INSIGHTS ON IRON-SULFUR CLUSTER ASSEMBLY DONOR PROTEINS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Eric M. Dizin, M.S.

٭ ٭ ٭ ٭ ٭

The Ohio State University 2008

Dissertation Committee: Approved by

Professor James Cowan, Advisor

Professor Claudia Turro

Professor Thomas J. Magliery Advisor Chemistry Graduate Program

ABSTRACT

Iron-sulfur clusters are an important class of prosthetic group involved in electron transfer, enzyme catalysis, and regulation of gene expression. Their biosynthesis requires a complex machinery located within the mitochondrion since free iron and sulfide are extremely toxic to the cell. This research has focused on the three central proteins dedicated to the assembly: a cysteine desulfurase, Nfs1, an iron donor protein, frataxin, and an iron-sulfur cluster scaffold protein, Isu1.

Human Nfs1, a PLP dependent enzyme, catalyzes the decomposition of cysteine to alanine and forms a persulfide bond with a conserved cysteine residue. To date, Nfs1 has only been partially characterized. Furthermore, its hyperproduction relies on yeast organisnm, Pichia pastoris, which is cumbersome and leads to quite low yields.

Therefore, we undertook to design a bacterial expression system by cloning and overexpressing the gene in different E. coli strain. This enabled only a partial characterization of the cysteine desulfurase.

Besides being an iron donor for iron-sulfur cluster assembly, frataxin has also been implicated in heme biosynthesis, and in iron storage in the mitochondrion with reported ferroxidase activity. We decided to further investigate its ability to bind to other metals,

ii such as magnesium, calcium, and zinc, and also studied its ferroxidase activity as a mature and as a truncated protein. We concluded that frataxin has negligible ferroxidase activity, comparable to iron, and quite distinct from ferritin. Moreover frataxin binds zinc besides iron, but with a different stoichiometry.

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Dedicated to Odile, Henri, Pierre, Colette, Dominique and Claude

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ACKNOWLEDGMENTS

I would like to express my gratitude to my advisor, Professor James A. Cowan, without whom I could not have completed this dissertation. Prof. Cowan made it possible for me to pursue my PhD in an intellectually stimulating and cheerful environment. With his broad scientific knowledge, patience, and enthusiasm, Prof. Cowan was an excellent guide.

My professional development at OSU was fostered by Professors Martin Caffrey and Dehua Pei. Prof. Caffrey taught me thoroughness, and Prof. Pei was a model of scientific inquiry. Thanks to both of them, and to all my teachers in the Department of

Chemistry.

Thanks also to Valerie Wright and Prof. Thomas Clanton for their attentive help with the oximetry device, to Prof. Timothy Stemmler for his help with the EXAFS samples and protein NMR, to the CCIC for the mass spectrometry, and to Dr. Gordon

Renkes for the circular dichroism. And thanks to the Chemistry Department for supporting me all these years.

I would also like to thank my labmates: Wen-I Luo, Jeff Joyner, Dr. Chun-An

Chen, Dr. Nikhil Gokhale, and Dr. Manunya Nuth.

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Last but not least, I thank for their warm support my parents, Claude and

Dominique, my grandparents, Henri and Odile, Anne-Sophie, Bruce, Christopher,

Nicolas and Rob.

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VITA

September 29, 1976 Born – Sèvres, France

2000-2001 Technician, Industrial Coating Department, Cognis GmbH, Düsseldorf

2003 M.S. Chemistry, ESCPE, Lyon, France

2001 – present Graduate Teaching and Research Associate, The Ohio State University

PUBLICATIONS

1. Kodapalli, K.C.; Dizin, E.; Cowan, J.A.; Stemmler, T.L. “Letter to the Editor: 1 H, 13 C and 15 N resonance assignments for full length mature human frataxin”, manuscript in preparation for J. Biomol. NMR.

2. Huang, J.; Dizin, E.; Cowan, J. A. Mapping Iron Binding Sites on Human Frataxin. Implications for Cluster Assembly on the ISU Fe-S Cluster Scaffold Protein. J. Biol. Inorg. Chem. 2008, 13, 0000.

3. Yoon, T.; Dizin, E.; Cowan, J. A. N-terminal iron-mediated self cleavage of human frataxin: regulation fo iron binding and complex formation with target proteins. J. Biol. Inorg. Chem. 2007, 12(4), 535-42.

4. Zhu, J.; Hu, X.; Dizin, E.; Pei, D. Catalytic mechanism of S- ribosylhomocysteinase (LuxS): Direct observation of ketone intermediates by 13C NMR spectroscopy. J. Am. Chem. Soc. 2003, 125(44), 13379-81.

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5. Zhu, J.; Dizin, E.; Hu, X., Wavreille, A.-S.; Park, J.; Pei, D. S- Ribosylhomocysteinase (LuxS) is a mononuclear iron protein. Biochemistry. 2003, 42(16), 4717-26.

FIELDS OF STUDY

Major Field: Chemistry

Specialization: Biological Chemistry

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TABLE OF CONTENTS

Page Abstract ...... ii Dedication ...... iv Acknowledgments ...... v Vita ...... vii List of Tables...... xiii List of Figures ...... xiv Abbreviations ...... xvii

Chapters:

1. Introduction 1

1.1 Iron...... 2 1.1.1 Reactivity ...... 2 1.1.2 Distribution ...... 5 1.1.3 Uptake ...... 5 1.1.4 Transport ...... 8 1.1.5 Import ...... 9 1.1.6 Storage ...... 9 1.1.7 Regulation of iron metabolism ...... 12 1.1.8 Iron-sulfur clusters cofactors ...... 14 1.2 Iron-sulfur clusters biogenesis ...... 19 ix

1.2.1 Iron import in mitochondria ...... 19 1.2.2 Frataxin ...... 19 1.2.3 Mitochondrial ferritin ...... 23 1.2.4 Cysteine desulfurase Nfs1 ...... 25 1.2.4.1 Sulfide toxicity and Activation ...... 25 1.2.4.2 PLP cofactor ...... 26 1.2.4.3 Mechanism ...... 27 1.2.4.4 Interacting partners ...... 31 1.2.5 Isu1: the scaffold protein ...... 32 1.2.6 Other scaffolding proteins ...... 33 1.2.7 Molecular chaperones ...... 34 1.2.8 Export machinery ...... 36

2. Cysteine desulfurase 38

2.1 Introduction ...... 38 2.2 Experimental procedures ...... 40 2.2.1 Nfs constructs ...... 40 2.2.2 Expression and Purification ...... 41 2.2.2.1 Full length and (1-31) HS-Nfs1 ...... 42 2.2.2.2 (1-58) HS-Nfs1 ...... 43 2.2.3 Mass spectrometry ...... 44 2.2.3.1 In Gel Digestion ...... 44 2.2.3.2 Protein identification ...... 45 2.2.3.3 Database search ...... 46 2.2.4 Western blots ...... 46 2.2.5 Factor Xa digestion ...... 47 x

2.2.6 U.V. Characterization...... 47 2.2.7 Activity Assays ...... 47 2.2.7.1 Cysteine binding ...... 47 2.2.7.2 Elemental sulfur determination assay ...... 48 2.2.7.3 Sulfide determination assay ...... 48 2.2.7.4 Iron-sulfur reconstitution kinetic ...... 49 2.3 Results & Discussion ...... 49 2.3.1 Full length Nfs1 ...... 50 2.3.2. (1-31) Nfs1 ...... 54 2.3.3. (1-58) Nfs1 ...... 56 2.4 Conclusion and future work ...... 62

3. Frataxin 64

3.1 Introduction ...... 64 3.2 Experimental procedures ...... 66 3.2.1 Truncated Frataxin constructs...... 66 3.2.2 Expression and Purification ...... 67 3.2.3 Mature frataxin expression and purification ...... 68 3.2.4 Thrombin cleavage of mature frataxin ...... 69 3.2.5 Metal binding studies by ITC ...... 69 3.2.6 Ferroxidase assay: Oximetry ...... 70 3.3 Results & Discussion ...... 71 3.3.1 Purification ...... 71 3.3.1.1 Mature frataxin (mFtx) ...... 71 3.3.1.2 Truncated frataxin (trFtx) ...... 72 3.3.2 Metal binding ...... 74 xi

3.3.2.1 Mature frataxin (mFtx) ...... 74 3.3.2.2 Truncated frataxin (trFtx) ...... 78 3.3.2.3 Conclusion ...... 85 3.3.3 Oximetry ...... 85 3.4 Conclusion ...... 90

Bibliography ...... 91

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LIST OF TABLES

Table Page

1.1 Composition of ferritins ...... 10

2.1 ITC titrations between human Isu/TM IscU and TM Nfs ...... 39

2.2 Nfs1 constructs...... 41

3.1 ITC binding parameters from the HS-mFtx-NTH and thmFtx titrations with iron (II)...... 77

3.2 ITC binding parameters from the HS-trFtx-NTH and HS-trFtx-Ø titrations with iron (II), zinc (II), calcium (II) and magnesium (II)...... 81

3.3 Ferritin kinetic parameters for recombinant H-ferritin (rHF), ferritin (HLF) and horse spleen ferritin (HoSF) ...... 86

3.4 The effects of ligands on the rate of oxidation of Fe(II) by atmospheric oxygen .... 86

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LIST OF FIGURES

Figure Page

1.1 General features of reactive oxygen species ...... 2

1.2 Haber-Weiss Cycle ...... 3

1.3 Iron absorption by the enterocyte ...... 7

1.4 Chemical pathways used by ferritin to store iron in its cavity ...... 11

1.5 Gene regulation by IRE-IRP system for (a) ferritin and (b) transferrin receptor. .... 13

1.6 Different type of Fe-S clusters found in prokaryotes, archae and eukaryotes ...... 17

1.7 Crystal structure of human frataxin (residues 91-210) at 1.8 Å ...... 21

1.8 Vitamin B6 family ...... 26

1.9 Ribbon diagram of monomeric Thermotoga maritima NifS ...... 28

1.10 Mechanism of cysteine desulfuration ...... 30

1.11 Solution NMR structure of Haemophilus influenzae IscU ...... 32

1.12 ISC export machinery ...... 36

2.1 Analysis of purified HS-Nfs1-NTH ...... 52

2.2 Analysis of the dissociation between GroEL and Nfs1 ...... 53

2.3 Immunoblots of EATP, WSCN, and overexpressed GroEL-ES crude cell lysate with

anti-His6 tag (top) and anti-GroEL ...... 53

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2.4 Kyte-Doolittle hydropathy plot of human Nfs1 ...... 55

2.5 UV-visible absorbance spectra of purified (1-58) HS-Nfs1-CTH and (1-58) HS- Nfs1-NMBP...... 57

2.6 (1-58) HS-Nfs1-NTH absorbance profile evolution over 25 min, at RT, in presence of 0.5 mM Cysteine...... 58

2.7 Iron-sulfur reconstitution kinetic ...... 59

2.8 10% SDS-PAGE of (1-58) HS-Nfs1-NMBP the purification on amylose resin. .. 61

2.9 Digestion of (1-58) HS-Nfs1-NMBP by Factor Xa...... 62

3.1 Purification of mature frataxin on TALON column seen on 15% SDS-PAGE ...... 72

3.2 20% SDS-PAGE of purified trFtx-Ø ...... 73

3.3 15% SDS-PAGE of purified trFtx-NTH ...... 73

3.4 Titration of 100 M thrombin cleaved mFtx with 2 mM iron (II) ...... 75

3.5 100 M mFtx-NTH titrated with 3 mM iron (II) ...... 76

3.6 60 M trFtx- Ø titrated with 5 mM iron (II) ...... 79

3.7 60 M mFtx-NTH titrated with 10 mM iron (II) ...... 80

3.8 50 M mFtx-NTH titrated with 2.5 mM zinc (II) ...... 82

3.9 100 M mFtx-NTH titrated with 5 mM calcium...... 83

3.10 100 M mFtx-NTH titrated with 5 mM magnesium ...... 84

3.11 Measurement of dioxygen consumption for mature frataxin ...... 88

3.12 Measurement of dioxygen consumption for truncated frataxin...... 89 xv

ABBREVIATIONS

BSA: Bovine Serum Albumin

CTH: C-terminal His6 tag

DPD: N, N-dimethyl-p-phenylenediamine

DT: Dithionite

DTT: Dithiothreitol

EDTA: Ethylene Diamine Tetraacetic Acid

EPR: Electron Paramagnetic Resonance

EtOH: Ethanol

EXAFS: Extended X-ray Absorption Fine Structure

FPLC: Fast Protein Liquid Chromatography

HCl: Hydrochloric acid

Hepes: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HS: Homo sapiens

I-AEDANS: N-iodoacetyl-N’-(5-sulfo-1-naphthyl)ethylenediamine

IPTG: Isopropyl--D-thiogalactoside

IMAC: Immobilized Metal Affinity Chromatography

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LB: Luria-Bertani

MBP: Maltose Binding Protein

MeOH: Methanol nH2O: nanopure water

NMR: Nuclear Magnetic Resonance

NTH: N-terminal His6 tag

PCR: Polymerase Chain Reaction

PLP: Pyridoxal 5’-phosphate

SDM: Site Directed Mutagenesis

SDS-PAGE: Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis

SP: Schizosaccharomyces pombe

TCEP۰HCl: Tris(2-Carboxyethyl) phosphine Hydrochloride

TM: Thermotoga maritima

Tris: 2-amino-2-hydroxymethyl-1,3-propanediol

WT: Wildtype

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CHAPTER 1

IRON HOMEOSTASIS

Transition metals are toxic to the environment at high concentration. However, in trace amounts some are widely used in living organisms as cofactors [1]. Iron is such a metal and is essential to life except for a few strains of Lactobacillus [2]. It is predominantly found in heme, ferritin and iron-sulfur clusters [3].

