reaction mixture in the absence of apo At Nfu2

(zero time point) and the broken line corresponds to the spectrum of At Nfu2 containing one

[4Fe-4S] cluster per dimer. The arrows indicate the direction of intensity change with time at selected wavelengths. The Δε values are expressed per [4Fe-4S]2+ cluster.

167

2

1 ↓ ) -1 cm -1

(M 0 Δε ↑ ↑

-1

350 400 450 500 550

Wavelength (nm)

168

CHAPTER 4

ANAEROBIC SULFATASE-MATURATING ENZYMES, FIRST DUAL SUBSTRATE

RADICAL S-ADENOSYLMETHIONINE ENZYMES1

1 This research was originally published in The Journal of Biological Chemistry. Sowmya Subramanian§, Alhosna Benjdia‡, Jérôme Leprince¶, Hubert Vaudry¶, Michael K. Johnson§, and Olivier Berteau‡. Anaerobic Sulfatase-maturating Enzymes, First Dual Substrate Radical S-Adenosylmethionine Enzymes. The Journal of Biological Chemistry. 2008; 283:17815-17826. © the American Society for Biochemistry and Molecular Biology. ‡INRA, UPR 910, Unité d’Ecologie et Physiologie du Système Digestif, F-78352 Jouy-en-Josas, France, the §Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, and the ¶INSERM U-413, IFRMP23, UA CNRS, Université de Rouen, 76821 Mont- Saint-Aignan, France. 169

1 Abbreviations. FGly, Cα-formylglycine; AdoMet, S-adenosylmethionine; anSME, anaerobic

sulfatase maturating enzyme; DNPH, 2,4-dinitrophenylhydrazine; Ni-NTA, nickel-nitrilotriacetic

acid; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometery.

170

Abstract

Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active- site cysteine or serine residue to Cα-formylglycine. This modification is catalyzed by at least

three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently

considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase active site and has been accordingly categorized as Cys-type and Ser-type sulfatase- maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type

or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in

vitro using synthetic substrates and the successful purification of both enzymes facilitated the

first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that

reconstituted anSMEs are radical S-adenosyl-L-methionine enzymes containing a redox active

[4Fe 4S]2+,+ cluster that initiates the radical reaction by binding and reductively cleaving S- adenosyl-L-methionine to yield 5′-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to Cα-formylglycine. Taken together, the results support a radical modification mechanism that is

initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate

target sequence. 171

Introduction

Bacterial sulfatases are widely distributed among the major bacterial phyla. Nevertheless, they have attracted only few studies compared with their eukaryotic counterparts (1). One of the difficulties in studying sulfatases is the requirement for post-translational modification of a critical active-site cysteine or serine residue to generate an active enzyme. Indeed, despite high homologies in the sulfatase domain (PFAM domain PF00884), sulfatases are divided into two sub-classes, Cys-type and Ser-type, according to the genetically encoded active site residue. In eukaryotes, only Cys-type sulfatases have been identified so far, whereas in bacteria, both types of sulfatases exist. Nevertheless, eukaryotic and prokaryotic sulfatases undergo the same post- translational modification involving oxidation of cysteine or serine to oxo-alanine, also called

Cα-formylglycine (FGly). To date, two distinct enzymatic systems have been identified for

catalyzing this reaction. One is the formylglycine generating enzyme system (FGE), an oxygen

dependent oxidoreductase that has been extensively studied in humans and is functional only on

Cys-type sulfatases (2). The other is AtsB, a putative member of the S-adenosyl-L-methionine

(AdoMet)-dependent family of radical enzymes, which is responsible for maturation of Ser-type sulfatases and was first identified in Klebsiella aeruginosa (3;4). Thus, the maturation of Cys-

type sulfatases, the most abundant type, in anaerobic environments such as the human gut, has

long remained unknown.

We recently identified an enzyme related to the AtsB enzyme from K. aeruginosa in the

obligate anaerobe Clostridum perfringens, which is surprisingly involved in Cys-type sulfatase

maturation (5). Although the K. aeruginosa enzyme, AtsB, appeared to be restricted to Ser-type

sulfatases harboring a signal peptide (4;6), we grouped these enzymes into a family called anaerobic sulfatase-maturating enzymes (anSMEs) (5). These results raised the possibility that 172 anSMEs could be divided into two sub-groups that differ in specificity for sulfatase FGly generation via oxidation of an active-site cysteine or serine residue.

Subsequent purification and characterization of C. perfringens anSME showed it to be a member of the radical-AdoMet superfamily of iron-sulfur enzymes (7). All the members of this super-family share a strictly conserved cysteine motif CX3CX2C, which coordinates an

unconventional [4Fe-4S] cluster. The [4Fe-4S] cluster is directly involved in the reductive

cleavage of AdoMet to generate a 5′-deoxyadenosyl radical, which then initiates various radical-

based reactions according to the functional specificity of the enzyme. In addition to the CX3CX2C

motif, all the anSMEs identified to date have two other strictly conserved cysteine clusters

(Figure 4.1), which could also be involved in the coordination of iron-sulfur centers. However,

no experimental data have been produced to substantiate the existence of additional iron-sulfur

clusters and, other than UV-visible absorption, no spectroscopic data have been reported for

these enzymes.

To get insights into this group of enzymes, we have investigated the in vitro and in vivo

activity of bacterial anSMEs that are responsible for modification of either Cys-type or Ser-type

sulfatases: anSMEcpe cloned from C. perfringens, which is responsible for maturation of a Cys-

type sulfatase and anSMEbt cloned from Bacteroides thetaiotaomicron, a prominent gut

symbiont, which possesses only Ser-type sulfatases. We report here the first detailed

characterization of purified anSMEs and demonstrate that both enzymes can maturate cysteine

and serine residues in vitro and in vivo. Furthermore, the ability to purify these unstable enzymes

allowed us to provide the first spectroscopic characterization of their iron-sulfur centers.

173

Experimental Procedures

Chemicals. p-Nitrophenylsulfate was purchased from Sigma. Enzymes for molecular bio- logy were obtained from New England Biolabs (Ipswich, MA). Oligonucleotides were purchased from Eurogenetec (Seraing, Belgium). Other chemicals and reagents were obtained from commercial sources and were of analytical grade.

Bacterial Strains, Plasmids, and DNA Manipulations. The B. thetaiotaomicron strain used in this study was the VPI-5482 strain. Escherichia coli DH5α was used for routine DNA mani-pulations. E. coli BL21(DE3) (Stratagene) was used for enzyme overexpression. The pET-

28(a) and pRSF plasmids were used to express the various proteins (Novagen, Inc.). T4 DNA ligase was from Promega, Inc. The plasmid DNA purification kit and QIAprep spin were from

Qiagen, Inc. DNA fragments were extracted from agarose gel and purified with the Wizard SV

Gel and PCR clean up system kit (Promega, Inc.). DNA sequencing was performed by MWG.

Cloning and Construction of the pET-6His-anSMEcpe and pET-6His-anSMEbt

Overexpressing Plasmids. B. thetaiotaomicron VPI-5482 was grown anaerobically in BHI medium, pH 7.0, and the cells were harvested to extract genomic DNA using the Wizard

Genomic Kit (Pro-mega). The BT_0238 gene encoding the putative anSME was amplified by a

PCR-based method using genomic DNA as a template. The following primers were used: 5′-cat

ATG aaa gca act act tat gca cct ttt gcc-3′ (NdeI site underlined, ATG codon in uppercase) hybridized to the non-coding strand at the 5′ terminus of the gene and 5′-ctc gag tta ata ttc tat ttt taa act tcc gtc-3′ (XhoI site underlined) hybridized to the coding strand. PCR was run as follows: genomic DNA (1 μg) in the presence of the primers (0.5 μM each) was mixed with the Hot Start

Kit (Promega) and 30 cycles of PCR were performed (1 min at 95 °C, 1 min at 50 °C, 2 min at

72 °C), followed by a final 10-min elongation step at 72 °C. The PCR product was digested with 174

NdeI and XhoI and then ligated with T4 DNA ligase into pET28(a) plasmid previously digested with the same re-striction enzymes. The entire sequence of the cloned gene was sequenced to ensure that no errors were introduced during PCR. The plasmid was designated pET-6His-an-

SMEbt. The pET-6His-anSMEcpe construction containing the CPF_0616 gene from C. perfringens ATCC 13124 has been previously reported (5).

anSMEcpe and anSMEbt Protein Expression and Purification. E. coli BL21(DE3) were transformed with pET-6His-anSMEcpe or pET-6His-anSMEbt, then grown aerobically overnight at 37 °C in LB medium (100 mL) supplemented with ampicillin (100 μg mL-1). An overnight

culture was then used to inoculate fresh LB medium (12 liters) supplemented with the same

antibiotic and bacterial growth proceeded at 37 °C until the A600 reached 0.6. The cells were induced by adding 500 μM isopropyl 1-thio-β-D-galactopyranoside and were collected after over-night growth at 25 °C. After resuspension in Tris buffer (50 mM Tris, 150 mM KCl, 10% glycerol, pH 7.5), the cells were disrupted by sonication and centrifuged at 220,000 × g at 4 °C

for 1 h. The solution was then loaded onto a Ni-NTA Sepharose column equilibrated with Tris buffer, pH 7.5. The column was washed extensively with the same buffer. Some of the adsorbed proteins were eluted by a washing step with 25 and 100 mM imidazole and the overexpressed protein was eluted by applying 500mM imidazole. Fractions containing the anSMEcpe or an-

SMEbt proteins were immediately concentrated in Ultrafree cells (Millipore) with a molecular cut-off of 10 kDa.

Reconstitution of Iron-Sulfur Clusters on anSMEbt and anSMEcpe. Reconstitution was carried out anaerobically in a glove box (Bactron IV). As purified anSMEs (200μM monomer)

were treated with 5 mM dithiothreitol and incubated overnight with a 7-fold molar excess of both

Na2S (Fluka) and (NH4)2Fe(SO4)2 (Aldrich) at 12 °C. The protein was desalted using a Sephadex 175

G-25 column (Amersham Biosciences) and the colored fractions were concentrated on Nanosep

10 (Amicon). Protein concentrations were determined by the DC protein assay (Bio-Rad), using bovine serum albumin as a standard. Iron concentrations were determined colorimetrically using bathophenanthroline under reducing conditions, after digestion of the protein in 0.8% KMnO4,

0.2 M HCl.

Construction of Plasmid Systems Expressing C. perfringens Sulfatase Alone or with anSMEbt or anSMEcpe. The CPF_0221 gene encoding C. perfringens Cys-type sulfatase was amplified by a PCR-based method using genomic DNA as a template. The following primers were used: 5′-gga tcc ATG aag cca aat att gtg tta atc atg gtt-3′ (BamHI site underlined and ATG codon in uppercase), which corresponded to the 5′ terminus of the gene, and 5′-ctg cag tta tta tct

tat atg ttt taa agt gct tac-3′ (PstI site underlined), complementary to the 3′-end of the gene. PCR

was run as follows. Genomic DNA (1μg) in the presence of the primers (0.5 μM each) was mixed with the Hot Start kit (Promega), and 30 cycles of PCR were performed (1 min at 94 °C, 1 min at 52 °C, and 1.5 min at 72 °C), followed by a final 10-min elongation step at 72 °C. The

PCR product was digested with BamHI and PstI and inserted into pRSF plasmid site one. The obtained plasmid was designated pRSF-Cys-Sulf. The anSMEbt or anSMEcpe genes previously

cloned were inserted into pRSF-Cys-Sulf plasmid (site two) previously digested by the NdeI and

XhoI enzymes. Each ligation step was performed with T4 DNA ligase. The entire sequences of the cloned genes were sequenced to ensure that no errors were introduced during the PCR. The plasmid obtained containing the C. perfringens sulfatase and anSMEbt or anSMEcpe were

designated pRSF-anSMEbt-Cys-Sulf and pRSF-anSMEcpe-Cys-Sulf.

Construction of the Serine Variant of C. perfringens Cys-type Sulfatase. Site-directed

mutagenesis was performed on plasmid pRSF-Cys-Sulf to mutate cysteine 67 into serine. The 176 cysteine to serine mutation of the C. perfringens sulfatase was performed with the Quik-

ChangeTM site-directed mutagenesis kit (Stratagene) using the following primers and a two-step

PCR method (8). Sulfatase Cys to Ser primers were: forward, 5′-aca gca gtt cca agt AGC att gca tct agg gca-3′; and reverse, 5′-tgc cct aga tgc aat GCT act tgg aac tgc tgt-3′ (mutated codon in uppercase). After verification of the correct mutation by sequencing, the plasmid obtained was

designated pRSF-Ser-Sulf. The anSMEbt or anSMEcpe genes previously cloned were inserted

into pRSF-Ser-Sulf plasmid (site two) previously digested by the NdeI and XhoI enzymes and,

after a ligation step, the following plasmids were obtained pRSF-anSMEbt-Ser-Sulf and pRSF-

anSMEcpe-Ser-Sulf.

Peptide Synthesis. The following 23-mer peptides (with the critical residue in bold): Ac-

FENAYTAVPSCIASRASILTGMS-NH2, Ac-FENAYTAVPSSIASRASILTGMS-NH2, and Ac-

FENAYTAVPSAIASRASILTGMS-NH2, were synthesized (0.1 mmol scale) by the solid phase

methodology on a Rink amide 4-methylbenzhydrylamine resin (Biochem, Meudon, France)

using a 433A Applied Biosystems peptide synthesizer (Applera-France, Courtaboeuf, France) and the standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) procedure from the manufacturer. The synthetic peptides were purified by reverse-phase HPLC on a 2.2 × 25-cm Vydac 218TP1022

C18 column (Alltech, Templemars, France) using a linear gradient (10–50% over 45 min) of

acetonitrile/trifluoroacetic acid (99.9:0.1; v/v) at a flow rate of 10 mL/min. Analytical HPLC,

performed on a 0.46 × 25-cm Vydac 218TP54 C18 column (Alltech), showed that the purity of

the peptides was >99.1%. The purified peptides were characterized by MALDI-TOF mass

spectrometry on a Voyager DE PRO (Applera, France) in the reflector mode with α-cyano-4-

hydroxycinnamic acid as a matrix. 177

Peptide Maturation. Samples containing 6 mM dithiothreitol, 3 mM sodium dithionite,

20 μM reconstituted anSMEbt or anSMEcpe were added with 500μM peptides and 1mM

AdoMet in Tris buffer, pH 7.5. The reactions were performed in an anaerobic glovebox (Bactron

IV) and incubated at 25 °C for 60, 120, 180, 240, and 360 min. Samples were divided in half, and one part was used to test the maturation activity by mass spectrometry, whereas the other half was diluted in 10 volumes of H2O, 0.1% trifluoroacetic acid and then loaded onto the C18 column to assay the reductive cleavage of AdoMet. Control samples were prepared without enzyme to verify the peptides and AdoMet stability over time.