Iron homeostasis has drawn attention because of its role in health and disease.

Indeed, an imbalance of body iron can ultimately lead to pathological conditions. One of the most common disorders is iron deficiency anemia, which affects over 30% of the world population and is prevalent in developing countries [4]. At the opposite end, there are iron overload disorders such as hemochromatosis and Friedreich’s ataxia [4]. In

Parkinson’s disease or Alzheimer’s disease, iron is believed to contribute to the pathogenesis [5]. Thus it is central to fully understand iron bioprocesses such as absorption, transport, regulation and utilization.

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1.1 Iron

1.1.1 Reactivity

Iron can easily change valence from +2 to +3 and vice versa. This ability can be finely tuned by a variety of ligands to offer electron transport proteins with a broad range of redox potentials, making iron an ideal redox prosthetic center [6]. As a result it plays a central role in numerous biological and cellular processes including electron transfer, oxygen transport and DNA synthesis [7]. However this chemical versatility enabling iron to toggle between ferrous and ferric states, paves the way for the generation of reactive oxygen species (ROS) (Figure 1.1) [8].

- - + - - + e .- e , 2 H e . e , H O2 O2 H2O2 OH H2O - 0.16 V 0.94 V 0.32 V 2.31 V

H+

OH-

Superoxide anion Hydrogen peroxide Hydroxyl radical Short half-life, 10-6 s (0.1 nM) Long half-life, 10-5 s (10 nM) Short half-life, 10-9 s (1 fM) Forms spontaneously, especially at the Easily passes through cellular membranes Derives from Fenton reaction mitochondrial level May generate other radical species Major known oxydant By-product of several redox reactions

Figure 1.1: General features of reactive oxygen species. Adapted from [8, 9].

Oxidative phosphorylation steadily generates toxic oxygen metabolites at low levels in cells and tissues. ROS actually modulate many physiological functions spanning inflammatory response, gene expression, and signal transduction [10, 11]. Up to 1% of the respiratory complex electron flow reduces oxygen to the superoxide anion which has

2 a short half life of 1 s and a concentration of 0.1 nM. This reaction is catalyzed by enzymes such as NADP(H) oxidases and xanthine oxidase, or by nonenzymatic redox- reactive compounds, like the semi-ubiquinone of the electron transport chain. Because superoxide anion is potentially toxic (Figure 1.2), superoxide dismutases (SODs) facilitate the dismutation of two superoxide anions into hydrogen peroxide and dioxygen.

-5 H2O2 which is the most stable (10 s) and abundant ROS (10 nM) of all, can freely diffuse through the mitochondrial membranes and so, can reach the cytosol. But its true power lies in its ability to react with reduced transition metals, via the Fenton reaction

(Figure 1.2), to produce the highly reactive hydroxyl radical. The latter’s effects are limited to its close environment because it has a very short half-life (1 ns) and a very low concentration of 1 fM [9]. Nonetheless, mitochondria have developed antioxidant defenses to minimize the impact of ROS; for instance, catalases and glutathione peroxidase convert hydrogen peroxide into water [12].

HO. + OH- Fe3+ O .- jlll 2 jlll llll Fenton llll llll reaction llll llll H O Fe2+ O llll 2 2 2

Figure 1.2: Haber-Weiss Cycle. The Fenton reaction is the left side of the cycle.

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According to Andreyev et al., “Oxidative stress is not the ROS generation per se but a spatiotemporal imbalance of ROS production and detoxification” [13]. Its impact on the cell metabolism is not necessarily negative. Indeed it can induce reversible as well as irreversible modifications, particularly on cysteine residues, to protect sensitive proteins from irreversible damage or to modulate protein function, a process known as redox regulation. However, irreversible modifications, like protein-protein cross-linking, di- tyrosine formation, lysine and arginine carbonylation or histidine oxidation, usually induce inactivation of the targeted protein [9]. Furthermore, they can lead to protein unfolding and degradation, or depositions of protein aggregates as observed in some neurological diseases. ROS, especially the hydroxyl radical, may also damage DNA by modifying the purine and pyrimidine bases, the deoxyribose backbone. It can also introduce single and double strand breaks as well as cross-links to other molecules [9].

Last but not least, lipid exposure to ROS often results in their peroxidation which ultimately affects respiration, oxidative phosphorylation, inner membrane barrier properties, maintenance of mitochondrial membrane potential and mitochondrial calcium buffering capacity [12].

Consequently, the human body has engineered many mechanisms to prevent a high concentration of cellular free iron under oxidative stress; thus iron is generally bound to storage or transport proteins.

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1.1.2. Distribution

In a normal human body, the amount of iron varies between 35 to 45 mg per kg body mass. But this amount greatly depends on gender, age, nutrition and health. Thus iron levels are usually lower in women due to smaller muscles, a reduced liver mass, and iron loss through menstruation [7]. Erythroid cells incorporate about two thirds of body iron into hemoglobin. 15% to 25% is stored in ferritin and haemosiderin. The remaining pool is shared between muscle myoglobin (~ 8%), reticuloendothelial macrophages, transferrin, cytochrome c, iron-containing enzymes and the labile iron pool (LIP). The exact composition of the LIP is unknown, but the presence of citrate, ascorbate, nucleotides, amino acids and pyrophosphates has been documented [14]. Humans do not possess specific mechanisms to excrete iron besides feces, sweat, urine and bleeding; most of the used iron is actually recycled [8]. Therefore absorption of iron is tightly regulated: only 1 to 2 mg of iron enters and leaves the body each day [4].

1.1.3 Uptake

Human acquires iron through food. Vegetables are a poor source of dietary inorganic iron because they contain phytates, polyphenols, and phosphates which tend to form insoluble complexes with iron and therefore limit its absorption. Since heme is readily absorbed, meat represents an excellent supply of iron [1].

Iron is mostly absorbed in the duodenum. A metalloporphyrin receptor on the brush border of intestinal cells, HCP1, takes up intact heme into the enterocytes [15], where

5 heme oxygenase decomposes it into free iron and biliverdin (Figure 1.3) [16]. The released iron then enters the labile iron pool. Non-heme iron is mainly in the ferric state in the gut lumen and needs to be first reduced to the ferrous ion by a ferrireductase

(DCYTB) at the apical surface of the brush border [17]. Next the divalent metal transporter 1, DMT1, achieves its transport in the enterocyte [18]. Inorganic iron then joins the LIP along with heme iron. From here iron is either stored in ferritin or is transferred into the portal blood by the iron exporter ferroportin, FPN [19]. The released metal ion is oxidized to the ferric state by hephaestin [20], and binds to transferrin, a major plasma protein that transports iron between the sites of absorption, storage, and utilization. Absorbed iron eventually ends up in the liver; more specifically in hepatocytes [4].

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Figure 1.3: Iron absorption by the enterocyte. Adapted from [4].

At least three mechanisms regulate iron absorption to meet daily needs. High iron ingestion triggers the first regulatory mechanism named mucosal block [21]. It immediately reduces dietary iron absorption for several days even though the body undergoes systemic iron deficiency. This is accomplished by rapidly downregulating the expression of DMT1 and DCYTB. Interestingly, ferroportin and hephaestin genes are not affected, signifying that the absorption is blocked at the apical surface of the enterocyte

[21]. However, this mechanism is not well understood. Increase in iron absorption occurs after a hemorrhage, when erythropoiesis is ineffective, or in case of anemia related illness. This leads to the idea of an existing erythropoietic regulator, which has not been

7 identified yet. It seems to involve a soluble signal directed from the bone marrow to the duodenum [22]. This second mechanism of regulation, known for about 15 years, remains poorly understood. The last regulatory mechanism senses the total body iron through the stores regulator. It changes the amount of absorbed iron based on the iron store level [22]. Two models confront each other in order to explain the store regulator mechanism: the crypt-programming model [23] and the liver hepcidin model [24], but none is satisfying.

1.1.4 Transport

Delivery of iron to different locations within the body through the plasma is handled by transferrin (Tf), an 80 kDa glycoprotein mainly synthesized by the liver, with small amounts produced by the brain, testis, and mammary gland. Tf is a bilobal protein which consists of a single polypeptide chain. Both lobes bind iron with high affinity (KD

= 10-23 M, pH 7.4) in a pH dependent manner [25]. Its binding extends to manganese, cobalt, copper, and cadmium with lower affinities. Coordination of iron involves tyrosine, aspartate, and histidine residues in both Tf binding sites. Transferrin is present in the plasma as a mixture of apotransferrin, monoferric transferrin, and diferric transferrin, depending on iron and tansferrin concentrations in the plasma. Tf concentration usually ranges from 22 to 35 M and is about 20% to 50% occupied by iron. The incomplete saturation of the binding sites of transferrin preserves against any

8 severe increase in plasma iron levels that may occur [26]. This is essential with regards to the toxicity of free iron.

1.1.5 Import

Internalization of diferric transferrin into cells takes place by binding with high affinity to specific Tf receptors (TfRs) on the outer face of the plasma membrane. TfRs are disulfide-linked homodimers of a 90kDa glycosylated polypeptide [27]. The short intracellular tail of TfRs directs rapid endocytosis of the Tf–TfR complex after ligand binding, mediating invagination of clathrin-coated pits and formation of endocytic vesicles. Once internalized, endosomes are acidified to pH 5.5–6.0 through the action of an ATP-dependent proton pump [28]. Endosomal acidification lowers the Tf binding affinity towards iron, and produces conformational changes in both Tf and TfR, strengthening their association. Then ferric iron is probably reduced to the ferrous state by a membrane-bound oxidoreductase that is yet to be identified. Iron is finally exported across the endosome membrane by DMT1 [29].

1.1.6 Storage

Absorbed iron in the duodenum inexorably ends up in the liver where it transits through the LIP prior to be storage into ferritin. A second role for the liver is to clear up the plasma of any iron excess. Even though hepatocytes are the main site of storage, any

9 cell possesses its own store made by ferritin, most likely to supply the metal for immediate needs and protect the environment against ferrous iron toxicity [4].

Human ferritin is composed of 24 polypeptide chains forming a cavity able to store up to 4500 atoms of ferric iron as ferrihydrite mineral (5 Fe2O3 •9 H2O) [1, 30]. Each subunit is composed of either two isoforms: H-chain (21 kDa) and L-chain (19 kDa). The proportion of H chains with respect to L chains depends on the type of organ; thus L-rich ferritins are found in liver and spleen (storage) which can house up to 1500 Fe/molecule,

H-rich ferritins are located in heart, red blood cells and brain, and only contain less than

1000 Fe/molecule (Table 1.1) [31].

Cell type Muscle Brain Lymphocytes Spleen Liver Serum H-Chain 20 16 10 4 2 0 L-Chain 4 8 14 20 22 24

Table 1.1: Composition of ferritins. Ratio of H-chain to L-chain in ferritin according to cell type. Adapted from [1].

Iron (II) reaches the buried ferroxidase centers in millisecond time frame through one of the eight pores of the ferritin cavity. The ferroxidase site, located on the H-chain, catalyzes the oxidation of iron (II) in the presence of oxygen to produce a diferroxo mineral precursor through a diferric peroxo intermediate (Figure 1.4, equation 1).When recombinant human H ferritin was incubated with ferrous iron at a ratio of 1:48, oxidation was 80% complete in 0.2 s and 99 % in 10 s [32]. Afterwards, hydrogen peroxide is released and the diferroxo precursor interacts with the L-chain to be 10 converted to hydrous ferric oxide and get to the ferritin core. Previously produced hydrogen peroxide is recycled by ferritin to keep oxidizing iron (II) and thereby detoxifying the cell environment (Figure 1.4, equation 2) [1, 33, 34]. Once the mineral core is sufficiently developed, ferrous iron undergoes autocatalytic oxidation at the surface of the core (Figure 1.4, equation 3) [1, 33, 34]. Cellular requirements dictate the release of iron stored in the inner core of ferritin through hydrophilic channels. This step is still obscure but it is thought to occur via oxidation of ferritin and then degradation by the 20S proteosome [35].

2+ 3+ 3+ 3+ 3+ 2 Fe(H2O)6 + O2 → Fe −O−O−Fe (H2O)2 → H2O2 + Fe −O−Fe → Mineral (1)

2+ 3+ 3+ 2 Fe + 2 H2O + H2O2 → Fe −O−Fe → Mineral (2)

2+ 3+ 3+ 4 Fe + 6 H2O + O2 → 2 Fe −O−Fe → Mineral (3)

Figure 1.4: Chemical pathways used by ferritin to store iron in its cavity.Adapted from [1, 33, 34]

Ferritin expression is regulated transcriptionally by cytokines, and post- transcriptionally by IRE-IRP system as described in the next section.

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1.1.7 Regulation of iron metabolism

As expected for a multi faceted metal such as iron, its homeostasis is tightly controlled. At least three levels of regulation have been elaborated by the cells, all being constantly active and sometimes targeting the same gene or protein; thereby unraveling an extraordinary complexity.

Several proteins implicated in iron metabolism are post-transcriptionaly regulated: an mRNA stem loop, called iron response element (IRE), is present in the untranslated region (UTR) of the protein’s mRNA. When iron is scarce, iron regulatory protein (IRP) loses its [4Fe-4S] cluster and is then able to bind to IRE. If IRE is located at the 5’-UTR as for ferritin and ferroportin, the binding would inhibit mRNA translation (Figure 1.5.a)

[36]. Conversely, if it is at the 3’ end as for transferrin receptor and DMT1, the IRE-IRP complex would stabilize the mRNA preventing its degradation from endonucleolytic cleavage and thus enhancing transcript and protein expression (Figure 1.5.b) [36].

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Figure 1.5: Gene regulation by IRE-IRP system for (a) ferritin and (b) transferrin receptor. –Fe indicate iron depleted cells and +Fe denotes iron replete cells. Adapted from [37].

Iron-related genes are transcriptionally regulated by iron levels, hypoxia and cytokines. It has been shown that DCYTB mRNA is noticeably increased in iron- deprived mice [17]. Similarly cytokines like interferon , interleukin 1 and interleukin 6,

13 modify H-ferritin, transferrin receptor, hepcidin, and ferroportin mRNA expression [38,

39]. Hypoxia also alters gene expression of transferrin receptor, transferrin, ceruloplasmin, DCYTB, ferroportin and hepcidin, by activating erythropoiesis [40].

Post-translational regulation has also been observed. High hepcidin concentration induces internalization of ferroportin leading to a decrease in cellular iron release [41]. In the same manner, high iron levels stimulate internalization of DMT1 in the duodenal enterocytes to cytoplasmic vesicles [42].

Many other proteins, such as the newly discovered hemochromatosis protein and hemojuvelin, participate in regulation of iron homeostasis. Although their action has not been firmly established, their discovery contributed to a better understanding of iron metabolism [4].

1.1.8 Iron-sulfur cluster cofactors

Besides heme proteins, iron-sulfur cluster proteins represent a major group of metalloproteins. The first characterizations came in the early 1960s [43]. In the 1970s Fe-

S cluster with different nuclearities were easily reconstituted in vitro by incubating the apo-protein with excess of inorganic sulfide, ferrous iron and DTT or 2-mercaptoethanol.

Those observations suggested that the maturation of iron sulfur cluster proteins could be a spontaneous process [43]. Due to the intrinsic toxicity of free iron and sulfide, dedicated proteins must mediate the assembly of Fe-S cluster. This lead to the identification of many proteins involved in the synthesis and maturation of Fe-S cluster

14 within the gene cluster of Azobacter vinelandii in 1989 [44]. Those proteins were dedicated to the assembly of the Fe-S cluster of nitrogenase which converts nitrogen to ammonia. This machinery was later called nitrogen fixing (NIF) because two more biosynthetic systems were identified in bacteria: sulfur utilization factor (SUF) and iron- sulfur cluster (ISC) [45]. The latter possesses a homolog, also termed ISC, in eukaryotes, which is interestingly found in mitochondria [46]. A cysteine desulfurase, an iron donor protein and a scaffolding protein are common to the three systems.

There are about 14 different Fe-S clusters. Up to now the mammalian NADH dehydrogenase is the biggest multi-Fe-S protein with 8-9 Fe-S clusters. The redox potential of Fe-S protein family spans a vast range of about 1 V [6]. Nonetheless with a single type of cluster, a protein is able to cover a wide array of potentials possibly by changing the local environment around the cluster. A second interesting feature is that sulfur exchanges with ease in Fe-S clusters as observed in both clusters of aconitase [47].

NMR and Mössbauer studies also showed that [2Fe-2S]+ fragments have mixed valence pair: there may be a jumping of the extra electron − that is abandoning one of the irons with which it was associated and jumping to one of the other irons, in this case in the lower layer − so that the mixed-valence pair can shift from one cluster face to another

[48].

Iron-sulfur cluster constitutes an excellent source of electrons as they are largely used in the respiratory chain [6]. Since electrons within an Fe-S cluster are delocalized, those cofactors can polarize their environment. This ability is illustrated in aconitase which converts citrate to isocitrate via the cis-aconitate formation. Citrate binds to the 15 iron sulfur cluster in the active site. Then one of the four irons which does not have any cysteine ligand acts as a Lewis acid by abstracting the hydroxyl group and a proton on the contiguous carbon [49]. Furthermore [4Fe-4S] have been recently observed to serve as electron donors in free radical reactions such as pyruvate formate lyase, lysine amino mutase or biotin synthase [6].

Nature has in addition taken advantage of the facile oxidation of Fe-S clusters that leads to their degradation for sensing and signaling as illustrated with the IRE/IRP system or in oxygen sensing [50].

In summary Fe-S-containing proteins are ubiquitous co-factors which participate in many cellular processes, for instance DNA synthesis, oxidative phosphorylation, DNA damage recognition and repair, oxygen/nitrogen sensing, and control of labile iron pool.

16

Figure 1.6: Different types of Fe-S clusters found prokaryotes, archae and eukaryotes. Adapted from [51].

[3Fe-4S] clusters where the fourth metal position is occupied by different transition metals (Mn, Co, Ni, Cu, Zn, Cd, Tl, Mo, V and Re) have been created and their properties analyzed in vitro. However such clusters have never been reported in nature

17 even though the affinity of zinc for the vacant position in [3Fe-4S] cluster is higher than that of iron. So one can ask why iron has been chosen amid all the transition metals [52].

In the cytoplasm, competing metals are bound to specific ligands which minimize their bioavailability compared to iron. Also the electronic resonance stabilization enhances the binding of iron in iron-sulfur clusters [53]. This question was also tackled by density functional calculation where the inner-sphere reorganization energy was estimated for 1Fe and 2Fe type iron-sulfur clusters and opposed to metal substituted analogues of Mn, Co, Ni, and Cu: results showed a value of 21 kJ/mol for 1Fe

(rubredoxin), 46 kJ/mol in 2Fe (ferredoxin), and 57-140 kJ/mol other metals clusters herein providing an interesting insight on why nature has chosen iron for those clusters

[54].

Nature certainly chose sulfur in such cluster because of its great flexibility. On one hand, sulfur is big enough to be able to populate 3d orbitals and has less inclination at forming hydrogen bonds, which marks it out from oxygen; on the other hand sulfur is small enough so that the effects of nuclear charge is not overpowering as it is in selenium and in Tellurium [6]. Moreover sulfur can assume many oxidation states, from -2 to +6.

In other words it is as susceptible to give off electrons to reach the neon configuration as to gain electrons to attain the argon configuration [6]. Also sulfur needs a very small amount of energy to switch between being a nucleophile (e.g. as in reduced cysteine), and being an electrophile (e.g. as in disulfide linkage): sulfur is able to form and break covalent bonds very easily.

18

1.2 Iron – Sulfur cluster biogenesis

1.2.1 Iron import in mitochondria

Ferrous iron crosses the inner mitochondrial membrane through two carrier proteins

Mrs3 and Mrs4. This process relies on the establishment of a membrane potential but does not require ATP [55]. Those proteins were initially identified as suppressors of mitochondrial RNA splicing defects in yeast and later in human [56]. Deletion of both genes lead to unusual elevated cellular iron uptake, iron content and increase sensitivity to reactive oxygen species [57, 58]. Further studies in isolated mitochondria and in vivo demonstrated a clear connection between the expression levels of these transporters and the efficiency of mitochondrial Fe/S proteins and heme biosynthesis [59]. However an alternative pathway to import iron might exist since the double deletion of MRS3 and

MRS4 genes is associated with mild phenotype [59].

Many questions remain open such as whether these proteins act as iron transporters or regulators and also whether iron is transported as a free or chelated metal. Their cognate partners are still unkown even though frataxin is a serious candidate.

1.2.2 Frataxin

Deficiency in human frataxin causes Friedreich’s ataxia (FRDA), an autosomal degenerative disorder characterized by limb ataxia, sensory loss, hypertrophic cardiomyopathy and diabetes [60]. Those symptoms usually result in confinement in a wheelchair and death in middle age [61]. Cell lines from patients show mitochondrial

19 iron accumulation, loss of Fe/S cluster-containing enzymes, reduce oxidative phosphorylation and increased oxidative damage [62]. Damages to the cells are thought to occur via Fenton reaction because of free labile iron. Supporting this theory in vitro data showed that idebinone protects heart muscle from iron-induced injury [62].

The majority of the patients have a GAA triplet repeat expansion (>120 repeats) in the first intron of the frataxin gene located on chromosome 9q13 in both alleles [63]. This expansion forms a triple helix non-B DNA structure which inhibits the transcription of frataxin mRNA [64]. Few patients are compound heterozygotes for the GAA expansion, or possess point mutations with typically the same phenotype [65].

Frataxin is nuclearly encoded as a 210 amino acid polypeptide precursor which contains a mitochondrial targeting sequence at the N-terminus [63]. Once transported within the mitochondrial matrix, the precursor undergoes a two-step cleavage to produce the mature frataxin (mFtx). Initial reports about mitochondrial processing of frataxin were based on the interaction between mouse frataxin preprotein and mouse mitochondrial processing peptidase (MPP) [66]. Those observations were completed by studying the two sequential cleavages of recombinant yeast MPP/rat MPP on human frataxin in vitro. The first cleavage was located between Gly41 and Leu42 producing an intermediate form of 19kDa; the second one was between Ala55 and Ser56 giving the mature form lacking the first 55 amino acids [67]. This product is prone to degradation to lead to a truncated form of 14 kDa (trFtx). Later experiments were performed in vivo with the frataxin precursor overexpressed in human cells along with mutants. This study revealed that the major form of mature frataxin was with a cleavage site between Lys80 20 and Ser81, which was obviously fully active. Indeed it was able to rescue aconitase defects in frataxin deficient cells. Furthermore those data suggested that the upstream site

55-56 is used in vivo when the 80-81 site is not available [68].

An important piece of information about the biological function of frataxin came from the crystal structure of its residues 88-210 at 1.8 Å resolution, which revealed a new protein fold. Overall frataxin looks like a compact  sandwich [69]. The secondary structure is made of two -helices (1 and 2) which encompass seven -strands (1 to

7). 1 and 2 are parallel with each other and supported by an antiparallel  sheet (1 to 7) [69]. Twelve solvent exposed acidic residues located on the 1 and 1 (Figure 1.5) form a contiguous anionic surface on the protein which is thought to participate in iron binding. Those features are well conserved from eukaryotes to prokaryotes [69].

D124 D122 E92 E96 E100 E111 E121 D104

N E101 D115 D112 E108

C

Figure 1.7: Crystal structure of human frataxin (residues 91-210) at 1.8 Å. Adapted from [69]. 21

As expected from its structure, truncated human frataxin has been reported to bind seven iron (II) with an apparent Kd of 55 M and six iron (III) with a KD of 10.2 M

[70]. Additional experiments have demonstrated by fluorescence quenching and ITC measurements that one holo frataxin forms a complex with one Isu1 with submicromolar binding constant. Those results suggested that frataxin was the iron donor involved in the

ISC machinery. This hypothesis was confirmed by following the increase velocity of Fe-

S cluster assembly on apo Isu1 in presence of frataxin compared with the velocity without frataxin [70]. Frataxin also mediates the delivery of iron to ferrochelatase in the last step of heme synthesis which occurs within the mitochondrion. Ferrochelatase catalyzes the insertion of iron (II) into the protoporphyrin IX to form heme. Calorimetric studies, fluorescence quenching and kinetic experiments indicated that frataxin binds to two ferrochelatase with an affinity of 17 nM [71]. Maping of iron coordination and ferrochelatase binding surface in presence of frataxin by NMR provided even more convincing data on frataxin role [72]. A third role attributed to frataxin is to store iron as ferritin protein [73, 74]. This was an appealing model until the discovery of a mitochondrial ferritin, discussed in the next section. Other functions of frataxin include iron chaperone delivery to aconitase and DNA protection against oxidative damage [75].