HPLC Detection of AdoMet Cleavage. Standards of 3 mM AdoMet and 3 mM 5′ deoxy- adenosine were run over a C18 column (LicroSphere, 5-μm, 4.6 × 150-mm) at 1 mL/min with the

following gradient: after a 1-mL step of Milli-Q H2O, 0.1% trifluoroacetic acid, a linear gradient

from 0 to 30% acetonitrile with 0.1% trifluoroacetic acid was used to elute the samples.

Detection was carried out at 260 nm.

Electron Paramagnetic Resonance (EPR). X-band EPR spectra were recorded on a

Bruker Instruments ESP 300D spectrometer equipped with an Oxford Instruments ESR 900 flow

cryostat (4.2–300 K). Spectra were quantified under non-saturating conditions by double

integration against a 1 mM CuEDTA standard.

Resonance Raman. Resonance Raman spectra were recorded using an Instruments SA U1000

spectrometer fitted with a cooled RCA 31034 photomultiplier tube with 90° scattering geometry.

Spectra were recorded digitally using photon counting electronics, and improvements in signal-

to-noise were achieved by signal averaging multiple scans. Band positions were calibrated using

-1 the excitation frequency and CCl4 and are accurate to ±1 cm . Lines from a Coherent Sabre 10-

W argon ion laser were used for excitation, and plasma lines were removed using a Pellin Broca 178 prism premonochromator. For each sample, ~140 milliwatts of incident laser power was used, and slit widths were adjusted to give 7.0 cm-1 spectral resolution. Scattering was collected from

the surface of a frozen 18-μl droplet of sample using a custom designed anaerobic sample cell (9)

attached to the cold finger of an Air Products Displex model CSA-202E closed cycle

refrigerator. This enables samples to be cooled down to 17 K, which facilitates improved spectral

resolution and prevents laser-induced sample degradation.

Sulfatase Protein Expression and Purification. E. coli BL21(DE3) were transformed with

pRSF-Cys-Sulf, pRSF-an-SMEbt-Cys-Sulf, or pRSF-anSMEcpe-Cys-Sulf to produce the Cys-

type sulfatase expressed alone or with the corresponding anSMEs but also with pRSF-Ser-Sulf,

pRSF-anSMEbt-Ser-Sulf, or pRSF-anSMEcpe-Ser-Sulf to obtain the corresponding Ser-type sulfatases. The six strains were grown overnight anaerobically in stoppered flasks under an

H2/CO2/N2 (5:5:90) atmosphere at 37 °C in LB medium (100 mL) supplemented with kanamycin

(50 μg mL-1). The overnight culture was then used to inoculate fresh LB medium (1 liter) supplemented with the same antibiotic and bacterial growth proceeded at 37 °C until the A600 reached 0.2. The cells were induced by adding 500 μM isopropyl 1-thio-β-D-galactopyranoside and were collected after overnight growth at 30 °C. After resuspension in Tris buffer, the cells were disrupted by sonication and centrifuged at 220,000 × g at 4 °C for 1 h. The solutions were then loaded onto Ni-NTA-Sepharose columns equilibrated with Tris buffer, pH 7.5. The columns were washed extensively with the same buffer. After two washing steps with 25 and 100 mM imidazole, the overexpressed proteins were eluted by applying 500 mM imidazole.

Sulfatase Assay. Sulfatase activity was assayed using the chromogenic substrate p-nitro- phenylsulfate as substrate. Standard assays were performed at 30 °C using 50 mM p-nitrophenyl- 179

sulfate in 100 mM Tris buffer containing 10 mM MgCl2, pH 7.15. Formation of p-nitrophenol

was measured spectrophotometrically at 405 nm (ε = 9,000 M-1 cm-1 at pH 7.15).

Sulfatase Treatment for MALDI-TOF Analysis. Purified sulfatases were dissolved (150

pmol μL-1) into 0.4 M urea, then digested overnight with trypsin (20 ng μL-1) in ammonium

carbonate buffer, pH 8.0, at 37 °C. 5 μL of digested sulfatase was further hydrolyzed with 5 μL

of CNBr (20 mg mL-1 in 0.1 M HCl) in the dark overnight at room temperature, then lyophilized

and purified on ZipTip (Millipore, Inc.) before MALDI-TOF mass spectrometry analysis.

MALDI-TOF MS Analysis. The α-cyano-4-hydroxycinnamic acid matrix was prepared at

4 mg mL-1 in 0.15% trifluoroacetic acid, 50% acetonitrile. The 2,4-dinitrophenylhydrazone acid

matrix (DNPH) was prepared at 100 mg mL-1 in 0.15% trifluoroacetic acid, 50% acetonitrile.

Equal volumes (1 μL) of matrix and sample were spotted onto the MALDI-TOF target plate.

MALDI-TOF analysis was then performed on a Voyager DE STR Instrument (Applied

Biosystems, Framingham,CA). Spectra were acquired in the reflector mode with: 20 kV

accelerating voltage, 62% grid voltage, and a 120-ns delay.

Results

Identification of Bacteroides thetaiotaomicron anSME. We previously reported the

identification of the C. perfringens anaerobic sulfatase maturating enzyme, anSMEcpe (5;7).

Using its sequence as probe, only one enzyme sharing 30% identity with anSMEcpe and

possessing the three strictly conserved cysteine clusters was identified by BLAST searching the

B. thetaiotaomicron genome which encodes only Ser-type sulfatases (10) (Figure 4.1). This

protein, encoded by the BT_0238 gene, annotated as a transcriptional regulator and named ChuR

for chondroitin sulfate/heparin utilization regulator (11), was amplified by PCR and named

anSMEbt. 180

Overexpression and Purification of anSMEbt and anSMEcpe with an N-terminal Hexa- histidine tag. Both anSMEcpe and anSMEbt gene were subcloned into the pET-28(a) plasmid and the resulting expression vectors, pET-6His-anSMEbt and pET-6His-anSMEcpe, were used to transform E. coli BL21(DE3) for overproduction of histidine-tagged anSMEbt and anSMEcpe.

Both enzymes were essentially expressed as inclusion bodies and induction under low tem- perature was necessary to obtain small amounts of the soluble proteins. Typically ~5 mg of pure proteins were obtained from 12-liter culture. Both proteins eluted as dark brown bands from Ni-

NTA affinity chromatography, which was consistent with the presence of iron-sulfur clusters, and each protein migrated according to its predicted molecular weight (at ~ 45 and 50 kDa for anSMEcpe and anSMEbt respectively) on SDS-PAGE (Figure 4.2A). Anaerobic reconstitution with Fe2+ and S2−, followed repurification, increased the Fe-S cluster content as evident by the increase in visible absorption at ~ 420 nm relative to the protein band at 280 nm. As shown in

Figure 4.2B, the UV-visible spectra of the reconstituted anSMEs were very similar and both exhibited the characteristic absorption bands associated with Fe-S clusters. The nature and stoichiometry of the Fe-S clusters in as isolated and reconstituted anSMEcpe were assessed by protein and Fe determinations and the combination of UV-visible absorption, electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies.

Nature and Stoichiometry of Iron-Sulfur Centers. In accord with previous studies (7), analytical data for the anSMEcpe samples used for spectroscopic analyses indicated 2.5 ± 0.3 iron/monomer for aerobically purified samples and 5.7 ± 0.5 iron/monomer for samples anaerobically reconstituted with Fe2+ and S2−. The form and visible extinction coefficients of

corresponding UV-visible absorption spectra, (see Figure 4.3), are consistent with approximately

one [2Fe-2S]2+ and one [4Fe-4S]2+ cluster in the as isolated and reconstituted samples, 181

respectively. As isolated anSMEcpe has resolved bands at 320 and 420 nm (A420/A280 = 0.19 ±

0.02) and shoulders centered at ~ 460 and ~ 550 nm that are characteristic of [2Fe-2S]2+ clusters

−1 −1 and the extinction coefficient at 420 nm (ε420 = 11.8 ± 1.2 mM cm ) is just above the high end

2+ −1 −1 of the range generally associated with a single [2Fe-2S] cluster (ε420 = 8-11 mM cm ) (12).

Reconstituted anSMEcpe exhibits a broad shoulders centered at ~ 320 and ~ 400 nm that are

2+ characteristic of a [4Fe-4S] cluster (A420/A280 = 0.29 ± 0.03) and the extinction coefficient at

−1 −1 400 nm (ε400 = 20 ± 2 mM cm ) is just above the range generally associated with a single

2+ −1 −1 [4Fe-4S] cluster (ε400 = 14-18 M cm ) (13).

More definitive information concerning the nature of the Fe-S clusters in as isolated and

reconstituted anSMEcpe and for AdoMet binding to the [4Fe-4S]2+ cluster in the reconstituted

enzyme was provided by RR spectroscopy (Figure 4.4). The resonance Raman spectrum of as

isolated anSMEcpe comprises broad bands centered at 286, 337 and 392 cm−1 (with the

possibility of an additional weak band underlying the glycerol band at 420 cm−1) (Figure 4.4A)

2+ and is characteristic of a cluster [2Fe-2S] that is derived from O2-degradation of a radical-

AdoMet-type [4Fe-4S]2+ cluster. For example, very similar spectra have been observed in as

purified pyruvate formate lyase activating enzyme and in [4Fe-4S]2+ cluster-containing forms of

2+ biotin synthase after exposure to O2 (14, 15). Hence we conclude that the [2Fe-2S] cluster in as

isolated anSMEcpe is wholly or predominantly derived from O2-induced degradation of a

radical-AdoMet type [4Fe-4S]2+ cluster during aerobic isolation. Reconstituted anSMEcpe has a

RR spectrum that is dominated by bands at 251, 336, 356 and 390 cm−1 that are characteristic of

a [4Fe-4S]2+ cluster (15) (Figure 4.4B). Bands associated with the [2Fe-2S]2+ cluster that is

present in the as isolated enzyme are still apparent, e.g. at 286 cm−1, but they tend to

overestimate the [2Fe-2S]2+ cluster content as resonance enhancement of [2Fe-2S]2+ clusters are 182

5-10 times greater than that of [4Fe-4S]2+ clusters with 458-nm excitation (15). Based on parallel

Mössbauer and resonance Raman studies of a range of radical-AdoMet enzymes with mixtures

2+ 2+ of [4Fe-4S] clusters and their [2Fe-2S] cluster O2 degradation products, we estimate the

cluster composition of reconstituted anSMEcpe to be 10-20% [2Fe-2S]2+ clusters and 90-80%

[4Fe-4S]2+ clusters. Hence, the majority of the [2Fe-2S]2+ clusters present in as isolated

anSMEcpe have been re-placed by [4Fe-4S]2+ clusters during anaerobic reconstitution.

Moreover, the [4Fe-4S]2+ cluster have been assembled in the radical-AdoMet cluster binding

site, as evidenced by changes in RR spectrum of re-constituted anSMEcpe on addition of excess

AdoMet (Figure 4.4C). Crystallographic studies have demonstrated that AdoMet binds to the

unique Fe of the active-site [4Fe-4S]2+ cluster in radical-AdoMet enzymes via the methionine

amide and carboxylate groups (16) and this is manifest by 3-4 cm−1 upshifts in both of the

dominant bands in the resonance Raman spectrum, i.e. the symmetric breathing mode of the

[4Fe-4S] core and the asymmetric stretching mode of the terminal Fe-S(Cys) ligands (17). These bands are observed at 336 and 356 cm−1, respectively, in reconstituted anSMEcpe and upshift to

339 and 360 cm−1, respectively, on addition of excess AdoMet.

EPR was used to investigate the type and properties of reduced forms of anSMEcpe that

were generated by anaerobic reduction with dithionite (Figure 4.5). The EPR properties of

dithionite-reduced reconstituted anSMEcpe were characteristic of a [4Fe-4S]+ cluster in a

radical-AdoMet enzyme (18,19) (Figure 4.5A, left panel), i.e. mixed spin system with a rhombic

S = 3/2 component (as evident by the broad low-field feature centered at g = 5.4, shown in the inset) and a near-axial, fast-relaxing S = 1/2 component (g|| ~ 2.05, g⊥ ~ 1.94) accounting for ~

0.2 spins/mol. A very weak S = 1/2 resonance (~ 0.03 spins/mol) with similar g-values and

relaxation behavior was observed on dithionite-reduction of the as isolated enzyme (Figure 4.5B, 183 left panel). We attributed this to [4Fe-4S]+ clusters formed either by reduction of residual

[4Fe-4S]2+ clusters in the as isolated sample or to [4Fe-4S] clusters that were reassembled in the

radical-AdoMet cluster binding site on reductive degradation of the [2Fe-2S]2+ cluster. The [4Fe-

4S]+ cluster in the reconstituted enzyme interacted with AdoMet as evidenced by partial conversion of the S = 1/2 EPR signal to a new species with significantly lower g-values on addition of AdoMet ( g|| ~ 1.99, g⊥ ~1.90) (Figure 4.5, right panel). Similar changes in the S =

1/2 [4Fe-4S]+ EPR signal on addition of AdoMet have been observed with many radical-SAM

enzymes (20-22). Because the radical-AdoMet active site can turnover on addition of dithionite in the presence of AdoMet, it is often difficult to get the [4Fe-4S]+ cluster in a fully AdoMet-

bound form.

Taken together, the spectroscopic and analytical data are consistent with the anSMEcpe

samples investigated in this work only having clusters assembled in the radical-AdoMet cluster

binding site involving the rigorously conserved CX3CX2C motif. Reconstituted samples have a

catalytically competent [4Fe-4S]2+,+ cluster assembled in this site that can interact with AdoMet in both the oxidized and reduced states. In aerobically isolated samples, the [4Fe-4S]2+,+ cluster

2+ degrades to an O2-stable [2Fe-2S] cluster that is rapidly and irreversibly degraded on reduction.

In Vitro Activity of anSMEbt and anSMEcpe. Purified and reconstituted anSMEs were

tested in vitro under anaerobic conditions for their ability to cleave AdoMet and to mature two

peptides mimicking the Cys-type and the Ser-type sulfatases. Each reconstituted protein (20 µM)

was incubated with 1 mM AdoMet in the presence or absence of peptides using dithionite as

reductant and in each case 5′-deoxyadenosine production was followed by HPLC analysis. In the

absence of peptides, anSMEcpe and anSMEbt exhibited different behaviors (Figure 4.6, open

square). After 6 hours incubation, anSMEcpe was able to produce only low amounts of 5′- 184 deoxyadenosine (8 µM) while anSMEbt produced 4 times more 5′-deoxyadenosine (33 µM). In contrast, both enzymes behaved similarly in presence of 23-mer peptides (500 µM). Both enzymes were sensitive to the nature of the first residue in the CXAXR motif, exhibiting substantially higher levels of 5′-deoxyadenosine production in the presence of the cysteine- containing peptide (23C) (35 µM for anSMEcpe and 17 µM for anSMEbt) (Figure 4.6, diamonds) compared to the serine-containing peptide (23S) (17 µM and 6 µM respectively)

(Figure 4.6, triangles). Furthermore, substitution of the cysteine residue by alanine (23A), resulted in inhibition of AdoMet cleavage for both enzymes and became almost undetectable for anSMEcpe.

In Vitro Peptide Maturation. Using MALDI-TOF MS, peptide maturation was followed both by direct measurement with a α-Cyano-4-hydroxycinnamic acid (CHCA) matrix and derivatization with 2,4-dinitrophenylhydrazine (DNPH) (23). AnSMEcpe efficiently converted the cysteine of peptide 23C as shown by the production of a peptide with an 18-Da mass shift

(Figure 4.7A, panel 1). The nature of the modification was assessed using DNPH which specifically reacts with the FGly aldehyde function leading to a hydrazone derivative with a

180.13-Da mass increment (Figure 4.7B, panel 1 and Figure 4.8). For the incubation with the

23S peptide, we were unable to directly observe a FGly-containing peptide which should have a

2-Da shift from the 23S peptide (Figure 4.7A, panel 2). Nevertheless, derivatization with DNPH allowed us to detect a product with the expected mass for the hydrazone derivative of the FGly- containing peptide (Figure 4.7B, panel 2 and Figure 4.8). Indeed DNPH not only gives a mass increment but also enhance peptide ionization which is then more easily detected.

To definitively demonstrate the formation of an FGly-peptide from the 23S peptide, the peptides from the incubation mixture were purified using C18-HPLC and the fractions obtained 185 were analyzed by MALDI-TOF MS. In our conditions, the 23S peptide elutes at 24 min as confirmed by MALDI-TOF MS analysis (molecular weight: 2415 Da - Figure 4.9A), and in a preceding fraction we were able to recover the FGly-containing peptide as assessed by its molecular weight (2413 Da) (Figure 4.9B).

Incubation with the peptide 23A did not lead to any new peptide despite the presence of four serine residues within the peptide sequence (Figure 4.7A, panel 3 & 4.7B, panel 3).

MALDI-TOF MS analysis performed on the peptides incubated with the B. thetaiotaomicron enzyme (anSMEbt) gave similar results (Figures 4.10 and 4.11).

Both, anSMEbt and anSMEcpe were thus active on cysteine and serine residue.

Nevertheless, MALDI-TOF MS experiment did not allow quantifying the efficiency of maturation. Notably, the 23S peptide ionizes much more efficiently than the 23C and the FGly containing peptides. Attempts to quantify peptide maturation using HPLC failed mostly because the 23C peptide proved to be unstable. We thus devised an in vivo strategy in order to compare anSME maturation efficiency on both residues.

In Vivo Maturation of C. perfringens Cys-type sulfatase. We used the Cys-type sulfatase from C. perfringens as reporter gene and constructed three pRSF plasmids containing the Cys- type sulfatase into the subsite 1 alone or with anSMEbt or anSMEcpe into the subsite 2. These plasmids were named pRSF-Cys-Sulf, pRSF-anSMEbt-Cys-Sulf and pRSF-anSMEcpe-Cys-Sulf.

Using these plasmids we obtained three strains expressing the Cys-type sulfatase alone as a His- tag protein or co-expressed with anSMEbt or anSMEcpe. These strains were grown under an- aerobic conditions as our previous investigations have demonstrated that E. coli constitutively matures Cys-type sulfatases but this maturation is partly repressed under anaerobic conditions

(24). After overnight induction with IPTG, the expressed sulfatases were purified (see 186

“Experimental Procedures”) and their maturation assessed by MALDI-TOF mass spectrometry.

The conversion of cysteine into FGly is expected to result in mass decrease of 18 Da which could be evidenced in the peptides obtained after trypsin/CNBr treatment of the sulfatases (7).

We also measured the specific activity of the purified sulfatases using the chromogenic substrate p-nitrophenyl-sulfate (pNP-S) as the sulfatase activity is directly proportional to the maturation level. As shown in Figure 4.12B, panel 1, when the Cys-type sulfatase was expressed alone under anaerobic conditions, one relevant peptide was obtained containing the critical cysteine

Cys-67 and encompassing the residues from 51 to 71: 51ATEGYNFENAYTAVPSCIASR71

(theoretical molecular weight 2264.03 Da). This peptide with an intact cysteine residue thus corresponds to unmaturated sulfatase. When the sulfatase was co-expressed with anSMEcpe

(Figure 4.12B, panel 2), the 2264 Da peptide disappeared and was replaced by a new peptide with a mass shift of 18 Da. This peptide (theoretical molecular weight 2246.03 Da) corresponded to maturated sulfatase. Surprisingly, when we expressed the Cys-type sulfatase with anSMEbt,

MALDI-TOF MS indicated principally the presence of unmaturated sulfatase (Figure 4.12B, panel 3).

The specific sulfatase activity assay of each enzyme confirmed the results obtained by

MALDI-TOF MS (Figure 4.12C). No differences in the sulfatases activity was evidenced between the Cys-type sulfatase expressed alone or in the presence of anSMEbt, while anSMEcpe significantly increased the sulfatase specific activity from 6 to 36 nmole min-1 mg-1. The basal

sulfatase activity in the absence of known maturation enzyme was probably due to an imperfect inhibition of the E. coli sulfatase maturation system as trace maturation could be seen in all the

MALDI-TOF MS spectra. 187

Construction of the Serine Variant of C. Perfringens Cys-type Sulfatase. To investigate the activity of both anSMEs toward the serine residue, we converted the C. perfringens Cys-type sulfatase into a Ser-type sulfatase by site directed mutagenesis. We obtained three strains harbouring the following plasmids named pRSF-Ser-Sulf, pRSF-anSMEbt-Ser-Sulf and pRSF- anSMEcpe-Ser-Sulf respectively. These three strains were also grown under anaerobic conditions (Figure 4.13) although identical results were obtained under aerobic conditions (data not shown). The MALDI-TOF MS analysis confirmed the mutation of the Cys-67 into Ser, as a

2248 Da peptide was observed consistent with the following sequence:

51ATEGYNFENAYTAVPSSIASR71 (Figure 4.13B). Like in the in vitro experiments, the

ionization properties of this peptide differed considerably from those of the cysteine and FGly-

containing peptide. As a result, this peptide dominated the whole spectrum and prevented the

direct measurement of maturation by MALDI-TOF MS.

The sulfatase activity of the serine mutant expressed alone in E. coli was almost

undetectable (Figure 4.13C, panel 1), also consistent with previous reports on Ser-type sulfatases

(3, 25). This result confirms that the unknown E. coli sulfatase maturation system is restricted to

the Cys-type sulfatases (24). Nevertheless, when the serine mutant was co-expressed with

anSMEcpe or an-SMEbt, we clearly measured sulfatase activity which was 25 times higher than

in the control experiment. anSMEbt and anSMEcpe are thus able to activate Ser-type sulfatases,

but with an apparent lower efficiency than on Cys-type sulfatase.

Discussion

Sulfatases are widely distributed in eukaryotes and prokaryotes (1) due to their biological

significance and their high substrate versatility. Sulfatases are involved in the metabolism of

various compounds ranging from small organic molecules to macromolecules such as mucins, 188 fucans and glycosaminoglycans (1). They are usually considered to be restricted to sulfate supply but they probably have also other functions which remain to be explored. Consequently, sulfatase genes are identified in many bacterial genomes and metagenomes notably from those arising from marine and gut environments (10; 26-28). Despite their functional importance, little is known about sulfatase maturation. Until recently, it was thought that an enzymatic system,

FGE, was dedicated to the maturation of Cys-type sulfatases (29; 30) and another, AtsB, to the

Ser-type sulfatases (3).

These assumptions were based on several observations. The specificity of FGE is based on experiments showing that eukaryotic cells are unable to maturate a serine sulfatase mutant

(31). In contrast, the data are more complex for AtsB. Co-expression experiments performed with a serine sulfatase mutant obtained from the Cys-type sulfatase of Pseudomonas aeruginosa failed to produce an active enzyme. Nevertheless, an active sulfatase was obtained if this serine mutant carried a signal peptide in addition of the serine critical residue (6). This lead to the assumption that AtsB recognizes only Ser-type sulfatase with the help of a signal peptide (4; 6).

We recently demonstrated that the C. perfringens AtsB ortholog is able to convert cysteine residue into FGly. In light of the homologies between the enzyme from K. pneumoniae and C. perfringens, these enzymes were gathered under the collective name of anaerobic sulfatase maturating enzymes (anSME) (5). This raised the possibility of enzymes with different substrate specificities within the anSME group, some enzymes being dedicated to maturation of

Ser-type sulfatases and others to the Cys-type sulfatases. To test this hypothesis, we decided to investigate the specificity of the anSMEs from C. perfringens and B. thetaiotaomicron, a prominent gut symbiont (10), that encodes only Ser-type sulfatases and only one enzyme related to the anSME group. The B. thetaiotaomicron anSME is currently called ChuR (chondroitin 189 heparin utilization regulator) and has been annotated as a transcriptional regulator as its inactivation prevents the bacterium to degrade various substrates (11). We cloned this enzyme and named it anSMEbt.

We successfully obtained both enzymes in soluble form after induction under low temperature and extraction in the presence of glycerol. Even though the yields were very low, sufficient amounts of anSMEcpe were obtained to enable characterization of the Fe-S centers in aerobically isolated and anaerobically reconstituted samples using the combination of UV-visible absorption, resonance Raman and EPR spectroscopies. The results indicate that anaerobic reconstitution generates a [4Fe-4S]2+,+ cluster in the radical-AdoMet cluster binding site

(CX3CX2C motif) that is able to bind AdoMet in both oxidation states. The aerobically isolated

enzyme contains stoichiometric amounts of a reductively labile [2Fe-2S]2+ cluster with resonance

Raman properties analogous to those established for [2Fe-2S]2+ clusters that form as a result of

2+ O2-induced degradation of radical-AdoMet active-site [4Fe-4S] clusters. Hence it seems likely

2+ that the active-site [4Fe-4S] cluster in anSME is not stable in air and degrades to yield an O2-

stable [2Fe-2S]2+ cluster in the radical-AdoMet cluster binding site during aerobic isolation.

Although the spectroscopic and analytical results presented in this work can be satisfactorily rationalized in terms of a single [4Fe-4S]2+ or [2Fe-2S]2+ cluster ligated by radical-AdoMet

CX3CX2C motif, it is not yet possible to definitively rule out the possibility that one or both of

the two other cysteine clusters in anSMEs (Figure 4.1) are involved in Fe-S cluster coordination as previously hypothesized (4). Notably the third cysteine cluster is also strictly conserved in the

AdoMet radical enzyme involved in quinohemoprotein amine dehydrogenase biosynthesis (32).

Further mutagenesis studies coupled with spectroscopic studies should bring definitive assessment of the role for these cysteine clusters. 190

The in vitro assays performed with both enzymes revealed that anSMEs were able to produce 5′-deoxyadenosine from AdoMet. Furthermore, in the presence of 23-mer peptides, the activity of both enzymes was modulated and surprisingly greatly influenced by the first amino acid present in the target sulfatase motif (CXAXR). When a cysteine or serine residue was present, both enzymes produced 5′-deoxyadenosine from AdoMet although at a different levels according to the amino acid residue. However when this residue was substituted with alanine, the

5′-deoxyadenosine production was strongly inhibited indicating a tight control of the anSME activity.

Maturation analysis of the different peptides showed that the serine residue of the 23S peptide and the cysteine residue of the 23C peptide were both converted into FGly by both anSMEbt and anSMEcpe. This result indicated that both enzymes were able to oxidize serine or cysteine residues to FGly. Furthermore, our results also definitively established that the enzyme encoded by the chuR gene is not a transcriptional regulator but a radical AdoMet enzyme involved in sulfatase maturation. We also showed that an alanine-containing peptide (23A), which contains four other serines in the sequence was not modified by either enzyme demonstrating a tight control of the anSMEs activity connected with the nature of the target residues. Nevertheless, the critical peptide determinants which direct the selectivity of these enzymes are not precisely known and are currently under investigation.

With these in vitro experiments we were not able to compare the maturation efficiency of both enzymes toward the cysteine and serine residues. We thus devised an in vivo strategy in order to compare their activity on cysteine and serine residues. Using the C. perfringens sulfatase and its serine mutant we measured the in vivo activation catalyzed by anSMEbt and anSMEcpe.

Both enzymes were able to activate the Cys-type and Ser-type sulfatases with sulfatase activity 191 increased 6 and 25 times, respectively. These in vivo experiments also demonstrated that anSME, contrary to previous reports (4;6), were able to maturate Ser-type sulfatases without the necessity of a signal peptide.

In conclusion, our data support the view that anSMEs are dual substrate radical-AdoMet enzymes rather than a class of enzymes divided into two subgroups dedicated to the maturation of either Ser-type or Cys-type sulfatases. Consistent with our experimental data, we propose herein the first mechanism for this fascinating class of enzymes (Figure 4.14). The reduced

[4Fe-4S]+ cluster in anSME interacts with AdoMet and reductively cleaves AdoMet to yield

methionine and a 5′-deoxyadenosyl radical (Ado•), which abstracts a proton from either a

cysteine or serine residue leading to the production of 5′-deoxyadenosine and to the formation of

FGly. In this scheme, it is not yet possible to determine if AdoMet is a co-substrate as is the case

for the majority of radical AdoMet enzymes, or a cofactor as demonstrated in the case of lysine

2,3-aminomutase or spore photoproduct lyase (33-35). Further investigations will thus be

required to fully elucidate the mechanism of this class of AdoMet radical enzymes.

Acknowledgements

We thank Dr. E. Mulliez (DRDC/CB, Grenoble) for providing S-adenosylmethionine.

Mass spectrometry experiments were performed at the Plateau d’Analyse Protéomique par

Séquençage et Spectrométrie de masse (PAPSS, INRA, Jouy-en-Josas).