22

1.2.3 Mitochondrial Ferritin

First described in 2001 mitochondrial human ferritin (mtFt) is encoded by an intronless gene located on chromosome 5q23. It encodes a 30 kDa precursor that is processed to a 22 kDa polypeptide upon import within the mitochondrion [76]. mtFt is essentially expressed in testis, neuronal cells, and islets of Langherans, but none was detected in iron storage organs such as liver or spleen. Conversely expression of cytosolic ferritin is not tissue specific [77]. Gene expression does not appear to be under control of the iron response element.

The mature mtFt has a high sequence homology with human H-ferritin with similar di iron ferroxidase centers. It also forms a homopolymer of 24 subunits [78]. To the contrary of human H-chain ferritin (HuHF), its ferroxidation is slower and its mineralization activity greatly ressembles to L-chain ferritin (HuLF) [79]. mtFt binds and oxidizes only 24 iron (II) signifying that half the ferroxidase centers is active, compared to HuHF. Furthermore mtFt displays sigmoidal kinetics of mineralization which is characteristic for an autocatalytic mineral surface mechanism as observed for HuLF [79].

Also mtFt produces small amounts of hydroxyl radical when oxidizing ferrous iron in presence of oxygen, and the detoxification reaction (Figure 1.4 equation 2) seen in cytosolic ferritin, lacks in mtFt [79]. Drysdale et al. showed in vitro with HeLa cells transfected with mtFt that mtFt incorporates iron as quickly as cytosolic ferritin does

[76]. Nie et al. demonstrated that the overexpression of mtFt in cells revealed an increase

IRP activity, and increase in transferrin receptor levels and decrease in cytosolic ferritin

RNA[80]. Also iron uptake was greatly increased, cytosolic ferritin iron was depleted and 23 most of the cellular iron was found into mtFt. Additionally iron from mtFt was less available to chelation than iron from cytosolic ferritin. All those results taken together indicate that mtFt is voracious of iron and could explain why levels of mtFt are kept so low within the cells. Moreover high expression of mtFt disturbs heme synthesis and compromises some non-heme proteins [80]. Therefore mtFt expression must be well regulated to ensure an adequate supply of iron to the mitochondrion.

The discovery of the mitochondrial ferritin provided more information on sideroblastic anemia. In patient erythroid cells, mtFt is highly expressed whereas healthy cells its expression is low. This high expression leads to the formation of erythroblast mitochondria ("ringed sideroblasts"); and it was shown that the iron in ringed sideroblasts is in mtFt [81] opening the way for new diagnosis tools.

In Friedreich’s ataxia, frataxin expression is highly reduced. This is thought to initiate iron accumulation in mitochondria of non-erythroid cells and thus increased oxidative damage. Affected cells are mainly neurons and cardiomyocytes which have no intense heme synthesis. Because those cells need iron to synthesize only iron-sulfur clusters, Napier et al. proposed that the excess of free iron was actively stocked in mtFt.

As a result mtFt would protect the cell from the deleterious effect of free ferrous iron, and would explain the observed delay in the pathogenesis [82].

24

1.2.4 Cysteine desulfurase Nfs1

Nfs1 is a pyridoxal 5’-phosphate (PLP) dependent -lyase. It decomposes cysteine to alanine and forms an internal persulfide bond with a conserved internal cysteine residue [83].

1.2.4.1 Sulfide toxicity and activation

At physiological pH, a third of sulfide exists as hydrogen sulfide, H2S, which is highly toxic to the cell [84]. Even though its reactivity resembles hydrogen cyanide, its cytoxicity is far different. Hydrogen sulfide is able to deplete glutathione, reduce intracellular bound ferric iron to produce unbound ferrous iron, and activate oxygen to form ROS. Ultimately this can lead to paralysis of the nervous and respiratory systems causing death [85]. Therefore, cells keep the concentration of free sulfide at about 90 M to allow cysteine synthesis [86]. Similarly, cysteine concentration falls in 100-200 M range because of its cellular toxicity as a free amino acid [87]. Thus, cells must use an activated form of sulfur for biosynthesis of cofactors such as biotin or glutathione. The characterization of the cysteine desulfurase NifS from A. vinelandii in 1993, provided strong evidence that sulfane sulfur, i.e. S0, in persulfide bond is the activated sulfur [88].

Since then, many enzymes have been identified using this kind of reactive sulfur.

25

1.2.4.2 PLP cofactor

PLP is a derivative of vitamin B6 which includes pyridoxamine 5’-phosphate and pyridoxine 5’-phosphate (Figure 1.6). Enzymes containing a vitamin B6 prosthetic group are conveniently referred as “B6 enzymes”. B6 enzymes acting on amino acid substrates are found in five classes of enzyme among the six as established by the Enzyme

Nomenclature Committee of the International Union of Biochemistry and Molecular

Biology [89]. All those enzymes share important mechanistic features: PLP is covalently bound to the -amino group of an internal conserved lysine residue, forming an internal aldimine. They also share the formation of an external aldimine when the -amino group of the substrate replaces the lysine [90]. B6 enzymes are subdivided into four families of paralogous proteins based on the available structural information: the  family, the  family, the D-alanine aminotransferase family, and the alanine racemase family. Nfs1 belongs to the former group and most likely shares the same overall fold of the family members as do its bacterial (IscS) and archae (NifS) counterparts [89].

Figure 1.8: Vitamin B6 family. Adapted from [91].

26

1.2.4.3 Mechanism

Only the crystal structures of E. coli IscS and its homolog Thermotoga maritima

NifS have been solved. They both appeared as homodimer of about 45 kDa. Although they have 40% sequence identity, their overall fold is similar as expected for two proteins belonging to the -family of PLP enzymes. Also in both polypeptides, PLP is covalently bound to a conserved lysine residue (K206 for E. coli, K203 for T. maritima), forming an internal aldimine [92].

T. maritima structure can be divided in two domains (Figure 1.9). The large domain comprises seven parallel -sheets (S2, S9, S8, S7, S5, S3, S4). This cluster is surrounded by helices H5 and H6 on the solvent exposed side, and, H3 and H4 which contain most of the residues involved in the dimer interface. Hydrophobic forces and hydrogen bonds dominate the interaction between the two dimers. The turn connecting H7 to S9 harbors the lysine 203 and so the PLP cofactor (Figure 1.9). Glutamate 257 forces H7 to make a

90° kink, and delimits the boundary between the large and small domains [93]. The small domain includes two parallel -sheets (S1, S12) and four antiparallel -sheets (S10, S13,

S11, S12). The latter is engaged in the formation of the active site. The twelve residues including the active cysteine 324, following S12, are unfortunately located in a disordered region [93]. However this region is resolved in E. coli IscS showing the active cysteine to be about 17 Å apart from the active site [92].

27

Figure 1.9: Ribbon diagram of monomeric Thermotoga maritima NifS. The last defined residues neighbouring the active site loop are represented with blue balls, and the internal aldimine Lys103-PLP is shown as ball and stick model. H: -helix; S: -strand. From [93].

The mechanism of desulfuration depicted on figure 1.10, is based on the crystal structure of T. maritima NifS and on biochemical data obtained for A. vinelandii NifS.

Once in the active site, the amino group of the cysteine substrate (1) conducts a nucleophilic attack on the C4’ of PLP to form an external aldimine (2). Next the lysine

203 abstracts the C proton of the substrate giving a quinonoid ketimine intermediate (3).

The ketimine is then protonated by the histine 99 (4) which in turn activate cysteine 325

(5). The nucleophile thiolate attacks the S of the substrate giving off the acrylic

28 intermediate (6). Then protonation of the C by the His99 occurs, leading to the ketimine intermediate (7). The ketimine C is now basic enough to deprotonate Lys203 (8) which now can attack the C4’ of PLP to release alanine and regenerate the internal aldimine (9).

29

(9) (1)

(2) (8)

(3) (7)

(4) (5) (6)

Figure 1.10: Proposed mechanism of Thermotoga maritima NifS. Adapted from [93].

30

1.2.4.3 Interacting partners

Because of its toxicity, free sulfide is unlikely to be transferred to the scaffold protein Isu1. Instead, Nfs1 is expected to hand it over directly from the persulfide formed on the cysteine residue to an accepting cysteine residue on Isu. Complex formation between bacterial Isu1 and bacterial Nfs1 has been observed both in vitro and in vivo, and therefore support this hypothesis [94-96]. But the transfer mechanism is still unclear: this could occur via the formation of a heterodisulfide bridged intermediate between Nfs1 and

Isu1 [97], or via the transpersulfuration as observed in SUF system [98]. A conserved cysteine on SufE binds to the sulfur released from SufS to form an intermediate persulfide bond before its transfer to the scaffold SufA [98].

However, only in eukaryotic ISC machinery, Nfs1 is associated with a small protein Isd11 to from a complex of about 200 kDa [99]. Furthermore, Nfs1 is prone to aggregation and degradation in the absence of Isd11. However the presence of Isd11 is not required for Nfs1 activity. Depletion of Isd11 greatly reduces the levels of aconitase proteins and Rieske protein, and impairs the formation of iron-sulfur clusters especially on Isu1 supporting an important role for Isd11 in Fe-S biogenesis [99, 100]. Isd11 is also thought to mediate the interaction between Isu1, Nfs1 and frataxin for iron-sulfur cluster assembly [101]. It is tempting to assume that Isd11 could be the counterpart of SufE in the ISC machinery, but this idea should be rejected as Isd11 does not contain any cysteine residue [102]. More studies are needed to address the exact mechanism of sulfide transfer.

31

Besides the mitochondrion, Nfs1 has been localized in the nucleus where it participates in thiouridine modification of tRNA but this process is still under investigation [103, 104].

1.2.5 Isu1: the scaffold protein

Isu1 is the recipient of the sulfide from Nfs1 [46, 105] and the iron from frataxin

[70], and thus serves as a molecular scaffold for iron-sulfur clusters. Among the most conserved proteins in evolution, those dimeric proteins are small, about 15 kDa. They share a compact core with an - sandwich architecture: three-stranded antiparallel - sheet and four -helices (Figure 1.6).

Figure 1.11: Solution NMR structure of Haemophilus influenzae IscU. Cysteine residues have been highlighted. Adapted from [106]

32

Three conserved cysteine residues are involved in the cluster formation and are partially or fully solvent exposed, near the surface [106, 107]. Indeed, they are close enough to each other to serve as ligand for iron and iron-sulfur clusters. Formation of one

[2Fe-2S]2+ cluster per monomer has been observed in vitro by incubating Isu from human, bacteria or yeast with inorganic sulfide and ferrous iron [45]. Isu1 homolog from

A. vinelandii was even able to assemble a [4Fe-4S]2+ cluster under reductive coupling

[108]. Subsequently, in vitro experiments have provided proofs of intact direct cluster transfer between Isu1 and a possible cognate partner, ferredoxin [109, 110].

But many open questions remain about the physiological nature of the cluster that is transferred to targeted proteins, and the specifity of the transfer to the interacting partners. Also the mechanism of cluster assembly has not been elucidated, biochemical experiments have not deciphered yet whether sulfide or iron comes first.

1.2.6 Other scaffold proteins

The mitochondrial human protein Nfu1 has been reported to bind a labile iron- sulfur cluster in vitro [111] but has eluded scientists in vivo so far. In yeast, NFU1 deletion has no effect on the phenotype whereas deletion along with other genes such as

ISU1 or SSQ1, is lethal, suggesting a secondary role in Fe-S biosynthesis [112]. Recently biochemical data points out that human Nfu1 might actually be mediating the sulfide transfer between Nfs1 and Isu1 in a [2Fe-2S] synthesis [113]. Interestingly, deletion of

Nfu1 homolog, NifU, in cyanobacteria is lethal [114], whereas in Arabidopsis thaliana, it

33 results in slow growth and impairment of chloroplast specific Fe-S proteins [115]. In vitro, isolated NifU assembles Fe-S cluster and subsequently transfers it to only apoferredoxin [115]. But sequence homology is low between Nfu1 and its homologs [46].

Therefore, the exact function of Nfu1 still has to be elucidated.