192

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195

Figure 4.1 Sequence alignment of the three anSME putative clusters in AtsB (Klebsiella pneumoniae), anSMEcpe (CPF_0616 - Clostridium perfringens) and anSMEbt (BT_0238 –

Bacteroides thetaiotaomicron). Sequence positions in the proteins are in brackets. In black are the conserved cysteines and in grey are the other conserved residues. 196

AtsB(29-49) KPIGPACNLACRYCYYPQDET anSMEcpe(9-29) KPASSGCNLKCTYCFYHSLSD anSMEbt(18-38) KPVGAVCNLACEYCYYLEKAN

AtsB(265-300) HTSGSCVHSARCGSNLVMEPDGQLYACDHLINAEHR anSMEcpe(250-285)GKSSSCGMNGTCTCQFVVESDGSVYPCDFYVLDKWR anSMEbt(271-306) EQPGVCTMAKHCGHAGVMEFNGDVYSCDHFVFPEYK

AtsB(327-364) RECQTCSVKMVCQGGCPAHLNAAGNNRLCGGYYRFF anSMEcpe(315-350)EECKKCKWFKLCKGGCRRCRDSKEDSDLELNYYCQS anSMEbt(337-372) TQCKECDFLFACNGECPKNRFSRTADGEPGLNYLCK

197

Figure 4.2 (A) Gel electrophoresis analysis of anSMEcpe (lane 1) and anSMEbt (lane2) (MW:

Molecular weight markers). (B) UV-visible absorption spectra of reconstituted anSMEbt (dotted line) and anSMEcpe (solid line). 198

MW 1 2 A B

175 kD

83 kD

62 kD

47.5 kD

O.D.

32.5 kD

25 kD

Wavelength (nm)

199

Figure 4.3 UV-visible absorption spectra of as isolated (dashed line) and reconstituted (solid line) anSMEcpe.

200

201

Figure 4.4 Resonance Raman spectra of anSMEcpe as isolated (A), reconstituted (B), and reconstituted, in the presence of a 20-fold excess of SAM (C). The spectra were recorded with

458-nm excitation, using samples that were ~3 mM in anSMEcpe frozen at 20 K, with 140 mW laser power at the sample. Each scan involved photon counting for 1 s at 1.0 cm-1 increments with

7 cm-1 spectral resolution, and each spectrum is the sum of ~100 scans. Bands resulting from the lattice modes of have been subtracted from each spectrum and the band at 420 cm-1 that is marked with as asterisk contains a contribution from glycerol.

202

203

Figure 4.5 EPR spectra of anSMEcpe after anaerobic reduction with a 10-fold excess of sodium dithionite. Spectra were recorded for samples of 0.2 mM anSMEcpe at 10 K with a microwave frequency of 9.603 GHz, a modulation amplitude of 0.63 mT, and a microwave power of 10 mW, unless otherwise indicated. Left panel: Reconstituted anSMEcpe (A) and as isolated anSMEcpe

(B). Inset shows the low field region of the spectrum for reconstituted anSMEcpe recorded at 4.3

K and 50 mM. Right panel: Reconstituted anSMEcpe in the presence (A) and absence (B) of a

20-fold excess of SAM. The difference spectrum corresponding to (A) minus 0.5×(B) is shown in

(C).

204

205

Figure 4.6 AdoMet reductive cleavage assayed by reverse phase HPLC. Incubation of 20µM of reconstituted anSMEbt (A) or anSMEcpe (B), with AdoMet alone () or with 23-mer peptides

(500 µM) containing a cysteine (‹), serine (▲) or alanine („) as target residue.

206

A

5’-deoxyadenosine (µM) (µM) 5’-deoxyadenosine

Time (hour)

B 5’-deoxyadenosine (µM) (µM) 5’-deoxyadenosine

Time (hour)

207

Figure 4.7 In vitro maturation of 23-mer peptides with reconstituted anSMEcpe. After 6 hours of incubation under anaerobic conditions in the presence of AdoMet and a cysteine- (1), serine- (2) or alanine-containing peptide (3), maturation was analyzed by MALDI-TOF MS either with a

CHCA (A) or a DNPH matrix (B).

208

HO

+ NH OH H2N DNPH 23C: Ac-FENAYTAVPSCIASRASILTGMS- [M+H]+ A B 2593 [M+H]+ + 1 [M+H] 1 2431 100 2431 100 [M+H]+ 2413 + + [M+K] [M+K]+ [M+K] 2451 2469 2469 0 0

23S: Ac-FENAYTAVPSSIASRASILTGMS- 2 + [M+H]+ [M+H]+ 100 2 2415 100 2415 + + + [M+K] [M+H] [M+K] 2453 2593 2453

0 0 Relative abundance Relative abundance Relative

23A: Ac-FENAYTAVPSAIASRASILTGMS- [M+H]+ 3 + 3 100 [M+H] 100 2399 2399 + [M+K] + 2437 [M+K] 2437 0 0 2375 2475 2375 2600 [M/Z] [M/Z]

209

Figure 4.8 In vitro maturation of 23-mer peptides with reconstituted anSMEcpe. After 6 hours of incubation under anaerobic conditions in the presence of AdoMet and a cysteine- (1), serine- (2) or alanine containing peptide (3), maturation was analyzed by MALDI-TOF MS with a CHCA matrix.

210

[M+H]+ 2431 100

[M+H]+ 50 2413 [M+K]+ 2469 [M+K]+ 2451 1 0 [M+H]+ 100 2415

50 [M+K]+ 2453

2 abundance Relative 0

100 [M+H]+ 2399

50 [M+K]+ 2437

3 0

2375 2600 [M/Z]

211

Figure 4.9 MALDI-TOF MS analysis of the serine (A) and FGly-containing peptide (B) purified by HPLC.

212

A [M+H]+ 2415

100

0 Relative abundance Relative

2310 2520 [M/Z]

B [M+H] + 2413

100

0 Relative abundance Relative

2310 2520 [M/Z]

213

Figure 4.10 In vitro maturation of 23-mer peptides with reconstituted anSMEbt. After 6 hours of incubation under anaerobic conditions in the presence of AdoMet and a cysteine- (1), serine- (2) or alanine-containing peptide (3), maturation was analyzed by MALDI-TOF MS either with a

CHCA (A) or a DNPH matrix (B).

214

HO

+ NH OH

H2N DNPH A Ac-FENAYTAVPSCIASRASILTGMS-NH2 B

[M+H]+ [M+H]+ 2431 2431 100 100 [M+H]+ + [M+H] [M+K]+ 2593 [M+K]+ 2413 2469 2469 1 1 0 0

+ Ac-FENAYTAVPSSIASRASILTGMS-NH2 + DNPH [M+H] [M+H]+ 2415 100 2415 100 [M+K]+ + [M+K] 2453 2453 2 2 0 0 abundance Relative abundance Relative

+ Ac-FENAYTAVPSAIASRASILTGMS-NH2 [M+H] + 2399 [M+H] 100 100 2399

+ [M+K]+ [M+K] 2437 2437 3 3 0 0

2375 2475 2375 2600 [M/Z] [M/Z]

215

Figure 4.11 In vitro maturation of 23-mer peptides with reconstituted anSMEbt. After 6 hours of incubation under anaerobic conditions in the presence of AdoMet and a cysteine- (1), serine-(2) or alanine-containing peptide (3), maturation was analyzed by MALDI-TOF MS with a CHCA matrix.

216

[M+H]+

2431 100

50 [M+K]+ [M+H]+ 2469 2413 1 0

[M+H]+ 2415

100

50 [M+K]+ 2453

2

Relative abundance Relative 0

[M+H]+ 2399 100

50 [M+K]+ 2437

3 0

2375 2600 [M/Z]

217

Figure 4.12 In vivo maturation of C. perfringens sulfatase expressed alone (1) or in the presence of anSMEcpe (2) or anSMEbt (3) - A: Maps of the plasmids used for the anaerobic production of the C. perfringens sulfatase. B: MALDI-TOF MS analysis of purified sulfatase digested with trypsin and CNBr (numbers indicate the amino acid residues). C: Specific activity of the purified sulfatases.

218

A B Unmaturated

Maturated sulfatase Cys-Sulf 100 sulfatase 371-391

+ plasmid [M+H] 2264 [M+H]+ 1 1 2246 0

100 Cys-Sulf

plasmid

2 anSMEcpe 2 0

Relative abundance Relative 100 Cys-Sulf

plasmid 3 anSMEbt 0 3 2235 2252.5 2270 [M/Z]

C

-1 mg -1 mole.min Sulfatase activity activity Sulfatase

1 2 3

219

Figure 4.13 In vivo maturation of the serine mutant of C. perfringens sulfatase expressed alone

(1) or in the presence of anSMEcpe (2) or anSMEbt (3) - A: Maps of the plasmids used for the anaerobic production of the Serine mutant of C. perfringens sulfatase. B: MALDI-TOF MS analysis of purified sulfatases digested with trypsin and CNBr (numbers indicate the amino acid residues). C: Specific activity of the purified sulfatases.

220

51-ATEGYNFENAYTAVPSSIASR-71 A B

Ser-Sulf 100

plasmid

1 1

0

Ser-Sulf 100

plasmid

anSMEcpe 2 2 0

Ser-Sulf abundance Relative 100

plasmid

anSMEbt 3 3 0 2235 2252.5 2270 [M/Z] C

-1 mg -1 mole.min Sulfatase activity activity Sulfatase

1 2 3

221

Figure 4.14 Schematic mechanism for the maturation reaction catalyzed by anSMEs.

222

H H SH

2+ N H O 5’-deoxyadenosine anSME Cys-type 2+ sulfatase Ado° H O anSME 1+ AdoMet H H OH R N H anSME O H FGly-sulfatase H OH N H O

N H O Ser-type sulfatase

223

CHAPTER 5

ANAEROBIC SULFATASE-MATURATING ENZYME: A MECHANISTIC LINK WITH

GLYCYL RADICAL ACTIVATING ENZYMES?1

1 Reproduced in full with permission of John Wiley & Sons from: Sowmya Subramanian†, Alhosna ‡ § § † ‡ ‡ Benjdia , Jérôme Leprince , Hubert Vaudry , Michael K. Johnson , Olivier Berteau FEBS Journal INRA, UPR 910, UEPSD, 78352 Jouy-en-Josas, France, § INSERM U413, IFRMP23,UA CNRS, Université de Rouen, 76821 Mont-Saint-Aignan, France, † Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA 224

1 Abbreviations. FGly, Cα-formylglycine; AdoMet, S-adenosylmethionine; anSME, anaerobic

sulfatase maturating enzyme; anSMEbt, Bacteroides thetaiotaomicron anaerobic sulfatase- maturating enzyme; anSMEcpe, Clostridium perfringens anaerobic sulfatase-maturating enzyme; anSMEkp, Klebsiella pneumoniae anaerobic sulfatase-maturating enzyme; DNPH, 2,4- dinitrophenyl-hydrazine; CHCA, α-cyano-4-hydroxycinnamic acid matrix IPNS, ; M1, C24A/C28A/C31A; M2, C276A/C282A; M3, C339A/C342A/C348A; WT, wild

type.

225

Abstract

Sulfatases form a major group of enzymes present in prokaryotes and eukaryotes. This

class of hydrolases is unique in requiring an essential post-translational modification of a critical

active-site cysteinyl or seryl residue to Cα-formylglycine (FGly). Herein, we report mechanistic

investigations of a unique class of radical-AdoMet enzymes, anSMEs (anaerobic sulfatase-

maturating enzymes) which catalyze the oxidation of Cys-type and Ser-type sulfatases and

possess three [4Fe-4S]2+,+ clusters. We were able to develop a reliable quantitative enzymatic assay which allowed the direct measurement of FGly production and AdoMet cleavage. The results demonstrate stoichiometric coupling of AdoMet cleavage and FGly formation using peptide substrates with cysteinyl or seryl active-site residues. Analytical and EPR studies of the reconstituted wild-type enzyme and cysteinyl cluster mutants indicate the presence of three almost isopotential [4Fe-4S]2+,+ clusters, each of which is required for in vitro FGly generation.

More surprisingly, our data indicate that the two additional [4Fe-4S]2+,+ clusters are required to

obtain efficient reductive cleavage of AdoMet suggesting their involvement in the reduction of

the radical AdoMet [4Fe-4S]2+,+ center. These results, in addition to the recent demonstration of

direct abstraction by anSMEs of the Cβ H-atom from the active site cysteinyl or seryl residue

using a 5′-deoxyadenosyl radical, provide new insights into the mechanism of this new class of

radical-AdoMet enzymes. 226

Introduction

Sulfatases belong to at least three mechanistically distinct groups, namely the Fe(II) α- ketoglutarate-dependent dioxygenases (1), the recently identified group of Zn-dependent

alkylsulfatase (2) and the broad family of arylsulfatases (3). This latter family of enzymes,

termed “sulfatases” in this article, is certainly the most widespread among bacteria with some of

them possessing more than one hundred sulfatase genes in their genomes (4). Nevertheless, their

biological function has almost never been investigated despite reports on their potential

involvement in pathogenic processes (5;6).

Among hydrolases, sulfatases are unique in requiring an essential catalytic residue, a 3-

oxoalanine usually called Cα-formylglycine (FGly) (7). In sulfatases, it has been proposed that

this modified amino acid is hydrated as a geminal diol in order to perform a nucleophilic attack

on the sulfur atom of the substrate. This leads to the release of the desulfated product and the

formation of a covalent sulfate-enzyme intermediate. The second hydroxyl group on the gem-

diol is essential for the release of the inorganic sulfate as demonstrated by the inactivation of sulfatase bearing a seryl residue instead of the FGly residue (8). This essential FGly residue results from the post-translational modification of a critical active-site cysteinyl or seryl residue

(Figure 5.1A). This has led to the classification of sulfatases into two sub-types, i.e. Cys-type and

Ser-type sulfatases. In eukaryotes, only Cys-type sulfatases have been identified so far, while in bacteria, both types of sulfatases exist. Nevertheless, eukaryotic and prokaryotic sulfatases undergo identical post-translational modification involving the oxidation of a critical cysteinyl or a seryl residue into 3-oxoalanine. In prokaryotes, 3-oxoalanine formation is catalyzed by at least three enzymatic systems but to date only two have been identified (9). The first one, termed formylglycine-generating enzyme (FGE), uses molecular oxygen and an unidentified reducing 227

agent in order to catalyze the aerobic conversion of the cysteinyl residue into FGly (10). The

second one, termed anaerobic sulfatase maturating enzyme (anSME), is a member of the S-

adenosyl-L-methionine (AdoMet)-dependent superfamily of radical enzymes (11-13).

We have recently demonstrated that anSMEs are dual substrate enzymes able to catalyze

the oxidation of cysteinyl or seryl residues making these enzymes responsible for the activation

of both types sulfatase under anaerobic conditions (12). Nevertheless, the mechanism by which

these enzymes catalyze the anaerobic oxidation of cysteinyl or seryl residues is still obscure.

2+,+ Furthermore, in addition to the Cx3Cx2C motif that binds the [4Fe-4S] cluster common to all radical AdoMet superfamily enzymes, anSMEs have two additional conserved cysteinyl clusters with unknown functions.

In the present study, we have carried out mutagenesis studies on the conserved cysteine clusters in anSME from Bacteroides thetaiotaomicron (anSMEbt) to elucidate their role in the anSME mechanism. Our data demonstrate that the additional conserved cysteinyl clusters bind two additional [4Fe-4S]2+,+ centers which are required for FGly generation and efficient

reductive cleavage of AdoMet suggesting that one or both of the additional [4Fe-4S]2+,+ centers play a role in mediating the reduction of the radical-AdoMet [4Fe-4S]2+,+ cluster. In addition, we

use a HPLC-based quantitative assay to quantify FGly formation using anSME from Clostridium

perfringens (anSMEcpe) and a 17-mer peptide substrate, and demonstrate a tight coupling

between AdoMet cleavage and FGly production.

Experimental Procedures

Chemicals. All chemicals and reagents were obtained from commercial sources and were

of analytical grade. S-Adenosyl-L-methionine (AdoMet) was synthesized enzymatically and

purified as described previously (14). 228

anSMEcpe and anSMEbt Protein Expression and Purification. Protein expression and

purification were performed as previously described (12). Briefly, E. coli BL21(DE3)

transformed with a plasmid bearing the anSMEcpe or the anSMEbt gene (pET-6His-anSMEcpe

or pET-6His-anSMEbt) were grown aerobically overnight at 37°C in LB medium (100 mL)

supplemented with kanamycin (50 μg mL-1). An overnight culture was then used to inoculate

fresh LB medium (15 L) supplemented with the same antibiotic. After overnight growth at 25°C

in the presence of IPTG, cells were collected and suspended in Tris-buffer (50 mM Tris, 150 mM

KCl, 10% glycerol pH 7.5). The cells were then disrupted by sonication and centrifuged at

220,000 × g at 4°C for 1 hour. The solution was then loaded onto a Ni-NTA Sepharose column

equilibrated with Tris-buffer, pH 7.5. The column was washed extensively with the same buffer.

Some of the adsorbed proteins were eluted by a washing step with 25 and 100 mM imidazole and

the over-expressed protein was eluted by applying 500 mM imidazole. Imidazole was removed

by gel filtration chromatography PD-10 columns (GE Healthcare) and fractions containing the

anSMEcpe or anSMEbt proteins were immediately concentrated using Ultrafree cells (Millipore)

with a molecular cut-off of 10 kDa.

Construction of Cysteinyl Cluster Mutants. anSMEbt mutants were obtained using the

QuikChange site-directed mutagenesis kit (Stratagene). For each mutant a two-step PCR method

was used (15). The following primers were used for C24A/C28A/C31A mutant : 5′-GCC GTA

GCC AAC CTC GCA GCC GAA TAC GCC TAT TAT-3′ and 5′- ATA ATA GGC GTA TTC

GGC TGC GAG GTT GGC TAC GGC-3′ for C276A/C282A mutant : 5′-GGC GTA GCT

ACA ATG GCG AAG CAT GCC GGA CAT-3′ and 5′-ATG TCC GGC ATG CTT CGC CAT

TGT AGC TAC GCC-3′ and for C339A/C342A/C348A mutant : 5′- ACC CAA GCC AAG

GAG GCC GAC TTT CTA TTT GCC GCC AAC GGA-3′ and 5′-TCC GTT GGC GGC AAA 229

TAG AAA GTC GGC CTC CTT GGC TTG GGT-3′ (in bold are indicated the changed codons). After verification of the correct mutation by sequencing, the plasmid obtained were transformed into E. coli BL21(DE3) and the mutated proteins produced using the same protocol as the wild-type enzyme.

Reconstitution of Fe-S Clusters on anSMEbt and anSMEcpe. Reconstitution was carried

out anaerobically in a glove box (Bactron IV). Anaerobically purified anSMEs (200 µM monomer) were treated with 5 mM DTT (SIGMA, St Louis, MO, USA) and incubated overnight

with a 10-fold molar excess of both Na2S (SIGMA, St Louis, MO, USA). and (NH4)2Fe(SO4)2

(SIGMA, St Louis, MO, USA) at 12 °C. The protein was desalted using a Sephadex G25 column

(GE Healthcare, WI, USA) and the colored fractions were concentrated on Amicon Ultra-4

(Millipore, Billerica, MA, USA). Protein concentrations were determined by the Bradford

protein assay (SIGMA, St Louis, MO, USA), using BSA as a standard. Iron concentrations were

determined colorimetrically using bathophenanthroline (SIGMA, St Louis, MO, USA) under

reducing conditions, after digestion of the protein in 0.8% KMnO4/0.2 M HCl.

Peptide Synthesis. The following 17-mer peptides (with the critical residue in bold): Ac-

TAVPSCIPSRASILTGM-NH2, Ac-TAVPSSIPSRASILTGM-NH2 and Ac-TAVPSAIPSRASIL-

TGM-NH2 were synthesized (0.1-mmol scale) by the solid phase methodology on a Rink amide

4-methylbenzhydrylamine resin (VWR, Fontenay-sous-Bois, France) by using a 433A Applied

Biosystems peptide synthesizer (Applera-France, Courtaboeuf, France) and the standard Fmoc

manufacturer’s procedure. The synthetic peptides were purified by reversed-phase HPLC on a

2.2 × 25-cm Vydac 218TP1022 C18 column (Alltech, Templemars, France) by using a linear

gradient (10-50% over 45 min) of acetonitrile/trifluoroacetic acid (99.9 : 0.1 ; v/v) at a flow rate

-1 of 10 mL min . Analytical HPLC, performed on a 0.46 × 25-cm Vydac 218TP54 C18 column 230

(Alltech), showed that the purity of the peptides was >99.1%. The purified peptides were

characterized by MALDI-TOF mass spectrometry on a Voyager DE PRO (Applera, France) in

the reflector mode with α-cyano-4-hydroxycinnamic acid as a matrix.

Peptide Maturation. Samples containing 6 mM dithiothreitol, 3 mM sodium dithionite,

500 µM peptides and 1 mM AdoMet in Tris-buffer, pH 7.5 were incubated with reconstituted

proteins. The reactions were performed in an anaerobic glove box (Bactron IV). The oxygen

concentration was monitored with a gas analyzer (Coy Laboratory). After incubation at 25°C,

samples were divided in half, one part was used to test the maturation activity by mass

spectrometry while the other half was used to quantify the reductive cleavage of AdoMet and

FGly formation. Control samples were prepared without enzyme to verify peptide and AdoMet

18 stability over time. Experiments performed in H2 O were made exactly as described above

18 except that the Tris-buffer was made in H2 O and the enzyme was exchanged twice with this

buffer before the experiments.

Peptide Maturation Analysis by MALDI-TOF MS. The α-cyano-4-hydroxycinnamic acid

matrix (CHCA) (SIGMA, St Louis, MO, USA) was prepared at 4 mg.mL-1 in 0.15%

trifluoroacetic acid, 50% acetonitrile. The 2,4-dinitrophenylhydrazone acid matrix (DNPH) was

prepared at 100 mg mL-1 in 0.15% trifluoroacetic acid, 50% acetonitrile. Equal volumes (1 µL)

of matrix and sample were spotted onto the MALDI-TOF target plate. MALDI-TOF analysis

was then performed on a Voyager DE STR Instrument (Applied Biosystems, Framingham, CA).

Spectra were acquired in the reflector mode with: 20 kV accelerating voltage, 62% grid voltage

and a 120 ns delay.

Peptide Maturation and 5′-Deoxyadenosine Production Quantification by HPLC. Peptide

modification and 5′-deoxyadenosine (5′-dA) production was measured by high-performance 231

liquid chromatography (HPLC) using a C18 column (LicroSphere, 5-µm, 4.6 × 150-mm)

equilibrated in solvent A (0.1% trifluoroacetic acid). A linear gradient from 0 to 80% acetonitrile

was applied at a constant flow rate of 1 mL min-1. Detection was carried out at 260 nm for

AdoMet and its derivative and at 215 nm to follow peptide modification.

Electron Paramagnetic Resonance (EPR). X-band EPR spectra were recorded on a

Bruker Instruments ESP 300D spectrometer equipped with an Oxford Instruments ESR 900 flow

cryostat (4.2–300 K). Spectra were quantified under non-saturating conditions by double integration against a 1 mM CuEDTA standard.

Results

Formylglycine and 5′-Deoxyadenosine Kinetics. The first step of the reaction catalyzed

by all radical AdoMet enzymes investigated thus far is the reductive cleavage of AdoMet, via

one-electron transfer from the enzyme [4Fe-4S]+ center to AdoMet, to yield methionine and a 5′- deoxyadenosyl radical (16;17). AdoMet is generally used as an oxidizing substrate with the noticeable exception of enzymes such as lysine 2,3-aminomutase (17;18) and spore photoproduct lyase (14;19-21) which use AdoMet catalytically. In other radical AdoMet enzymes, AdoMet is a co-substrate and as such one equivalent of AdoMet is used to oxidize one molecule of substrate. The only known exceptions are coproporphyrinogen III oxidase (HemN) which uses two AdoMet molecules per turnover for the decarboxylation of two propionate side chains

(22;23) and the radical AdoMet enzymes which catalyze sulfur insertion such as lipoyl synthase, biotin synthase and MiaB (16;17).

Recently Grove et al. characterized the K. pneumoniae anSME (anSMEkp) and investigated the maturation of a 18-mer peptide, derived from the K. pneumoniae sulfatase sequence, containing the seryl residue target of the modification (24). Quantitative data were 232

extracted from HPLC and MALDI-TOF mass spectrometry analysis of the products. With the

18-mer peptide substrate, three uncharacterized products and 5′-dA were observed using HPLC

analysis and two peptide products were identified by mass spectrometry analysis. The expected

FGly product (i.e. a 2-Da mass decrease, see Figure 5.1A) was found to be a minor product in the

mass spectrometry analysis, while the major product exhibited a 20-Da mass decrease and was

tentatively attributed to the loss of a water molecule from the FGly product as a result of

formation of a Schiff base via interaction between the aldehyde carbonyl of FGly and the N-

terminal amino group. The three products observed in the HPLC analysis were not further

characterized and it is not currently possible to state whether or not they are FGly-containing

peptides, side reaction products or reaction intermediates. Nevertheless, based on the assumption

that all three products observed by HPLC corresponded to or were derived from the FGly

product, the authors concluded that anSMEs use one mole of AdoMet to produce one mole of

FGly-containing peptide. While this is the most likely scenario based on mechanistic studies of

other radical AdoMet enzymes, this result must be viewed as preliminary in light of the

undetermined nature of the multiple peptide products.

Intrigued by the possibility that some of the peptides produced could be reaction

intermediates, we have performed similar experiments with the Clostridium perfringens enzyme

(anSMEcpe) that was recently characterized in our laboratory (11;12). In our previous studies, we used 23-mer peptides as substrates (11;12). Although these substrates proved to be satisfactory to demonstrate that anSMEs are able to catalyze the anaerobic oxidation of cysteinyl or seryl residues, the instability of these peptides prevented accurate quantifications of the enzymatic reaction. We thus investigated several peptides in order to find a more stable substrate and finally chose a 17-mer peptide closer in size to the 18-mer substrates used by Grove et al. 233

+ (24). The substrate peptides used were Ac-TAVPSCIPSRASILTGM-NH2 (17C) ([M+H] =

+ 1745) and Ac-TAVPSSIPSRASILTGM-NH2 (17S) ([M+H] = 1729). Upon incubation with anSMEcpe, both peptides were converted into a new species with a mass [M+H]+ of 1727 Da

(Figure 5.1B,C & 5.2). This molecular mass was precisely the one expected for the conversion of the cysteinyl or the seryl residue into FGly. To further ascertain the nature of the modification, labeling experiments with DNPH were performed (25). A hydrazone derivative with a mass increment of 180 Da was formed demonstrating the presence of an aldehyde functional group in the newly formed peptide (Figure 5.3). Thus, in our experiments, only the substrate and the expected product were evident in the mass spectra and no other species appeared even after extended incubation (i.e. 12 hours with peptide 17S) (Figure 5.1, 5.2 and 5.3).

We thus developed an HPLC-based assay that could provide reliable and direct quantitative data regarding the anSME activity. During incubation with each peptide, one new peptide appeared with a retention time of 20.4 min (Figure 5.4A,B). The purification of this product and its MALDI-TOF MS analysis confirmed the nature of the product formed and kinetic experiments demonstrated that in both cases (i.e. with a cysteinyl or seryl containing peptide) a strict 1:1 coupling between AdoMet cleavage and FGly production occurred (Figure

5.4C,D). anSMEcpe exhibited a specific activity of 0.07 nmol min-1 mg-1 with the 17S substrate, while the specific activity increased more than 15-fold (1.09 nmol min-1 mg-1) for the 17C substrate.

Peptide 17A was initially included as a control to demonstrate that FGly production occurred on the target cysteinyl or seryl residue. As expected, in presence of enzyme, no modification of the peptide 17A occurred (Figure 5.4 and 5.2C). Interestingly, AdoMet cleavage analysis in presence of this peptide showed that no 5′-dA was produced (Figure 5.4D). This 234

result is surprising because we previously showed that anSMEcpe alone, is able, under reducing

conditions using sodium dithionite as electron donor, to produce 5′-dA from AdoMet (11). This

result suggests that non-productive peptides such as 17A bind near the active site and prevent

either direct reduction of the [4Fe-4S]2+,+ center or interaction with new AdoMet molecules.

Analytical and Spectroscopic Evidence for Multiple Fe-S Clusters in anSME. We previously demonstrated that anSMEs possess a typical radical AdoMet [4Fe-4S]2+,+ center likely

coordinated, as in all radical AdoMet enzymes, by the Cx3Cx2C motif (12). Interestingly, in

addition to this first conserved cysteine motif, anSMEs have seven other strictly conserved

cysteinyl residues and an additional cysteinyl residue in the C-terminus part of the protein

(Figure 5.5A). We and other groups (11;12;26;27) have proposed that additional iron-sulfur

cluster(s) may be coordinated by the remaining conserved cysteinyl residues. Nevertheless, in

our previous analytical and spectroscopic studies of as purified and reconstituted samples of WT

anSMEcpe, we did not succeed in obtaining definitive evidence to support this proposal (11;12).

To address this issue, we used the anSMEbt enzyme which proved to be more stable and

produced three mutants in which groups of conserved cysteinyl residues were mutated to alanyl

residues. The following mutants were generated: C24A/C28A/C31A variant (named mutant M1),

C276A/C282A (named mutant M2) and C339A/C342A/C348A (named mutant M3). Mutants

were purified as previously described starting from a 15 L culture (12). Purification of the

mutants M1 and M2 proved to be satisfactory while mutant M3 exhibited a major contamination

which probably occurred from proteolytic cleavage (Figure 5.6). All purified enzymes exhibited

the typical brownish color of [4Fe-4S] 2+ cluster-containing enzymes and a broad shoulder

centered near 400 nm (Figure 5.5B).