Humans also own an ortholog of both yeast ISA1 and ISA2 [116]. In gene deletion experiments in Saccharomyces cerevisiae, maturation of several Fe-S proteins was defective but not aggravated by the double deletion (Mühlenhoff, Ulrich, unpublished data). Worth noticing is the presence of three conserved cysteines that are essential for protein activity in vivo. Schizosaccharomyces pombe Isa1 binds a [2Fe-2S] cluster in vitro [117], whereas Saccharomyces cerevisiae Isa1 and Isa2 bind only iron in vivo

(Mühlenhoff, unpublished data). Furthermore the latter two proteins are also required for the biosynthesis of aconitase-like proteins [46]. On the other hand, bacteria homolog,

IscA, has been observed to bind iron and iron-sulfur clusters in vitro [118]. The crystal structures of many apo IscA did not help deciphering the exact role of this protein: iron donor or scaffold protein [119-121]. Much work is needed to decode Isa role in iron- sulfur clusters biogenesis.

1.2.7 Molecular chaperones

Genetic and biochemical studies have identified an essential chaperone system involved in the maturation of iron-sulfur cluster protein maturation. It comprises an

Hsp70 chaperone and a J-type protein co-chaperone: HscA/HscB in bacteria, Ssq1/Jac1

34 in yeast and mtHsp70/homolog to HscB/Jac1 in higher eukaryotes [122]. Furthermore in yeast, an ADP-ATP nucleotide exchange factor, Mge, plays a central role by enhancing the release of ADP from Ssq1. This reaction is the rate limiting step for Ssq1 [123].

The only reported substrate for Ssq1/HscA is the scaffold protein Isu1/IscU. Both form a complex through the interaction with a conserved recognition motif (LPPVK), located on a solvent exposed loop within Isu1 [124]. The crystal structure of HscA in presence of the peptide has been solved. The motif binds to a solvent accessible hydrophobic gap in the  subdomain of HscA [125]. Thus the mechanism is as follow:

[Fe-S] Isu1 binds the ATP loaded Hsp70, then ATP undergoes hydrolysis which in turn stabilizes the interaction, eventually Isu1 releases its substrate to target partner, and ADP is exchanged to ATP favoring complex breakup. HscB stimulates HscA ATPase activity by involving its N-terminal J-domain while its C-terminal domain binds to IscU [126].

In vivo, mutation in the LPPVK motif did not perturb iron-sulfur cluster formation on Isu but annihilated Fe-S cluster transfer to apoproteins. Those findings constrast with in vitro studies that support a Fe-S transfer without the presence of chaperones [124].

Therefore more biophysical data are needed to solve this discrepancy and to extent those conclusions to mtHsp70.

35

1.2.8 Export machinery.

In 1995, the first ABC transporter in mitochondria, Atm1, was identified in yeast by the polymerase chain reaction. It was shown to localize in the mitochondrial inner membrane with its C-terminal ATPase domain facing the matrix [127]. Furthermore, in

Atm1 depleted yeast, iron accumulates in mitochondria, and the maturation of iron-sulfur proteins is impaired only in the cytosol and the nucleus. Therefore Atm1 acts as an exporter involved in the transport of iron-sulfur clusters (Figure 1.12). Its deletion was not lethal, and Mdl1, an ABC peptide exporter, could partially rescue the function of

Atm1 in ATM1 null yeast [128]. Defects in its human homolog, ABCB7, is responsible for the X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A) whose phenotype closely resembles to Atm1 deleted strain [129].

Figure 1.12: ISC export machinery. Adapted from [46].

36

The purified yeast Atm1 has typical kinetic parameter for an ABC transporter, a Km

-1 of 0.1 mM and a kcat of 2 s . Interestingly, its activity was stimulated by peptides containing cysteine residues and in general by thiol compounds [130]. Also, upon ATP binding, the transporter dimerizes [131]. Since Atm1 is a member of the peptide- translocating ABC transporters along with Mdl1, its natural substrate could be a sulfur containing peptide or a small protein bound with iron-sulfur cluster. Consistent with this assumption is the second protein, Erv1, which is a sulfhydryl oxidase situated in the intermembrane space [132]. This dimeric protein shuttles electrons into cytochrome c and introduces disulfide bonds into cognate proteins such as Mia40 involved in import and folding of TIM proteins [133]. Erv1 was identified as essential for cytosolic Fe-S cluster assembly in 2001 [134]. Its human counterpart, ALR, can functionally substitute the yeast protein showing the conservation of export mechanism among eukaryotes [134]. In addition it was reported that glutathione (GSH) depletion in yeast would provoke the same phenotype as in Atm1 null and Erv1 null strains even in presence of DTT or under anaerobic conditions; thereby GSH is a valid player in ISC export machinery [135].

Additional studies are necessary to understand the interaction between the different actors and to determine the physiological substrate of Atm1 and Erv1.

37

CHAPTER 2

STUDIES ON HOMO SAPIENS CYSTEINE DESULFURASE

2.1 Introduction

Nfs1 is a PLP-based cysteine desulfurase which provides sulfide for iron-sulfur cluster biosynthesis on the scaffold protein Isu1. It catalyzes the removal of sulfur from cysteine, releasing alanine and forming a persulfide bond with a conserved internal cysteine residue. This simplistic picture is firstly complicated by the need for electrons in order to release the sulfane: S0 to S2-. This role might be fulfilled by ferredoxin reductase,

Arh1, and ferredoxin, directing electrons from NADH to Nfs1 [46]. Indeed their depletion impairs the maturation of Fe/S proteins [136]. However, alternatively Liu &

Cowan proposed Nfu1 as the provider of electrons [113] although its deletion hardly affects iron-sulfur cluster assembly [112]. In eukaryotes, an additional protein, Isd11, is present as a direct partner of Nfs1. It prevents Nfs1 aggregation and degradation, but barely affects Nfs1 activity [99]. Isd11 interacts with Isu1 and frataxin [100, 101] apparently facilitating Fe/S synthesis in vivo.

38

Furthermore, the role of Nfs1 goes beyond iron sulfur cluster biosynthesis [46] and extends to thionucleosides synthesis [103, 137] and could participate in thiamin and

NAD synthesis as does its bacterial counterpart E. coli IscS [138, 139], thereby acting a general sulfur donor.

Most of the available data is from studies on yeast, fungi, bacteria and archaea. A few are available for higher eukaryotes such as human or mouse. Even though, the ISC system is highly conserved in evolution, differences have already arisen and more could come. However previous ITC work performed by Dr. M. Nuth showed that HS-Isu1 and

TM-IscU could bind with the similar affinity to TM-NifS (Table 2.1) (unpublished data).

Table 2.1: ITC titrations between human Isu/TM IscU and TM Nfs. The table summarizes ITC titrations conducted by Dr. Manunya Nuth between human Isu/TM IscU and TM Nfs. Experiments were conducted at 25 °C in 50 mM HEPES, pH 7.5 supplemented with 1 mM TCEP. Typically, 30 M TM-NifS-Ø were titrated with 29 injections of 10 M 1.2 – 1.5 mM Isu/IscU solution spaced by 6 min at 300 rpm. Deconvoluted data were fitted to a one-site model with the included Origin 7 software to obtain the thermodynamic parameters. (Manunya, Nuth, unpublished data).

Since additional experiments would require large amount of protein Nfs1, and to date no E. coli system has been designed for overexpression of human cysteine desulfurase, only the cumbersome yeast expression system has been established without

39 very good yield [140]. Therefore the objective of this work was to clone, isolate, characterize Homo sapiens Nfs1 and then study its interaction with cognate partners

(human Isu, TM IscU and maybe Isd11) by ITC.

2.2 Experimental Procedures

2.2.1 Nfs1 constructs

Nfs1 gene was cloned by the polymerase chain reaction using a human heart cDNA library, the 5’ primer 5’-CTA GCT AGC ATG CTG CTC CGA GTC GCT TGG A-3’ the

3’ primer 5’-GGA ATT CCT AGT GTT GGG TCC ACT TGA TGC TCT-3’ and Taq polymerase. The PCR product was digested with NheI and EcoRI and subsequently ligated in either pET 21b(+) or pET 28b(+). Chemically competent E. coli DH5 cells were transformed with the ligation products. Next, plasmid DNA was isolated by miniprep on bacterial cells grown from single colony and sequenced.

Nine other clones were generated the same way as described above by using different primers to delete residues at the N-terminus either in the pET vectors mentioned above or in pPIC9, pMAL-p2x and pMAL-c2x (Table 2.2).

40

Table 2.2: Nfs1 constructs. The first column indicates the number of amino acids that have been deleted at the N-terminus. The second column shows which vector has been used for expression. The third column indicated the restriction sites for gene insertion in the chosen vector. The fourth column indicates the tag attached to the protein: N-terminal His6 tag (NTH), C-terminal His6 tag (CTH), N-terminal MBP (NMBP), and no tag (Ø). The fifth column shows the primers used for PCR.

2.2.2 Expression & Purification

Miniprep DNAs were used to transform chemically competent E. coli BL21-

CodonPlus (DE3)-RIL, BL21(DE3) GroEL-ES, and Rosetta pLys. Cells were grown in

LB medium supplemented with the adequate antibiotic and 2 g/L of glucose in the case of the pMAL vector, at 37 °C and 220 rpm. Expression of the protein was induced with 100

M IPTG and 1 mM PLP when the OD600 had reached about 0.6. The strain containing the GroEL-ES plasmid also required 0.5 g/L of L-arabinose and 5 g/L of tetracycline.

The cells were then incubated for 4 to 16 hours at 30 °C or 15-18 °C. After induction,

41 cells were harvested by centrifugation at 5,000 rpm and 4 °C for 10 min. The cell pellet was then stored at -80 °C and used within a week.

2.2.2.1 Full length and (1-31) HS-Nfs1

These proteins formed inclusion bodies when overexpressed with or without His6 tag independent of the induction temperature. The frozen cell pellet was resuspended in buffer C (50 mM Tris, 25% sucrose, 1 mM EDTA, 10 mM DTT, 5 mM imidazole, pH

8.0). The mixture was then sonicated for 45 min (10 s pulse / 2 min at 50% output) on ice. Then lysozyme, DNase and MgCl2 were added. The mixture was vortexed for 20 s and an equal volume of buffer D was added (50 mM Tris, 1% Triton X-100, 1% deoxycholate, 100 mM NaCl, and 10 mM DTT, pH 8.0). Again the mixture was vortexed for 20 s and incubated at RT for 60 min. 350 L 0.5 M EDTA, pH 8.0, were then added and the mixture was frozen in liquid nitrogen. Then it was thawed in a water bath for 30 min at 37 °C. A 200 L volume of 0.5 M MgCl2 was added and another 60 min was used to let the viscosity decrease. Again 350 L 0.5 M EDTA, pH 8.0, were added and the pellet was centrifuged 20 min at 14,000 rpm and 4 °C. The supernatant was discarded and the pellet was resuspended by sonication in buffer E (50 mM Tris, 0.5% Triton X-100,

100 mM NaCl, 1 mM EDTA and 10 mM DTT, pH 8.0). Again the mixture was centrifuged 20 min at 12,000 rpm and 4 °C. The supernatant was discarded and the pellet was resuspended by sonication in buffer F (50 mM Tris, 100 mM NaCl, 1 mM EDTA

42 and 10 mM DTT, pH 8.0). Again the mixture was centrifuged for 20 min at 10,000 rpm and 4 °C.

The inclusion bodies were dissolved in a buffer containing 50 mM Tris, 2 M Gnd-

HCl, pH 11.0. Sonication was used to help solubilizing the inclusion bodies. Refolding was performed by adding protein solution dropwise to refolding buffer (50 mM Tris, 400 mM L-Arginine, 2 mM EDTA, 5% sucrose, 10% glycerol, 1 mM PLP, 5 mM DTT, pH

11) with constant stirring at 4 °C. This resulted in a dilution of 10-fold of the protein solution. Afterward the protein was concentrated with an Amicon equipped with a

YM30K membrane and finally dialyzed against the desired buffer, depending on the experiment.

2.2.2.2 (1-58) HS-Nfs1

Clones harboring a His6 tag were purified on Ni-NTA resin. Frozen cell pellet was resuspended in buffer A (50 mM phosphate, 500 mM NaCl, 5 mM imidazole, pH 8.0) supplemented with 75 ng / mL lysozyme, 1 mM TCEP, 0.1% Triton X-100, DNase. The mixture was then sonicated for 30 min (10 s pulse / 2 min at 80% output) on ice. The lysate was then centrifuged at 15,000 rpm and 4 °C for 30 min. The supernatant was then loaded on a pre-equilibrated resin. The loaded resin was then washed with 20 column volumes. Elution was performed with buffer A supplemented with 400 mM imidazole.

MBP fusion proteins were purified on amylose resin. Frozen cell pellet was resuspended in buffer B (100 mM Pi, 200 mM NaCl, pH 7.4) supplemented with DTT

43 and EDTA. The mixture was then sonicated for 45 min (10 s pulse / 2 min at 80% output) on ice. The lysate was then centrifuged at 15,000 rpm and 4 °C for 30 min. The supernatant was then loaded on pre-equilibrated resin. The loaded resin was then washed with 20 column volumes. Elution was performed with buffer B supplemented with 10 mM maltose.

Protein purity was checked by SDS-PAGE and concentration determined by absorbance peak at 280 nm and / or by the Bio-Rad protein assay kit.

2.2.3 Mass Spectrometry

2.2.3.1 In Gel Digestion

Gels were digested with sequencing grade trypsin from Promega (Madison WI) using the Montage In-Gel Digestion Kit from Millipore (Bedford, MA) following the manufacturers recommended protocols. Briefly, bands were trimmed as close as possible to minimize background polyacrylamide material. The gels were washed in 50% methanol/5% acetic acid for several hours. The gel bands were dried with acetonitrile are reconstituted with dithiothreitol (DTT) solution to reduce the cysteines. Iodoacetamide was added to alkylate the cysteines and the gel was washed again with cycles of acetonitrile and ammonium bicarbonate. Trypsin was added and digested at room temperature overnight. The peptides were extracted from the polyacrylamide gel with

50% acetonitrile and 5% formic acid several times and pooled together. The extracted pools were concentrated in a speed vac to ~25 mL.

44

2.2.3.2 Protein Identification

Capillary liquid-chromatography nanospray tandem mass spectrometry (Nano-

LC/MS/MS) was performed on a Thermo Finnigan LTQ mass spectrometer equipped with a nanospray source operated in positive ion mode. The LC system was an

UltiMate™ Plus system from LC-Packings A Dionex Co (Sunnyvale, CA) with a

Famous autosampler and Switchos column switcher. The solvent A was water containing

50mM acetic acid and the solvent B was acetonitrile. 5 L of each sample was first injected on to the trapping column (LC-Packings A Dionex Co, Sunnyvale, CA), and washed with 50 mM acetic acid. The injector port was switched to inject and the peptides were eluted off of the trap onto the column. A 5 cm 75 μm ID ProteoPep II C18 column

(New Objective, Inc. Woburn, MA) packed directly in the nanospray tip was used for chromatographic separations. Peptides were eluted directly off the column into the LTQ system using a gradient of 2-80%B over 30 minutes, with a flow rate of 300 nl/min and a total run time was 58 minutes. The scan sequence of the mass spectrometer was based on the TriplePlay™ method; briefly the analysis was programmed for a full scan, a zoom scan to determine the charge of the peptide and an MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive instrument scans of the most abundant peak in the spectrum. Dynamic exclusion was used to exclude multiple MS/MS of the same peptide.

45

2.2.3.3 Database Search

Sequence information from the MS/MS data was processed using Turbo SEQUEST algorithm in BioWorks 3.1 Software. Data processing was performed following the guidelines in Molec. Cell. Proteomics. Assigned peaks have a minimum of 10 counts

(S/N of 3). The mass accuracy of the precursor ions were set to 1.5 Da to accommodate accidental selection of the C13 ion and the fragment mass accuracy was set to 0.5 Da.

Considered modifications (variable) were methionine oxidation and carbamidomethyl cysteine (fixed).

2.2.4 Western blots

A protein estimation was performed using a modified Lowry assay from BioRad

(Bio-Rad DC Protein Assay) and 20 g of proteins from each protein sample, or 5 g of

GroEL cell lysate were boiled in SDS sample buffer (60 mM Tris pH 6.8, 2.3% SDS,

10% glycerol, 0.01% bromophenol blue, and 1% 2-mercaptoethanol) for 5 min, and the proteins were separated by SDS-PAGE. Following separation, proteins were transferred to nitrocellulose membranes, which were blocked for 1h at RT in 5% milk TBST (Tris buffer saline with 1% Tween 20), and then incubated with anti-His tag (Cell Signaling, mouse monoclonal, 27E8), or anti-GroEL (Sigma, rabbit polyclonal) antibodies overnight at 4 °C. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies, washed again, and briefly incubated in ECL (Amersham

Biosciences) for chemiluminescent detection on x-ray film.

46

2.2.5 Factor Xa digestion

(1-58) HS-Nfs1-NMBP was twice dialysed against 20 mM Tris-HCl, 100 mM

NaCl, 2 mM CaCl2, pH 8.0. Then 200 g of dialyzed protein was incubated with 4 g of

Factor Xa for 6 h at 23 °C. Reaction products were analyzed by SDS-PAGE.

2.2.6 U.V. Characterization

The presence of PLP was determined spectrophotometrically by scanning the 250 –

500 nm region. Generally, PLP generates a characteristic absorption band when bound to proteins around 420 nm[91].

2.2.7 Activity Assays

2.2.7.1 Cysteine binding

Reaction was performed in Ni-NTA elution buffer (50 mM phosphate, 500 mM

NaCl, 500 mM imidazole, pH 8.0). 15 M (1-58) HS-Nfs1-CTH was incubated with 0.5 mM L-cysteine for 25 min at RT. Decrease in intensity of the PLP band at 420 nm and increase in external aldimine band at about 330 - 340 nm were monitored by recording the absorption spectra between 250 nm and 500 nm at 0, 5, 10, 15, and 25 min[90, 141].

47

2.2.7.2 Elemental sulfur determination assay

The formation of human Nfs1 persulfide bond was detected by cold cyanolysis

[142]. 40 M of (1-58) HS-Nfs1-CTH were placed in a sealed vial which was then alternatively vacuumed and argon purged for 30 min. Then 80 M of L-cysteine were injected through the septum. The reaction was incubated at room temperature or 37 °C for 2 hours. Then, 0.4 mL of 1 M ammonium hydroxide, 3.6 mL of water and 0.5 mL of

0.5 M potassium cyanide were added. After 45 min at RT, 1 mL of Goldstein’s reagent

(25 g/L ferric nonahydrate in 4.15 M nitric acid) was added. Samples were centrifuged to remove any protein precipitation. Supernatant absorbance was determined at 460 nm.

2.2.7.3 Sulfide determination assay

This assay was developed by Siegel in 1965 [143] and later slightly modified for biochemistry application [88, 141]. 1 M to 40 M of Nfs1 were mixed along with 10

M PLP, 10 mM MgCl2, and 5 mM DTT in a sealed vial. Then the mixture was alternatively vacuumed and argon purged for 30 min. Then 80 M L-cysteine or sodium sulfide nonahydrate solution was injected through the septum. The reaction was incubated at room temperature or at 37 °C for 2 h 30 min. Many different buffers were used:

 - (1-58) HS-Nfs1-CTH: 50 mM Pi, 500 mM NaCl, 500 mM imidazole, pH 8.0

- (1-58) HS-Nfs1-NMP: 20 mM Tris, 200 mM NaCl, 10 mM maltose, pH 8.0

20 mM Tris, 200 mM NaCl, 10 M PLP, pH 8.0 48

10 mM Hepes-KOH, pH 7.9

After 20 mM DPD in 7.2 N HCl and 30 mM FeCl3 in 1.2 mM were injected into the vials which were left at RT for 20 min away from any light. Samples were centrifuged for

10 min at 15,000 rpm to remove any precipitate that could have formed. Finally, the absorbance of the supernatant containing the newly formed methylene blue was measured at 670 nm.

2.2.7.4 Iron-sulfur reconstitution kinetic

Purified human Isu D37A in 50 mM Tris, 100 mM NaCl, pH 8.0 was incubated with 200 M iron (III) chloride, 4.3 mM DTT, 2.4 mM Na2S•9H2O or 1 M Nfs1 and 2.4

M L-cysteine. The time course for [2Fe-2S]2+ formation was monitored by UV at 450 nm for 30 min.

2.3. Results and discussion

Primers design was based on the sequence reported by Land et al. in 1998 [144].

However four amino acids differ from the sequence reported by Stanchi et al. in 2001

[145]. Consequently the full length clones of human Nfs1 have one mutation located in the 5’ primer and one silent mutation situated elsewhere. Thus, since all the other clones have been made from the full length gene of human Nfs1, they all bear this silent mutation.

49

2.3.1. Full length Nfs1

Protein expression was first checked as well as protein solubility. Surprisingly, the protein was actually not soluble and formed insoluble aggregates. Often undesirable, inclusion bodies (IB) formation may be advantageously exploited because IB consist of a very high level of protein, quite pure; they are easy to isolate from bacterial cells, they are less prone to self degradation and to proteolysis by cellular proteases. Thus IB were washed in the presence of detergents to remove membrane-bound proteins and lipids.

Some insoluble aggregate proteins retain their secondary structure [146] which, in turn, favors proper refolding. Therefore, mild conditions were used to solubilize those inclusion bodies. The solubilized proteins were refolded using many buffer conditions: different detergents (Tween 20, Triton X-100, sodium deoxycholate, sodium cholate), different combination of salt (NaCl, KCl, ammonium sulfate), presence of amino acids

(L-arginine and L-glutamate), different pHs. The refolding process was always easily achieved at pH 10 or 11, but when the pH was later lowered to physiologically relevant values (6.5 to 8.0), the protein would always fully precipitate. Even the presence of DTT did not prevent the phenomenon to happen. This instability could be explained by many factors: hydrophobic interactions, cysteines sensitive to oxidation, aggregation due to ionic interactions, post-translational modification essential to its structural integrity.

However enough was isolated for identification by LC-MS.

One way to circumvent misfolding in bacterial expression system is to use an E. coli strain containing a vector harboring indudible GroES-EL gene [147]. Indeed GroEL-

GroES are prokaryotic chaperones mediating protein folding in an ATP dependent 50 process. A non-native substrate protein in a distorted, largely amorphous state binds

GroEL-GroES-ADP asymmetric complex through hydrophobic interactions. This action may as well favor further unfolding of the polypeptide. Binding of ATP and then GroES to the same ring as the substrate follows. As a consequence GroEL domains undergo large-scale conformational changes: hydrophobic patches move away from the hollow surface allowing GroES to cap GroEL loop. Simultaneously, the protein is expelled into the central cavity where it starts folding because chamber walls are now hydrophilic. This promotes burial of hydrophobic sites of the polypeptide and so exposes its hydrophilic side chains, typical of the native state. Once ATP is hydrolyzed, the complex is weakened and folding stops. The substrate is subsequently released from the cavity independently of its folding state. “The released polypeptide may have folded to its native state (N) or one committed to it (Ic), or it may have failed to reach the native state (Iuc), in which case it can be bound to GroEL again for another attempt at folding” [148]. So this cell line was used in an attempt to isolate soluble Nfs1. The protein was successfully isolated through IMAC purification on Ni-NTA resin. But on SDS PAGE, GroEL (57 kDa) and

HS-Nfs1-NTH (53 kDa) would co-migrate and could not be distinguished (Figure 2.1.a).

Hence two western blots, anti-His6 tag and anti-GroEL, confirmed the presence of Nfs1 along with GroEL (Figure 2.1. b & c).

51

SDS-PAGE -Histag -GroEL a b c Nfs1 M Nfs1 M Nfs1 250 150 100 75

50 37

25 20

Figure 2.1: Analysis of purified HS-Nfs1-NTH: (a) Purified HS-Nfs1-NTH analysis on 10% SDS PAGE. (b) Immunoblot with anti-His6 tag of purified HS-Nfs1-NTH. (c) Immunoblot with anti-GroEL of purified HS-Nfs1-NTH. M: Protein ladder.

Nonetheless an attempt was made to dissociate the complex GroES-GroEL from

Nfs1 by incubating the cell lysate with 5 mM ATP after sonication and centrifugation, for

10 min at 37 °C, then 15 min on ice as described by Thain et al.[149]. Unfortunately this method did not prevent the co-elution of GroEL (Figure 2.3). An another method described by Roy et al. to dissociate very tightly bound proteins is the use of sodium thiocyanate in the cell lysate to minimize the salting out effect of NaCl and decrease the tendency of aggregation [150]. Unfortunately, this technique barred binding to the Ni-

NTA resin (Figure 2.2).

52

SDS-PAGE

M S FT W M 94 67 43 30

Figure 2.2: Analysis of the dissociation between GroEL and Nfs1. Overexpressed Nfs1 in the presence of the GroES-EL complex was purified on a Ni-NTA resin. Cell lysate was split in two. One (A) was incubated with 5 mM ATP as described by Thain et al. [149], and subsequently purified as usual. The elution portion (EATP) was analyzed on 15% SDS-PAGE. One (B) was applied to resin after centrifugation, wash (W) normally and then washed with NaSCN (WSCN) [150], and finally eluted (ESCN). Those fractions were compared to a crude cell lysate of only GroEL-ES.

-histag

-GroEL

Figure 2.3: Immunoblots of EATP, WSCN, and overexpressed GroEL-ES crude cell lysate with anti-His6 tag (top) and anti-GroEL (bottom).