235

The iron-sulfur cluster content of as-purified and reconstituted samples of WT and M1 mutant anSMEbt were assessed by iron and protein analysis coupled with UV-visible absorption studies of oxidized and dithionite-reduced samples (Figure 5.7) and EPR studies of dithionite reduced samples in the absence or presence of AdoMet (Figure 5.8). Samples of as-purified WT and M1 mutant anSMEbt contained 6.3 ± 0.5 and 4.3 ± 0.5 Fe/monomer, respectively, which

increased to 12.0 ± 1.0 and 10.8 ± 1.0 Fe/monomer, respectively, in reconstituted samples. In all

cases the absorption spectra were characteristic of [4Fe-4S]2+ clusters, i.e. broad shoulders

centered at ~ 320 and ~ 400 nm. Moreover, the extinction coefficients at 400 nm mirror the Fe

determinations and indicate 1.6 ± 0.2 and 1.1 ± 0.2 [4Fe-4S]2+ clusters per monomer for the as-

2+ purified WT and M1 mutant samples, respectively, and 2.8 ± 0.4 and 2.6 ± 0.4 [4Fe-4S] clusters per monomer for the reconstituted WT and M1 mutant samples, respectively, based on

2+ -1 -1 the published range observed for single [4Fe-4S] clusters (ε400 = 14-18 mM cm ) (28). The

2+ [4Fe-4S] cluster content is likely to be an overestimate for the reconstituted M1 mutant sample

due to the increased absorption in the 600 nm region which generally indicates a contribution

from adventitiously bound polymeric Fe-S species. While more quantitative analyses will require

Mössbauer studies, the analytical and absorption data are consistent with WT and M1 mutant anSMEbt being able to accommodate up to three and two [4Fe-4S]2+ clusters per monomer,

respectively. Hence the additional seven or eight conserved cysteinyl residues (see Figure 5.5A)

have the ability to coordinate two additional clusters. A similar conclusion was recently

published for the homologous K. pneumoniae AtsB protein based on definitive analytical and

Mössbauer studies (24).

Based on the absorption decrease at 400 nm on reduction, compared to well characterized

[4Fe-4S]2+,+ clusters, we estimate that ~20% and ~30% of the [4Fe-4S] clusters are reduced by 236

dithionite in the reconstituted WT and M1 mutant forms of anSMEbt, respectively, (see Figure

5.7). Both samples exhibited weak, fast-relaxing EPR signals in the S = 1/2 region accounting for

0.12 spins/monomer for WT and 0.07 spins per monomer for the M1 mutant (Figure 5.8). The

relaxation behavior (observable without relaxation broadening only below 30 K) is characteristic

of [4Fe-4S]+ clusters rather than [2Fe-2S]+ clusters. The origin of the low spin S = 1/2

quantitations for dithionite-reduced WT and M1 mutant anSMEbt, relative to the extent of

reduction estimated based on absorption studies, is unclear at present. Probably, it is a

consequence of [4Fe-4S]+ clusters with S = 1/2 and 3/2 spin state heterogeneity as dithionite- reduced reconstituted samples of WT anSMEcpe with substoichiometric cluster content (~6

Fe/monomer) exhibit weak features in the g = 4-6 region indicative of the low field components

of the broad resonances spanning ~ 400 mT that are associated with S = 3/2 [4Fe-4S]+ clusters

(12). As shown in Figure 5.9, WT anSMEcpe exhibits well-resolved low-field S = 3/2 resonances in the g = 4-6 region that are perturbed in the presence of AdoMet suggesting that radical-

AdoMet [4Fe-4S]+ cluster contributes at least in part to the S = 3/2 EPR signal. In contrast, the

fully reconstituted WT and M1 mutant anSMEbt samples do not exhibit well-resolved resonances

in the g = 4-6 region (data not shown). However, as indicated below, the lack of clearly observable S = 3/2 [4Fe-4S]+ cluster resonances may well be a consequence of broadening due to

intercluster spin-spin interaction involving the strongly paramagnetic S = 3/2 clusters in cluster

replete samples of reduced anSMEbt.

The S = 1/2 resonance for the reduced M1 mutant cannot be simulated as a single species

and arises either from two distinct magnetically isolated [4Fe-4S]+ clusters with approximately axial g tensors or as a result of weak magnetic interaction between two [4Fe-4S]+ clusters. We suspect the latter, as two S = 1/2 resonances with different relaxation properties cannot be 237

resolved based on temperature and power-dependence studies. Such magnetic interactions would be expected to be greatly enhanced for clusters with S = 3/2 ground states resulting in additional broadening that would render the resonances unobservable except at inaccessibly high enzyme concentrations. However, irrespective of the explanation of the origin for the complex EPR signal exhibited by the dithionite-reduced M1 mutant anSMEbt, the EPR data support the

presence of two [4Fe-4S]2+,+ clusters in addition to the radical-AdoMet [4Fe-4S]2+,+ cluster in

anSMEbt. Moreover, subtraction of the reduced M1-mutant EPR spectrum from the reduced WT

spectrum affords an axial resonance, g|| = 2.04 and g⊥ = 1.92, that is readily simulated as a

magnetically isolated S = 1/2 [4Fe-4S]+ cluster (accounting for 0.05 spins/monomer) and is

attributed to the reduced radical-AdoMet [4Fe-4S]+ cluster. This is confirmed by changes in the g values (g = 1.98, 1.90, 1.84) and increased spin quantitation (0.05 to 0.15 spins/monomer) for the S = 1/2 form of the radical-AdoMet [4Fe-4S]+ cluster on addition of excess AdoMet (Figure

5.8B). Similar changes in the EPR properties of radical-AdoMet S = 1/2 [4Fe-4S]+ clusters on

binding AdoMet have been reported for many radical-AdoMet enzymes (29;30) and the increase

in spin quantitation is likely to be a consequence of the increase in redox potential that results from AdoMet binding (31). In contrast, within the limits of experimental error, the EPR spectra and spin quantitation of the two additional S = 1/2 [4Fe-4S]+ clusters that are present in the

reduced M1 mutant are not significantly perturbed by AdoMet.

Overall, the EPR and absorption results are best interpreted in terms of three [4Fe-4S]2+,+ clusters in anSMEbt. Each is likely to be mixed spin (S = 1/2 and S = 3/2) in the reduced state and only one is capable of binding AdoMet at the unique Fe site. Since each is only partially reduced by dithionite at pH 7.5, their midpoint potentials are all likely to be in the range of -400 to -450 mV. 238

Function of anSMEs Cysteinyl Clusters. Dierks and co-workers carried out pioneering

studies to assess the function of the anSMEs cysteinyl clusters (26). They made single amino

acid variants in the three conserved cysteinyl clusters of anSMEkp and co-expressed these

mutants in E. coli, along with the sulfatase from K. pneumoniae. All mutants failed to mature the co-expressed sulfatase as no sulfatase activity could be measured. Nevertheless, it was not possible to conclude whether the mutated enzymes were unable to catalyze any reaction or led to the formation of reaction intermediates like spore photoproduct lyase (SPL), another radical

AdoMet enzyme, for which it has been elegantly demonstrated that a cysteinyl mutant, while inactive in vivo (32), efficiently catalyzes in vitro AdoMet cleavage with substrate H-atom abstraction leading to the formation of a side product (19).

We thus assayed the in vitro activity of anSMEbt wild type and mutants after reconstitution in presence of iron and sulfide. All proteins exhibited UV-visible spectra compatible with the presence of [4Fe-4S] centers (Figure 5.5B). Enzymatic assays were conducted using 17C peptide as a substrate and reactions were analyzed by HPLC and MALDI-

TOF MS. The results demonstrate that WT anSMEbt is able to mature the substrate peptide, but

that none of the mutant forms (i.e. M1, M2, or M3) were able to catalyze peptide maturation or to

produce a peptidyl intermediate, as no other peptide was observed by HPLC or MALDI-TOF

MS analysis (Figure 5.10A,B). Even after derivatization with DNPH, which strongly enhances

the signal of the FGly-containing peptide, no trace of modified peptide could be detected using

the M1, M2, or M3 mutants (Figure 5.11).

AdoMet cleavage was assessed for WT and M1, M2, or M3 variants of anSMEbt using the

HPLC assay. As expected, the results showed that mutant M1, which lacks the radical AdoMet

cysteinyl cluster, is unable to produce 5′-dA contrary to the wild type enzyme (Figure 5.10C). 239

More surprisingly, HPLC analyses revealed that the reductive cleavage of AdoMet was also strongly inhibited in the M2 and M3 mutants with a 50 to 100-fold decrease compared to the WT

enzyme (Figure 5.10D).

The variant proteins were also incubated with AdoMet under reducing conditions in the

absence of substrate, as we previously reported that anSMEbt is able to produce 5′-dA efficiently

under these conditions (12). In the absence of substrate, the AdoMet reductive cleavage activity

of all mutants was identical to the one obtained in presence of peptide again indicating that all

three clusters are required for effective reductive cleavage of AdoMet. This observation is most

readily interpreted in terms of a role for the two additional [4Fe-4S]2+,+ clusters in mediating

electron transfer to the radical-AdoMet [4Fe-4S]2+,+ cluster. A similar interpretation was made to explain the strong inhibition of AdoMet reductive cleavage that was observed in the 4- hydroxyphenylacetate decarboxylase activating enzyme, a radical AdoMet enzyme possessing three [4Fe-4S] centers, when cysteinyl residues in its two additional cysteinyl clusters were mutated to alanines (33). However, in the absence of detailed spectroscopic characterization of the clusters in the M2 and M3 mutant anSMEbt samples, we cannot rule out the possibility that the loss of one of the additional [4Fe-4S] clusters affects the ability to reductively cleave

AdoMet by perturbing the redox potential, AdoMet-binding ability or assembly of the radical-

AdoMet [4Fe-4S]2+,+ cluster.

Sequences Comparison With Other Radical AdoMet Enzymes. Primary sequence

comparisons with previously studied radical AdoMet enzymes did not reveal significant

homologies, but several other radical AdoMet enzymes catalyzing post-translational protein

modifications contain conserved cysteinyl clusters involved in the coordination of additional

[4Fe-4S] centers. These enzymes are B12-independent glycyl radical activating enzymes (GRE- 240

AE), i.e. benzylsuccinate synthase (34), glycerol dehydratase (35;36) and 4-hydroxyphenyl-

acetate decarboxylase (33) activases, which catalyze the formation of a glycyl radical on their

respective cognate enzyme using 5′-deoxyadenosyl radical. The role of these additional clusters

has still to be established, but preliminary mutagenesis studies for a hydroxyphenylacetate

decarboxylase activating enzyme indicated a role in mediating electron transfer to the radical-

AdoMet [4Fe-4S] cluster (33).

Further examination of radical AdoMet enzymes involved in protein or peptide

modification lead to the identification of several proteins sharing the third cysteinyl cluster,

Cx2Cx5Cx3C, located in their C-terminal parts while the second cysteinyl cluster found in

anSME could only be tentatively assigned in the central part of these proteins (Figure 5.12).

These proteins are the activating enzyme involved in quinohemoprotein amine dehydrogenase biosynthesis (QHNDH-AE) which is involved in the cross-linking of cysteinyl residues with glutamate and aspartate residues (37) and a new radical AdoMet enzyme involved in the biosynthesis of a cyclic peptide through a lysine-tryptophan linkage (ST protein) (38). Although not strictly conserved, we also identified this cluster in PqqE, an enzyme involved in pyrroloquinoline quinone biosynthesis and proposed to catalyze the linkage of glutamate and tyrosine moieties (39). All these proteins, despite not being homologous, have conserved cysteinyl clusters and catalyze various amino acid modifications. It is thus likely that all these enzymes share common features with anSMEs and notably the presence of additional [4Fe-4S] centers as demonstrated for PqqE (40).

Discussion

We recently demonstrated that sulfatase maturation catalyzed by the radical AdoMet enzyme anSME is initiated by Cβ H-atom abstraction (41). Nevertheless, the entire mechanism of 241

this enzyme has not yet been deciphered. The results presented herein using a new anSME

substrate facilitate more definitive conclusions concerning the catalytic mechanism of anSME

and the AdoMet requirement. Indeed, using a HPLC-based quantitative assay, we have

demonstrated tight 1:1 coupling between AdoMet cleavage and FGly production using both

cysteinyl- and seryl- containing peptides. We also demonstrate tight inhibition of AdoMet

reductive cleavage when the target residue is substituted by an alanyl residue contrary to what

happens in absence of the substrate. Our interpretation is that the peptide binding at the enzyme

active site prevents AdoMet access to the active site. The recently solved crystal structure of

another radical AdoMet enzyme (PFL-AE) (42), has demonstrated that such a hypothesis is

structurally valid. In PFL-AE, the [4Fe-4S] cluster and AdoMet are deeply buried, thereby

preventing uncoupling between AdoMet cleavage and glycyl radical generation.

A longstanding question about anSMEs concerns the function of the conserved additional

cysteinyl clusters originally identified by Schrimer and Kolter (27). In this bioinformatics study,

it was suggested that these clusters were involved in [Fe-S] center coordination. The mutagenesis

of these conserved residues in the K. pneumoniae enzyme subsequently revealed that they are

essential for in vivo activity (26). Nevertheless, their function remained elusive. Grove et al.

provided the first definitive evidence that they are involved with coordinating two [4Fe-4S]

centers in addition to the radical AdoMet [4Fe-4S] center (24). Based on the inferred AdoMet

requirement, a mechanism was proposed involving site-specific ligation of one of the additional

[4Fe-4S]2+ centers to the target cysteinyl or seryl residue resulting in substrate deprotonation.

The 5′-deoxyadenosyl radical generated by reductive cleavage of AdoMet bound at the unique

2+,+ site of radical AdoMet [4Fe-4S] cluster would then abstract a Cβ H-atom from the target

residue and an aldehyde product is generated by using the cluster as the conduit for removal of 242

the second electron (24). The proposed mechanism is reminiscent of the isopenicillin N synthase

(IPNS) which catalyzes the Cβ H-atom abstraction from a cysteinyl residue after its coordination

by a mononuclear non-heme iron center. Following H-atom abstraction, a postulated thioaldehyde intermediate is formed leading to peptide cyclization (43;44). Interestingly, using substrate analogues it has been reported that IPNS can oxidize its target cysteinyl residue into a hydrated aldehyde which is virtually the same as the reaction catalyzed by anSME (45).