53

Human Nfs1 was successfully cloned and overexpressed. However insoluble aggregates formation rendered the purification cumbersome and lead to a refolded soluble protein at high pH values. The presence of bacterial chaperones (GroEL-ES) during hyperproduction of Nfs1 enhanced its solubility and facilitated its isolation.

Unfortunately, this method was impaired by the co-elution of GroEL.

2.3.2. (1-31) Nfs1

Deleting or substituting amino acids can highly improve soluble protein expression

[151]. The analysis of the primary sequence by MitoProt [152] indicated that the first 31 amino acid are most likely the mitochondrial targeting sequence. The hydropathy plot of the protein sequence based on Kyte-Doolittle plot with a window size of 9 amino acids established by ProtScale (http://ca.expasy.org/tools/protscale.html) showed that this segment was hydrophobic (Figure 2.4). Consequently the first 31 amino acids were deleted; however the protein remained expressed as inclusion bodies.

54

3

2

1

0

Score

-1

-2

-3 0 50 100 150 200 250 300 350 400 450 Residue Position

Figure 2.4: Kyte-Doolittle hydropathy plot of human Nfs1. The hydrophobicity profile was obtained using ProtScale with a window size of nine residues.

Formation of inclusion bodies upon hyper production in a foreign organism such as

E. coli, are thought to occur because the relevant binding partners are absent [151] and/or stress activities (heat shock, stringent, or SOS responses) are triggered upon induction

[153]. But those mechanisms are still poorly understood. Hence, the gene was cloned into a yeast vector pPIC9 for further incorporation into Pichia pastoris yeast genome thus allowing the isolation of the posttranslational modified Nfs1. Unfortunately, PCR never confirmed the integration of the gene within the yeast genome.

55

2.3.3. (1-58) Nfs1

However mitochondrial targeting sequences can be longer, up to 60 amino acids.

Their features include: predicted formation of an amphiphilic -helix; overall positive charge and presence of an arginine residue at the -2, -3 or -10 position from the cleavage site. However those motifs are found in only 65% of the presequences from S. cerevisiae with a similar result with plants. As a result the confidence level to predict the cleavage site is quite low, this is furthermore complicated by the presence of additional cleavage sites for the mitochondrial intermediate peptidase or the inner membrane peptidase [154].

So it was decided to delete more amino acids at the N-terminus which led to the removal of 58 residues total at the N-terminus. (1-58) Nfs1 would now be close in sequence to its cytosolic counterpart. As done previously, three recombinant proteins (C-terminus

His6 tag, N-terminal His6 tag, no tag) were engineered but only Nfs1 with a His6 tag at the C-terminus was slightly soluble when cells were induced at 18 °C. So the protein was isolated but was unstable; indeed, Nfs1 was precipitating when the buffer did not contain enough imidazole (400 mM) or salt (500 mM). This high salt requirement might be explained by the fact that at physiological pH, histidine residues can form salt bridges with negatively charged amino acids and thereby mediate protein aggregation. Also histidine residues can be subject to oxidation, triggering subsequent protein aggregation and degradation [155]. In addition, the protein could not be concentrated to a concentration higher than 40 M. Yet, enough was isolated to confirm the presence of

PLP at 394 nm (Figure 2.5) and characterize its activity by detecting sulfide formation

56 according to Flint procedure [141], adapted from Zheng et al. [88], sulfane sulfur, and external aldimine formation.

0.8

0.7

0.6

0.5

0.4

0.3 Absorbance

0.2

0.1

0.0

250 300 350 400 450 500 Wavelength (nm)

Figure 2.5: UV-visible absorbance spectra of the purified (1-58) HS-Nfs1-CTH (red) and (1-58) HS-Nfs1-NMBP (blue). PLP absorbs at 394 nm (1-58) HS-Nfs1-CTH for and 414 nm for (1-58) HS-Nfs1-NMBP.

57

1.0 0.9 0.8 0.7 0.6

0.5

0.4 External Aldimine Absorbance 0.3 PLP 0.2 0.1 0.0 250 300 350 400 450 500 Wavelength (nm)

Figure 2.6: (1-58) HS-Nfs1-NTH absorbance profile evolution over 25 min, at RT, in presence of 0.5 mM Cysteine. Black line represents (1-58) HS-Nfs1-NMBP only, in red after 30 s, in green after 10 min, in blue 15 min, in cyan, 25 min.

Cold cyanolysis failed to detect any sulfane sulfur formation on (1-58) HS-Nfs1-

CTH. In addition the methylene blue assay did not detect any sulfide when the reaction was conducted at room temperature. However the activity was measurable at 37 °C: barely 7% of the substrate was converted to alanine which corresponds to only one turn- over. But in E. coli or T. maritima, multiple turnovers were observed. Also it failed to generate iron-sulfur cluster on human Isu (Figure 2.7) [93, 141].

58

0.20

0.15

0.10

0.05 Normalized OD change 0.00

0 10 20 30 40 50 TIme (min)

Figure 2.7: Iron-sulfur cluster reconstitution kinetic. Purified human Isu D37A in 50 mM Tris, 100 mM NaCl, pH 8.0, was incubated with iron (III), DTT, Na2S (red) or Nfs1 and cysteine (green). A control experiment was done by incubating sodium sulfide. The time course for [2Fe-2S]2+ formation was monitored by UV at 450 nm for 30 min.As a control experiment, only sodium sulfide and iron(III) were incubated (black).

Those results could be explained by the conclusions of the study on Cysteine

Desulfurase Slr0387 from Synechocystis sp. PCC 6803 by Behshad et al. They demonstrated the necessity of a complex formation between the PLP cofactor and DTT prior to the cleavage of the persulfide bond by DTT. However once the persulfide bound is formed, a second molecule of L-cysteine can bind to the cofactor in the persulfide of

Slr0387 explaining why several cysteine desulfurase are subject to potent inhibition by L- cysteine during turnover with DTT [156].

59

The protein was more soluble although it was limited to buffer with high ionic strength containing a high concentration of imidazole. Also precipitation would occur if the temperature was raised to 37 °C.

With advances in cloning, fusion tags able to solubilize proteins have begun to surface, offering a way to circumvent the inclusion body problem. Fusion partners that have been successfully used include glutathione S-transferase (GST), thioredoxin (TRX), and maltose binding protein (MBP), Protein A, ubiquitin, and DsbA. In a comparative study, MBP showed a higher propensity to solubilize proteins which are otherwise forming inclusion bodies. It was even helping in the folding process compared to TRX or

GST [157]. pMAL-p2x vector contains the malE signal sequence located before the multiple cloning site, resulting in periplasmic expression of the fusion polypeptide. This allows the formation of disulfide bonds if necessary. It also includes a sequence coding for the recognition site of Factor Xa (Glu/Asp-Gly-Arg). pMAL-c2x is identical to pMAL-p2x except the malE signal sequence has been removed to permit cytoplasmic expression of the desired protein. Nfs1 was fused with MBP at N-terminus in both vectors and expressed in E. coli Rosetta pLys at 15 °C overnight, however, the periplasmic expression lead to insoluble aggregates. In the case of cytoplasmic expression, the protein was successfully isolated in 50 mM Tris, 200 mM NaCl, pH 8.0.

60

M S FT W E

94 67 43

30

20

Figure 2.8: 10% SDS-PAGE of (1-58) HS-Nfs1-NMBP the purification on amylose resin. M: protein ladder, S: supernatant, FT: flow-through, W: column wash, E: elution.

The solubility and stability were greatly increased: higher concentrations were reached, different buffers, salts and pHs were successfully tested and no more precipitates were observed at 37 °C, even after 2 h. But unfortunately its activity was no better than

Nfs1-CTH, only one turnover was accomplished.

The maltose binding protein is a large protein of about 40 kDa and as such it could impair Nfs1 catalytic activity by steric hindrance. So (1-58) HS-Nfs1-NMBP was digested by Factor Xa to remove the MBP tag and test the solubility of the digested recombinant protein. This was successfully applied to the TEV protein in vivo [157].

(1-58) Nfs1 does not contain any primary cleavage site for Factor Xa, i.e. Ile-

Glu/Asp-Gly-Arg. Yet it possesses two secondary cleavage sites at R220 and R412. As observed on SDS-PAGE (Figure 2.9), MBP was removed but Nfs1 was also cut at one of the two secondaty sites, giving two fragments: ~ 18 kDa and ~ 27 kDa. In addition they

61 were not soluble. There seems to be a correlation between proteins that are unstable in E. coli and those that are cleaved by Factor Xa at secondary sites: this may indicate that these proteins are in a partially unfolded state (Walker, I., Riggs, P., unpublished data).

S P ND 94 67 43

30

20

14.4

Figure 2.9: Digestion of (1-58) HS-Nfs1-NMBP by Factor Xa. S represents the soluble part of digested (1-58) HS-Nfs1-NMBP; P is the insoluble part of digested (1-58) HS- Nfs1-NMBP; ND is non-digested (1-58) HS-Nfs1-NMBP.

MBP possesses many clusters of hydrophobic patches on its surface [158], and therefore could be interacting with the partially unfolded Nfs1 by hydrophobic interactions thereby enhancing its solubility [157].

62

2.4 Conclusion and future work

In summary, when expressed in E. coli, the full length of Nfs1 formed insoluble aggregates, but the presence of the co-chaperones GroEL made it soluble most likely because of hydrophobic interactions between the two proteins. This means that most likely Nfs1 has solvent exposed hydrophobic segments promoting its aggregation. The 31 amino acids truncation did not solve the solubility issue whereas the 58 amino acids truncation did. However the later protein was not stable at low salt concentration or at low imidazole concentration which means salt bridges were favoring its aggregation in presence of a His6 tag. The fusion with MBP tag improved its solubility and stability, but the digestion with Factor Xa and activity assays proved that Nfs1 was most likely in a misfolded state.

Future work would include optimization of Nfs1-NMBP purification and co- expression of Nfs1 and its cognate partner Isd11. Also the arginine residue of the secondary cleavage site of Factor Xa could be mutated (R220) to an glutamic acid as a way to enhance its stability and maybe its solubility, and to make it active. Also the leucine residue 304 could be mutated to aspartate in order to lower the hydrophocity of the segment it is involved in, and make it almost as hydrophobic as its bacterial counterpart. Finally expression in E. coli C41 and C43 hosts should be tested as they have been successfully applied to solve those kinds of matters.

63

CHAPTER 3

STUDIES ON HOMO SAPIENS FRATAXIN

3.1 Introduction

Frataxin is a ubiquitous protein in mitochondrial iron homeostasis. Found mainly in heart, spinal cord, liver, skeletal muscles and pancreas, it fulfills many different roles: iron storage protein, iron delivery protein for de novo iron sulfur cluster synthesis and heme synthesis, and iron chaperone activity [159, 160]. Despite its involvement in those very important processes its deletion in yeast is not lethal and has rather mild effects indicating that its function can be bypassed. However in humans, frataxin deficiency give rise to the Friedreich’s ataxia, a neurodegenerative disorder characterized by progressive neurological disabilities as such sensory loss, sometimes assiociated with diabetes, and heart abnormalities that may eventually be fatal [46].

The first controversy lies in the active form of this protein. Indeed, frataxin was reported to be synthesized as a 210 amino acid precursor protein. In vitro studies showed it was subsequently cleaved upon import in the mitochondrial matrix by the mitochondrial processing peptidase  to form an intermediate polypeptide (amino acids

64

41 to 210) that is futher processed to give a mature protein comprising residues 56 to 210.

It was also found to be associated with membranes on the matrix side [65, 66]. But Yoon et al. showed that frataxin 56-210 was prone to self cleavage in presence of transition metal ions such as iron (II) or manganese (II) to lead to a “truncated” frataxin (residues

76 to 210) that is able to bind 6-7 iron (II), interact with Isu1 to form a [2Fe-2S]2+ cluster and deliver iron to ferrochelatase in heme synthesis pathway [70, 71, 161]. Consequently it was proposed this product was the active form of frataxin. This hypothesis is further supported by a recent study in vivo on the maturation of frataxin concluding that the active form comprises amino acids 81 to 210 but could be 56-210 if the cleavage site

(residues 80-81) is not available[68].

A second conflicting role is its ability to polymerize to store iron by catalyzing the oxidation of iron (II) as do ferritin in the cytoplasm but with a lower capacity: about 10 iron atoms per monomer compared with 190 iron atoms per monomer for human ferritin

[73]. However this is in contradiction with the study conducted by Becker et al. on Friend cells. Indeed they showed that level of frataxin was not sensitive to increase amount of intracellular iron. Thus this observation is not consistent with an iron storage role [162].

In order to improve our understanding of frataxin maturation and function, we have studied the metal affinity and the ferroxidase activity for both forms.