Thus, it is conceivable that one of the two additional clusters binds and deprotonates the target cysteinyl or seryl residues and provides a conduit for removal of the second electron (24).

If such a mechanism is correct, our recent demonstration that the 5′-deoxyadenosyl radical produced by anSME directly abstracts one of the cysteinyl Cβ H-atoms (41), coupled with the

results reported herein, indicate that deprotonation occurs prior to or simultaneously with

AdoMet cleavage. Indeed, using an alanyl containing peptide we observed complete inhibition of

AdoMet cleavage.

Although the mutagenesis studies reported herein suggest that both of the two additional

[4Fe-4S] clusters are required for AdoMet cleavage using dithionite as an electron donor, we

cannot rule out the possibility that this is a consequence of perturbation of the redox or AdoMet-

binding properties of the radical-AdoMet [4Fe-4S]2+,+ center that are induced by loss of either

the two additional clusters. Hence it is possible that one of the additional [4Fe-4S] clusters

(Cluster II) is involved with binding the peptide substrate and providing a conduit for removal of

the second electron. The other [4Fe-4S] cluster (Cluster III) could function in mediating electron

transfer from the physiological electron donor to the radical-AdoMet [4Fe-4S] cluster or from

Cluster II to the physiological electron acceptor, see Figure 5.13A. The former mechanism is

analogous to that recently proposed by Grove et al. (24). 243

Nevertheless, the data presented herein suggests an alternative mechanism. Indeed, the

primary sequence analyses discussed above indicate that the two additional clusters are likely to

be ligated by the eight conserved cysteinyl residues and hence both [4Fe-4S] clusters may have

complete cysteinyl ligation, one cysteinyl residue from the last motif being involved in the

coordination of the second cluster (Figure 5.5A). Furthermore, the preliminary observation that

these clusters are almost isopotential with the radical-AdoMet cluster, together with the mutagenesis studies reported herein (which indicate that both additional [4Fe-4S] clusters are required for productive reductive cleavage of AdoMet), suggest that the additional [4Fe-4S]2+,+ clusters play a role in facilitating electron transfer to the radical-AdoMet cluster, as appears to be the case in some B12-independent glycyl radical-activating enzymes (33). Finally, sequence

analysis revealed that these cysteinyl clusters are also found in other radical AdoMet enzymes

involved in protein or peptide modification. These enzymes catalyze the modification of amino

acids such as glutamate or tyrosine which are not known to bind [Fe-S] centers. Moreover,

another radical AdoMet enzyme, BtrN, has recently been demonstrated to use AdoMet

stoichiometrically to catalyze the two electron oxidation of a hydroxyl group to a ketone without

additional Fe-S centers, a reaction formally analogous to the one catalyzed by anSME (46).

However, the absence of additional Fe-S clusters in BtrN clearly requires confirmation using

Mössbauer spectroscopy.

Based on the above considerations, we propose an alternate mechanism for anSME

(Figure 5.13B). In our proposed mechanism, the initial step is the reduction of the radical-

AdoMet [4Fe-4S]2+ cluster via electron transfer from the two additional [4Fe-4S]2+,+ clusters.

Following this reduction, the Cβ H-atom of the substrate is abstracted by the 5′-deoxyadenosyl

radical generated by the reductive cleavage of AdoMet bound at the radical-AdoMet [4Fe-4S]2+,+ 244 cluster as recently demonstrated (41). Simultaneously, deprotonation of the thiol or hydroxyl group occurs catalyzed by an amino acid side chain. The substrate radical intermediate formed by Cβ H-atom abstraction is then further oxidized to yield an aldehyde or a thioaldehyde. In this scenario, the radical would be transferred back to the radical-AdoMet [4Fe-4S]2+ cluster by outer-sphere electron transfer. The implication is that the reaction would have a substrate radical intermediate, as recently demonstrated for BtrN (46), and would be self-sustaining once the initial electron has been supplied by an exogenous electron donor. Both possibilities are currently under investigation in our laboratories. For Cys-type sulfatases, both mechanisms shown in

Figure 5.13 result in the formation of a thioaldehyde intermediate as is also the case in IPNS (45) and cysteine decarboyxlases (47; 48). Hydrolysis of the thioaldehyde by a water molecule is the likely next step. In accord with this hypothesis 18O incorporation into the FGly is observed when

18 the reaction is carried out in H2 O buffer (see Figure 5.14).

Although further work needs to be carried out to clarify the catalytic mechanism of anSMEs and the role of the two additional [4Fe-4S] clusters, the present report suggests that anSMEs possess common features with some glycyl radical-activating enzymes and that radical

AdoMet enzymes possessing additional [4Fe-4S] clusters are likely to be found, notably in enzymes catalyzing protein post-translational modifications. It remains to be seen if the function of these additional clusters involves mediating electron transfer and ⁄ or binding and activating the peptidyl substrates.

Acknowledgement

This work was supported by grants from Agence Nationale de la Recherche (Grant ANR-

08-BLAN-0224-02) and the NIH to M.K.J. (GM62524). Mass spectrometry experiments were performed at PAPSSO, INRA, Jouy-en-Josas. 245

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250

Figure 5.1 Sulfatase maturation scheme leading from a cysteinyl or seryl residue to a FGly in sulfatase active site (A) – MALDI-TOF MS analysis of maturation of peptide 17C (B) and 17S

(C) incubated 2 and 12 hours with anSMEcpe respectively. AnSMEcpe was incubated with each peptide (500 µM) under reducing conditions in the presence of AdoMet (1 mM). 251

A H H H SH H OH

N N H H O O - 18 Da - 2 Da Cys-type Ser-type

sulfatase H O sulfatase

N H O

FGly-sulfatase

B C

17C: Ac-TAVPSCIPSRASILTGM-NH2 17S: Ac-TAVPSSIPSRASILTGM-NH2 + 100 [M+H] 100 [M+H]+ 1745 1729

) ) % % ( ( T0 T0 18 Da 2 Da

[M+H]+ [M+H]+ 1727

Relative abundance Relative abundance 1727

Relative abundance T2H T12H 0 0

1715 [M/Z] 1760 1715 1745 [M/Z] 252

Figure 5.2 MALDI-TOF MS analysis of 17-mer peptides after incubations with anSMEcpe.

Incubations of the17-mer peptide 17C (A), 17S (B) and 17A (C) with anSMEcpe were performed in reducing conditions in the presence of SAM (1mM), DTT (6 mM) and dithionite (3 mM) in Tris HCl buffer (pH 7.5 – 50 mM). Samples were analyzed with a MALDI-TOF mass spectrometer (Voyager DE STR Instrument- Applied Biosystems, Framingham, CA, USA) using an α-cyano-4-hydroxycinnamic acid matrix (CHCA). 253

[M+H]+ A 100 1745

[M+K]+ 1783 [M+Na]+ T0 1767 0 100

+ [M+H] + 1727 [M+K] 1783 [M+K]+ Relative abundance (%) Relative abundance T2H 1765 0 1500 1900 [M/Z] [M+H]+ 1729 100 B [M+K]+ 1767 [M+Na]+ 1751 T0 0 100

[M+H]+ Relative abundance (%) Relative abundance 1727 T12H 0

1500 1900 [M/Z]

[M+H]+ 100 1713 C [M+K]+ 1751 [M+Na]+ T0 1735 0 100 Relative abundance (%) Relative abundance T12H 0

1500 1900 [M/Z] 254

Figure 5.3 MALDI-TOF MS analysis of 17C and 17S peptides after incubation with anSMEcpe.

Incubations of the 17-mer peptide 17C (A) and 17S (B) with anSMEcpe were performed under

reducing conditions in the presence of SAM (1mM), DTT (6 mM) and dithionite (3 mM) in Tris

HCl buffer (pH 7.5 – 50 mM). Samples were analyzed with a MALDI-TOF mass spectrometer

(Voyager DE STR Instrument - Applied Biosystems, Framingham, CA, USA) using a 2,4- dinitrophenyl hydrazine (DNPH) as matrix. 255

A

+ DNPH [M+H]+ 1907 100

[M+K]+ 1945 [M+H]+ 1745 0

1700 2000

B + DNPH [M+H]+ 1907 100

[M+K]+ 1945 [M+K]+ 1767

[M+H]+ 1729

0

1700 2000 [M/Z] 256

Figure 5.4 HPLC analysis of incubation reactions of anSMEcpe with peptide 17C (A) and 17S

(B) and time-dependant formation of FGly-containing peptide (C) and 5′-deoxyadenosine (D). anSMEcpe was incubated with 17C (‹), 17S (▲) or 17A (●) peptide (500 µM) under reducing conditions in the presence of AdoMet (1 mM), DTT (6 mM) and dithionite (3 mM). 257

A B

17C : Ac-TAVPSCIPSRASILTGM-NH2 17S : Ac-TAVPSSIPSRASILTGM-NH2

T12H T2H

T0 T0

16 25 16 25 Time (min.) Time (min.)

C D

250 250 17C 17C

125 125 FGly (µM) 17S 17S 5’ deoxyadenosine (µM) (µM) 5’ deoxyadenosine

17A 17A 0 0 0 720 0 720 Time (min.) Time (min.) 258

Figure 5.5 (A) Sequence alignment of the three anSMEs putative clusters in AtsB (anSMEkp from Klebsiella pneumoniae), anSMEcpe (CPF_0616 from Clostridium perfringens) and anSMEbt (BT_0238 from Bacteroides thetaiotaomicron). Sequence positions in the proteins are in brackets. The conserved cysteinyl residues are indicated in black boxes and the other conserved residues are shadowed – (B) UV visible absorption spectra of reconstituted wild-type

(WT) and M1, M2 and M3 variants of anSMEbt.

259

A

AtsB(29-49) KPIGPACNLACRYCYYPQDET anSMEcpe(9-29) KPASSGCNLKCTYCFYHSLSD anSMEbt(18-38) KPVGAVCNLACEYC YYLEKAN

AtsB(265-300) HTSGSCVHSARCGSNLVMEPDGQLYACDHLINAEHR anSMEcpe(250-285)GKSSSCGMNGTCTCQFVVESDGSVYP CDFYVLDKWR anSMEbt(271-306) EQPGVCTMAKHCGHAGVMEFNGDVYSCDHFVFPEYK

AtsB(327-364) RECQTCSVKMVCQGGCPAHLNAAGNNRLCGGYYRFF anSMEcpe(315-350)EECKKCKWFKLCKGG CRRCRDSKEDSDLELNYYCQS anSMEbt(337-372) TQCKECDFLFACNGECPKNRFSRTADGEPGLNYLCK

B

WT M1

M 2 M 3

260

Figure 5.6 Gel electrophoresis analysis (SDS PAGE 12.5%) of wild-type (WT) and the M1, M2 and M3 variants of anSMEbt. Row 1 corresponds to molecular weight markers.

261

MW anSMEbt M1 M2 M3

121 kDa

74 kDa

48 kDa

37 kDa

25 kDa

18 kDa 262

Figure 5.7 UV-visible absorption spectra of oxidized (solid line) and dithionite-reduced (broken

line), as purified and reconstituted forms of WT and M1 mutant anSMEbt. Samples of WT and

M1 mutant anSMEbt (each 0.4 mM) in Tris-HCl buffer, pH 7.5, were anaerobically reduced with a 10-fold stoichiometric excess of sodium dithionite. Extinction coefficients are based on protein concentrations and asterisks indicate bands resulting from excess dithionite. 263

160 WT as purified * 120 WT reconstituted

120 ) ) -1 80 -1 cm cm -1 -1 * 80 mM mM ε ( ε ( ε (

40 40

0 0 400 600 800 400 600 800 Wavelength (nm) Wavelength (nm)

160 120 M1 mutant as purified M1 mutant reconstituted

120 * ) ) -1 -1 80 cm cm -1 -1 80 (mM (mM ε ε * 40 40

0 0 400 600 800 400 600 800 Wavelength (nm) Wavelength (nm) 264

Figure 5.8 X-band EPR spectra of dithionite-reduced reconstituted samples of WT and M1 mutant anSMEbt in the absence (A) and presence (B) of a 20-fold stoichiometric excess of

AdoMet. The WT minus M1 mutant difference spectrum at the bottom of each panel corresponds to the EPR spectrum of the S = 1/2 [4Fe-4S]+ radical-AdoMet cluster with (B) and without (A)

AdoMet bound at the unique Fe site. EPR spectra were recorded at 10 K with 20 mW microwave

power, 0.65 mT modulation amplitude and a microwave frequency of 9.603 GHz. The

spectrometer gain was 2-fold higher for the samples prepared without AdoMet. Samples of WT

and M1 mutant anSMEbt (each 0.4 mM) in Tris-HCl buffer, pH 7.5, were anaerobically reduced

with a 10-fold stoichiometric excess of sodium dithionite.

265

266

Figure 5.9 Low field X-band EPR spectra of dithionite-reduced WT anSMEcpe in the presence and absence of a 20-fold excess of AdoMet. The samples (each 0.2 mM and containing ~6

Fe/monomer) were in Tris-HCl buffer, pH 7.8, were anaerobically reduced with a 10-fold stoichiometric excess of sodium dithionite conditions. EPR spectra were recorded at 4.2 K with

50 mW microwave power, 0.65 mT modulation amplitude and a microwave frequency of 9.603

GHz. 267

g = 5.4 g = 4.3 Without AdoMet

g = 5.4 g = 4.6 With AdoMet

100 150 200 Magnetic Field (mT) 268

Figure 5.10 HPLC (A) and MALDI-TOF MS (B) analysis of the peptide maturation catalyzed by WT and M1, M2 and M3 mutants of anSMEbt. The WT and mutant forms of anSMEbt (each

60 µM) were incubated with 17C peptide (500 µM) under reducing conditions in the presence of

AdoMet (1 mM), DTT (6 mM) and dithionite (3 mM) for 4 hours under anaerobic and reducing conditions. (C) HPLC analysis of AdoMet cleavage catalyzed by WT or M1, M2 and M3 mutants of anSMEbt in presence of 17C peptide. (D) Relative production of 5′-dA compared to the WT enzyme with or without substrate peptide (inset: magnified picture of the results obtained for the mutants). 269

A B

C D 270

Figure 5.11 MALDI-TOF MS analysis of 17C peptide after incubations with wild-type (WT) and M1, M2 and M3 mutants of anSMEbt. Incubations of the 17-mer peptide (17C) with variants of anSMEbt were performed in reducing conditions in the presence of SAM (1mM), DTT (6 mM) and dithionite (3 mM) in Tris HCl buffer (pH 7.5 – 50 mM). After 4 hours of incubation samples were analyzed with a MALDI-TOF mass spectrometer (Voyager DE STR Instrument-

Applied Biosystems, Framingham, CA, USA) using 2,4-dinitrophenyl hydrazine (DNPH). Equal volumes (1 µL) of matrix and sample were spotted onto the MALDI-TOF target plate. MALDI-

TOF analysis was then performed. Spectra were acquired in the reflector mode with the following parameters: 20 kV accelerating voltage, 62% grid voltage, 120 ns delay. The arrow indicates the hydrazone derivative that is characteristic of FGly formation.

271

[M+H]+ 100 [M+K]+ [M+Na] +

M3 0 100

M2 0 100 + DNPH Relative abundance (%) Relative abundance

M1 0 100

[M+H]+ [M+K]+ 1745 1907

WT 0

1710 1830 1940

[M/Z] 272

Figure 5.12 Sequence alignment of anSMEcpe, quinohemoprotein amine dehydrogenase, PqqE and the ST protein. The positions of the sequences in the proteins are shown in parentheses. The percentage of similarity between the corresponding region of anSME and the different enzymes is indicated in brackets. 273

anSMEcpe (9-27) PASSGCNLKCTYCFYHSL ST (112-129) YPSMYCDLKCGFCFLANR (55.6%) QHNDH-AE (117-124) NVNTGCNLACTYCYKEDL (66.7%) PqqE (17-34) ELTYRCPLQCPYCSNPLD (39%)

anSMEcpe (250-282) GKSSSCGMN------GT-----CTCQF----VVESDG-SVYPC-DFYV ST (316-371) FATEGCHLFTAYPELINNSIEFSEFGEMYYG-CRAKYTK—MEIMSNGDIL-PCIAFLG (30%) QHNDH-AE (321-377) YVEAACRGENIGFSNMHQLLTDIAQGTKKAVPCGAGLGM—LAVDKDGELH-LCHRFVG (32%) PqqE (219-271) QKMAASGNLTNLLFVTPDYYEERPKGERPKG-CMGGWGSIFLSVTPEGTALPCHSARQ (25%)

anSMEcpe (312-338) KVHEECKKCKWFRLCKGGCRRCRDSKEDSALEL-NYYCQS ST (401-439) TKNSKCLSCGLLKICEGGCYVNLI-KEKSPKYFRDPVCNL (41%) QHNDH-AE (404-446) RSAYGCKTCRIRSICAGGCYHESYARQGDPFAPVWHYCDL (45%) PqqE (301-342) WMPEPCRSCDEKEKDFGGCRCQAFMLTGSADNAD-PVCSK (38.5%)

274

Figure 5.13 Two possible mechanisms for anSMEs with Cys-type sulfatases substrate – (A)

After reduction of the radical AdoMet [4Fe-4S] center, AdoMet is reductively cleaved and the resulting 5′-deoxyadenosyl radical abstracts a Cβ hydrogen atom from cysteinyl residue of the substrate peptide that is ligated to a unique site of a [4Fe-4S] center (Cluster II). Cluster III is proposed to play a role in mediating electron transfer from the physiological electron to the

radical AdoMet [4Fe-4S] cluster or from Cluster II to the physiological electron acceptor. (B)

The peptidyl substrate is first deprotonated and AdoMet is reductively cleaved. The resulting 5′-

deoxyadenosyl radical abstracts a Cβ hydrogen atom from cysteinyl residue to generate a

substrate radical that is converted to the thioaldehyde intermediate via outer-sphere electron

transfer to the radical AdoMet cluster. In this scheme, the additional [4Fe-4S] centers, Clusters II

and III, have a key role in mediating the initial electron transfer from the physiological electron

to the radical AdoMet [4Fe-4S] cluster. In both mechanisms, a thioaldehyde intermediate is

formed and further hydrolyzed to form the FGly residue with the release of hydrogen disulfid 275

A

276

B

277

Figure 5.14 MALDI-TOF mass spectrometry analysis of 17C peptide before (1) and after a

16 18 4 hour incubation with anSMEcpe in H2 O (2) or H2 O (3) buffer. AnSMEcpe (30 µM) was incubated with peptide 17C (500 µM) under reducing conditions in the presence of AdoMet

(1 mM). (B) Potential mechanism for isotope exchange via hydrolysis of the thioaldehyde intermediate.

278

17C A [M+H+] : 1745 1 100

Relative abundance (%) 0 17FGly + [M+H ] : 1727 2 100

16 H2 O

Relative abundance (%) 0

17FGly-18O

17FGly-16O

3 100

18 H2 O Relative abundance (%) 0

1715 [M/Z] 1760

B

279

CHAPTER 6

CONCLUSIONS AND FUTURE WORK

The papers presented herein address different aspects of the nature and properties of Fe-S

centers in three different classes of proteins, ferredoxins, glutaredoxins and radical SAM

enzymes. In chapter 2, the results of experiments directed towards understanding the electronic

properties and the origin of valence-delocalized [2Fe-2S]+ species in the Cys-to-Ser variants of thioredoxin-like Fds, CpFd and AaeFd4 have been presented. Through redox and CD spectrophotometric titrations together with EPR and VTMCD studies, we were able to ascertain that serinate ligation to the reducible iron at pH > pKa is one of the major determining factors for

the observation of valence-delocalized, S = 9/2 [2Fe-2S]+ clusters in the variants. The negative

shift in redox potential upon serinate coordination is proposed to render the two Fe sites

equipotential, thereby favoring electron delocalization between the two iron sites.

One of the major advances in understanding the electronic properties due to the valence- delocalized [2Fe-2S]+ unit reported in this work is the assignment of VTMCD bands in the NIR

region to transitions arising from Fe-Fe interaction. This was accomplished by assigning the

polarization of electronic transitions in the NIR region by VHVT MCD magnetization studies.

The assignment of the VTMCD band observed at 1070 nm to the z-polarized, σ→ σ*

intervalence transition enabled the measurement of the double exchange parameter (B),

B = 930 cm-1, which is in excellent agreement with the estimated value for [2Fe-2S]+ cluster

(B = 965 cm-1) based on extrapolation from the Fe-Fe electronic coupling of the best

2+ characterized, valence delocalized S = 9/2 diiron synthetic complex, [Fe2(OH)3(tmtacn)2] (1;2). 280

Thus, the Cys-to-Ser variants of the thioredoxin-like Fds presented an unique opportunity to characterize the ground state and excited state electronic properties of the valence-delocalized

[2Fe-2S]+ unit, and also facilitated the identification of the NIR-VTMCD bands due to valence-

delocalized [2Fe-2S]+ units in higher-nuclearity Fe-S clusters (See Figure 2.21).

Comparison of the RTMCD and VTMCD spectra of the Cys-to-Ser variants in both CpFd

and AaeFd4 at high pH, indicated that the [2Fe-2S]+ center is completely valence delocalized in

solution at room temperature, but is partially or exclusively converted to valence-localized

species upon freezing. The observation of a valence-delocalized versus valence-localized state is

the result of a delicate balance between the relative contribution of the Heisenberg exchange

parameter (J) which tends to localize the extra electron, the double exchange parameter (B)

which tends to delocalize the extra electron, and the difference in site potential, which again

tends to localize the extra electron. We speculate that these determinants are affected by solvent

rearrangement and the resultant change in H-bonding interactions near the solvent exposed,

reducible Fe site, as the samples are frozen and promote the conversion of valence-delocalized

fraction to valence-localized state.

Fe-Fe distance is an important determinant of the value of B and is well exemplified in

2+ the synthetic complex, [Fe2(OH)3(tmtacn)2] , which has a Fe-Fe distance of 2.51 Å and a B

value of 1350 cm-1 (1;2). Based on the atomic resolution crystallographic studies of the oxidized

C55S and C59S AaeFd4 variants which revealed a shortening of the Fe-Fe distance by 0.04 Å

(Fe-Fe distance in WT is 2.73 Å) (3), and the observation of valence delocalized species at room

temperature, crystallographic studies of the reduced variants at room temperature are planned to

determine the Fe-Fe distance and understand its correlation to the value of B in the valence-

delocalized variants. 281

Chapter 3 focused on determining the nature of Fe-S centers assembled on GrxS16, a

monothiol glutaredoxin localized in the chloroplast, and its function. We were able to show

through spectroscopic and analytical studies that under anaerobic conditions, recombinant

GrxS16 can be purified with an all-cysteinyl ligated [2Fe-2S] cluster. On anaerobic in vitro

reconstitution, it can assemble an all-cysteinyl ligated [2Fe-2S]2+, linear [3Fe-4S]+ and [4Fe-

4S]2+ clusters. Although, no structural information is available for any of the cluster-bound forms of GrxS16, the spectroscopic and analytical gel filtration studies provided a good picture of the coordination properties of the clusters. Close similarities in the spectroscopic properties of [2Fe-

2S]-GrxS16 to [2Fe-2S]-GrxS14, and the structurally characterized E. coli [2Fe-2S]-Grx4 (4) indicates that the [2Fe-2S] cluster in GrxS16 is coordinated by the active-site cysteines of two

Grx molecules and two trans GSHs. This was also borne out by the results of analytical gel filtration studies, which indicated that [2Fe-2S]-GrxS16 is a dimer. Based on analytical gel filtration studies of [4Fe-4S]-GrxS16, the in vivo results from complementation studies using yeast grx5 mutant that lacks the second conserved cysteine, and the inability of GrxS14 to reconstitute a [4Fe-4S] cluster under similar reconstitution conditions used for GrxS16

(Bandyopadhyay, S. unpublished results), we propose that the [4Fe-4S] cluster in GrxS16 is

coordinated at the tetramer interface by the active site (CGFS) cysteine of four Grx molecules.

Cysteine mutagenesis experiments are underway to test this hypothesis.

In contrast to its counterpart in chloroplast, GrxS14, the ability of GrxS16 to assemble a

[4Fe-4S] cluster and transfer it to the scaffold protein, Nfu2 at rates comparable to what has been

reported for [4Fe-4S] cluster transfers (5;6) indicates a primary role for S16 in trafficking [4Fe-

4S] clusters in the chloroplast. The slow and incomplete transfer of [2Fe-2S] cluster from

GrxS16 to apo Fd on the other hand suggests that S16 is competent at being a [2Fe-2S] cluster 282

donor, and probably works with a yet unidentified partner protein or it may not have a primary

role in the trafficking of [2Fe-2S] clusters. In either case, further in vitro cluster transfer studies

to a wider range of chloroplastic [2Fe-2S] and [4Fe-4S]-containing proteins, coupled with in vivo

gene knock out studies in A. thaliana would be required to identify the physiological partner/s and the exact role of the cluster bound forms of GrxS16.

The biological relevance of the ability of monothiol Grxs such as GrxS16 (this work) and

Grx5 and GrxS14 (Bandyopadhyay, S. unpublished results) to accommodate linear [3Fe-4S]+ clusters is unclear, as there is no precedence of a role for linear [3Fe-4S]+ clusters in biology.

The presence of linear [3Fe-4S]+ clusters may thus be an artifact resulting from oxidative

denaturing conditions, but they do have the potential to serve as intermediates, which can

synthesize [2Fe-2S]2+ clusters under oxidizing conditions with the loss of Fe3+ ions or [4Fe-4S]2+ clusters under reducing conditions in the presence of Fe2+ ions. However, it remains to be seen if

nature uses such a route, which undoubtedly provides an efficient way of synthesizing/recycling

Fe-S centers.

The final chapters presented in this dissertation, 4 & 5 focused on determining the

number and nature of Fe-S clusters coordinated by anSMEs and elucidate their role in the

catalytic mechanism. The wild-type and site-directed variant anSMEs from Clostridium

perfringens and Bacteriodes thetatiotamicron were used for these studies. Based on the

analytical and EPR data for the reconstituted WT anSMEbt (see chapter 5), we were able to

conclude that anSMEbt is able to coordinate three almost isopotential [4Fe-4S] clusters, which

includes the radical SAM cluster and two additional [4Fe-4S] clusters coordinated by the

conserved clusters of cysteines, similar to what has been reported for AtsB (7). These results also

suggest that the reconstituted samples of anSMEcpe (see chapter 4) were deficient in clusters, as 283

the results were rationalized in terms of the presence a single [4Fe-4S] cluster coordinated by the

radical SAM motif, based on the analytical determinations. Moreover, Fe determinations on the

reconstituted samples of the WT and the M1 mutant of anSMEbt (lacking the radical SAM

motif) indicated the presence of 2.8 ± 0.4 and 2.6 ± 0.4 [4Fe-4S]2+ clusters, respectively. While

the analytical data could be well rationalized with the observed EPR results indicating the

presence of three [4Fe-4S]2+ clusters in the WT, the number of clusters in the mutant was clearly

an overestimate, which could be due to adventitiously bound polymeric Fe-S species and is identified by an increased absorption in the 600 nm region. A more quantitative analyses on well-purified (with less adventitious iron content), cluster-replete samples of anSMEcpe and anSMEbt WT and variants using Mössbauer studies are planned to address these discrepancies.

In vivo and In vitro MALDI-TOF and HPLC analyses of the enzymatic products obtained

by using both Cys-type and Ser-type sulfatases, and the 23-mer and 17-mer, synthetic peptide

substrates with active site cysteine/serine residue, indicated the ability of anSMEcpe and

anSMEbt to maturate both Cys-type and Ser-type sulfatases. A 1:1 coupling between AdoMet

cleavage and FGly production was also demonstrated through quantitative HPLC analyses, using

anSMEcpe and the 17C/17S peptide substrates.

To clarify the role of the additional Fe-S clusters, enzymatic assays using the anSMEbt

variants were performed, which indicated that Adomet cleavage and FGly production were

strongly dependent on the presence of the two additional Fe-clusters. Based on the presence of

three almost isopotential [4Fe-4S]2+ clusters identified in anSMEbt by EPR studies and the

requirement of the additional [4Fe-4S]2+ clusters for efficient Adomet cleavage, we propose that

the two additional Fe-S clusters are involved in mediating electron transfer from the

physiological electron donor to the radical-Adomet [4Fe-4S] cluster (See Figure 5.13B). A 284 similar argument was also made to explain the strong inhibition of Adomet reductive cleavage in the 4-hydroxyphenylacetate decarboxylase activating enzyme, a radical SAM enzyme with two additional [4Fe-4S] centers (8). Moreover, the presence of conserved cysteinyl clusters and their ability to coordinate additional [4Fe-4S] clusters is also a common feature in the B-12 independent glycyl radical activating enzymes and other radical SAM enzymes involved in peptide/protein modification, and their roles remain to be determined. Further studies are certainly needed to determine the exact role of these additional [4Fe-4S] clusters in activating the substrate (See Figure 5.13A) as suggested by Grove, et al., and/or in serving as an electron relay

(See Figure 5.13B) in the anSMEs, and is part of ongoing research.

285

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