65

3.2 Experimental procedures

3.2.1 Truncated Frataxin constructs

Truncated frataxin (trFtx) was subcloned from pET-28b(+) containing the full- length frataxin gene obtained from Taejin Yoon. PCR. Amplication of the gene was accomplished by using DeepVent polymerase, 5’-GGA AGG CCA TAT GTT GAG

GAA ATC TGG AAC TTT G as 5’ primer and 5’-AGA ATT CAA GCA TCT TTT CCG

GAA TAG GCC AA as 3’ primer. The underlined motifs correspond to NdeI and EcoRI cleavage sequence respectively. PCR product was subsequently purified with the QIAgen

PCR purification kit and analyzed on agarose gel. PCR product and pET-21b(+) were separately double digested at 37 °C. The vector was furthermore treated with CIAP. Then digested insert and vector were mixed in an equimolar ratio and ligated with T4 DNA ligase overnight at 14 °C. Chemically competent E. coli DH5 were transformed with the ligation product. Afterwards the DNA sequence was confirmed by DNA sequencing.

Addition of a His6 tag at the N-terminus of this protein was accomplished by subcloning the gene previously inserted into pET-21b(+) and insert it into pET28b(+). It was PCR amplified using Pfu Turbo, and the previous primers. Double digestion, ligation and transformation were identical as above. Again the DNA sequence was confirmed by

DNA sequencing.

66

3.2.2 Expression & Purification

E. coli BL21 (DE3) strain was used to express the truncated frataxin. After being grown in LB media supplemented with the proper antibiotic overnight at 37 °C, the culture was diluted 100 fold into fresh LB media to be shaken at 220 rpm at 37 °C until

OD600 reached ~0.6. At that point the cells were induced with an IPTG solution (final concentration 300 M) for 4 h at 30 °C and at 220 rpm. Cells were harvested by centrifugation for 5000 rpm at 4 °C for 10 min. The cell pellet was stored at -80 °C if not used immediately.

The cell pellet containing the pET 21b(+) frataxin was resuspended in buffer A (50 mM Hepes, 50 mM NaCl, pH 8.0) supplemented with 1 mM TCEP, 0.1% Triton X-100,

75 ng/mL lysozyme, DNase I, 1 mM EDTA. The cells were disrupted by sonication (10s pulse/2 min at 80% output) on ice. The lysate was centrifuged at 15,000 rpm and at 4 °C to remove cellular debris. The supernatant was loaded on a preequilibrated DE52 resin

(Whatman) with buffer A. The loaded resin was washed with 2 column volumes of buffer

A. Afterwards the protein was eluted off the resin by a continuous gradient from 50 mM

NaCl to 1 M NaCl. The fractions containing the frataxin were pooled and concentrated by

Amicon with YM10K filter. The concentrate was then filtered with an YM30K to yield pure frataxin in the filtrate.

The cell pellet containing the pET 28b(+) frataxin was resuspended in buffer B (50 m sodium phosphate, 500 mM NaCl, 10% glycerol, pH 7.0) supplemented with DNase I, lysozyme, TCEP, 0.1% Triton X-100, 1mM PMSF. The cells were disrupted by sonication (10s pulse/2 min at 80% output) on ice. The cell debris were removed by 67 centrifugation at 15,000 rpm and 4 °C for 30 min. The supernatant was then loaded on pre-equilibrated TALON resin. The loaded resin was then washed with 10 column volumes of buffer B with 5 mM imidazole and 10 column volumes of buffer B with 10 mM imidazole. Elution was performed with buffer B supplemented with 150 mM imidazole.

3.2.3 Mature frataxin expression and purification

Cell pellet was resuspended in buffer C (50 mM Pi, 300 mM NaCl, 10% glycerol, pH 7.0) supplemented with 1 mM TCEP, 0.1% Triton X-100, 75 ng/mL lysozyme,

DNase I. Cell lysis was achieved by sonication (10 s pulse / 2 min) for 30 min at 4 °C.

Then the lysate was centrifuged at 15,000 rpm for 30 min at 4 °C. Next, the supernatant was loaded onto a TALON resin preequilibrated with buffer A. Subsequently the loaded resin was washed with 10 bed volumes of buffer A, 5 bed volumes of buffer A with 5 mM imidazole and 5 bed volumes of buffer A with 10 mM imidazole. Protein was eluted with buffer A with 200 mM imidazole, pH 7.0. Protein purity was assessed by SDS-

-1 PAGE. Concentration was determined by absorbance at 280 nm (280nm = 33920 M cm-1).

68

3.2.4 Thrombin cleavage of mature frataxin

Human mature frataxin with an N–terminal His6 tag (HS-FLFx-NTH) was purified on TALON as previously described. Then the purified protein was incubated with 100 mM EDTA and 100 M TCEP for 4 h. Removal of the His tag by thrombin was done according to the manufacturer instructions. Afterwards, the thrombin digested HS-mFtx-

NTH (thrmFtx) was dialysed against 40 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole. After that, it was loaded on Ni-NTA resin to remove undigested protein and cleaved His6 tag. The loaded resin was subsequently washed with 5 column volumes to recover most of the thrFLFx. Flow through and wash were combined and concentrated by

Amicon with an YM10K membrane. This solution was next filtered through an YM30K membrane to remove the thrombin. Buffer exchange of pure thrmFx was performed on desalting column G-25 (Aldrich). Concentrations were measured by UV using the calculated ε at 280 nm (33260 M-1cm-1).

3.2.5 Metal binding studies by ITC

Protein buffer was exchanged to 50 mM Hepes, 150 mM NaCl, pH 7.5 on desalting

G25 resin. Subsequently titrant and sample were prepared using the G25 buffer. Both preparations were thoroughly degassed and argon purged before each titration. ITC measurements of metal ion binding to apo frataxin were carried out at 25 °C on a MicroCal

OMEGA ultrasensitive titration calorimeter. Metal stock solutions were prepared in argon purged water. If the transition metal was iron (II), titrant and sample preparation

69 contained 10 mM dithionite to keep the system anaerobic. Typically 10 L of titrant was injected to a sample solution of 50-100 M frataxin over a period of 10 s with an interval of 10 min between injections to allow complete equilibration. All binding isotherms were subtracted of heats of dilution from injections of titrant into buffer for the specific conditions. The data were collected automatically and subsequently analyzed with a one- site binding model by the Windows-based Origin software package supplied by

MicroCal. The Origin software uses a nonlinear least-squares algorithm (minimization of

2) along with the concentrations of the titrant and the sample, to fit the heat flow per injection to an equilibrium binding equation, providing best fit values of the stoichiometry (n), change in enthalpy (ΔH), and binding constant (K).

3.2.6 Ferroxidation assays: Oximetry

Ferroxidase activity was monitored by measuring the oxygen concentration in reaction mixture for 15 min with a Clark electrode plugged to an Oxygraph System

(Hansatech Instruments). Sets of data were acquired on a personal computer via oxygraph software (Hansatech Instruments). The electrode was calibrated with air-saturated buffer and and nitrogen purged buffer. Oxygen consumption was monitored in 0.5 mL reaction volume. After addition of buffer with or without protein, the chamber was capped. The reactions were started by adding 10 L of anaerobically prepared iron (II) stock solution to the sample using a gas-tight syringe through the cap. During measurements, the sample was rapidly stirred with a microspin bar, and the temperature was maintained to 30 °C by

70 use of a water jacket connected to a circulating water bath. Two buffers were used: 50 mM Tris, 100 mM NaCl, pH 7.0 and 50 mM Tris, 100 mM NaCl, pH 7.5.

3.3 Results & Discussion

3.3.1 Purification

3.3.1.1 Mature frataxin (mFtx)

Purification of the mature frataxin on Ni-NTA resin was not satisfactory as some degradation product, namely truncated frataxin, was co-purified along with the desired protein. Besides, some higher molecular weight polypeptides were present as well. The second step of the purification involved an anionic exchange column. However this step promoted the degradation of the mFtx to trFtx thereby lowering the yield. Therefore cobalt affinity purification was tested. It usually gives purer proteins because of a better selectivity compared to nickel based IMAC. Indeed mFtx was purified to a purity of 98% as judged on SDS-PAGE (Figure 3.1). Moreover no degradation product was detected.

71

M S FT W1 W2 W3 E 94 67 43 30

20

14.4

Figure 3.1: Purification of mature frataxin on TALON column seen on 15% SDS-PAGE. M: marker; S: supernatant; W1: Wash with 10 bed volumes of buffer A; W2: wash with 5 bed volumes of buffer A with 5 mM imidazole; W3:wash with 5 bed volumes of buffer A with 10 mM imidazole; E: elution.

Finally, mFtx-NTH has a tendency to precipitate when its concentration exceeds

200 M. At pH 7.5, the protein concentration can reach 1.5 mM if in an appropriate buffer, e.g. 100 mM sodium phosphate, or 5 mM if in a buffer containing 1% sodium deoxycholate.

3.3.1.2 Truncated frataxin (trFtx)

The truncated frataxin was successfully cloned as attested by the DNA sequencing results. Both recombinant proteins (His6 tag and non-His6 tag) were successfully isolated to 98% purity (Figure 3.2 & 3.3).

72

M S E 94 67 43

30

20

14.4

Figure 3.2: 20% SDS-PAGE of purified trFtx-Ø. M:Marder; S: Amicon supernatant; E: Amicon flow-through containing purified trFtx-Ø.

M E 94 67 43 30

20

14.4

Figure 3.3: 15% SDS-PAGE of purified trFtx-NTH. M:Marker; E: Elution of trFtx-NTH from TALON resin.

The bands located above the purified trFtx-NTH from TALON resin are most likely artifacts from overloading the SDS-PAGE.

73

In early purifications, DNA binding molecules like protamine sulfate and streptomycin sulfate were used to clarify the cell lysate and decrease its viscosity [163].

The antibiotic streptomycin precipitates ribonuclear proteins whereas protamine precipitates DNA and RNA molecules. Numerous proteins bind specifically to protamine, and trFx was such protein [163]. Indeed, it did not bind any iron when protamine sulfate was used during the purification. This indicates that the site of iron is highly negative, and is big enough to accommodate a molecule such as protamine or solvent exposed.

This confirms the conclusion drawn from the crystal structure of frataxin [69], as discussed in chapter 1.

3.3.2 Metal binding

3.3.2.1 Mature frataxin

Next we looked at the binding of iron to HS-mFtx-NTH and thrombin cleaved HS- mFtx-NTH (thmFtx) which does not have an N-terminal His6 tag anymore.

HS-mFtx-NTH binds 2 iron (II) atoms in presence of a His6 tag (Figure 3.4) with an overall binding affinity of 17.1 M, and one iron atom when the six histidine residues tag was removed by thrombin digestion (Figure 3.5) with a Kd of 9.1 M.

74

Time (min) -20 0 20 40 60 80 100 120 140 160 0.08 0.07 0.06 0.05 0.04 0.03

0.02 µcal/sec 0.01 0.00 -0.01 0.20

0.15

0.10

0.05

0.00 kcal/moleof injectant

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Molar Ratio

Figure 3.4: Titration of 100 M thrombin cleaved HS-mFtx-NTH with 2 mM iron (II). 5 -1 The best fit was obtained for the following: N = 1.00; Ka = 1.10x10 ; H = 167.9 J.mol ; S = 23.6 J.K-1.mol-1.

75

Time (min) 0 20 40 60 80 100 120 140 160 0.1

0.0

-0.1

-0.2

-0.3 µcal/sec

-0.4

-0.50.1

0.0

-0.1

-0.2 Data: FLFXFe2_NDH Model: OneSites Chi^2/DoF = 401.2 -0.3 N 1.94 ±0.059 K 5.84E4 ±1.3E4 H -378.5 ±15.9

kcal/moleof injectant -0.4 S 20.5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Molar Ratio

Figure 3.5: 100 M mFtx-NTH titrated with 3 mM iron (II)

The difference in stiochiometry between thmFtx and mFtx, is easily explained by the His6 tag tail that would be able to stabilize the binding of a labile iron to the carboxylates of the negative charges patch via coordination of the nitrogen from imidazole residues. For both proteins the stoichiometry is lower than for the truncated frataxin which binds about 6 to 7 iron per monomer. According to the crystal structure, about 30 amino acids at the N-terminus represent a disordered region, meaning this part

76 of the polypeptide is flexible and could possibly protect a segment of the anionic patch

(Figure 1.7). The other thermodynamic binding parameters are summarized in table 3.1.

n Kd H TS G No. of sites 10-6 M cal mol-1 cal mol-1 kcal mol-1 HS-mFtx-NTH 1.94 17.1 -378.5 6109 -6.5 th HS-mFtx-NTH 1.00 9.1 167.9 7033 -6.9

Table 3.1: ITC binding parameters from the HS-mFtx-NTH and thmFtx titrations with iron (II).

Iron binding to thmFtx is slightly endothermic whereas it is exothermic with mFtx.

Enthalpy change also represents the evolution of the organization of the hydrogen bond network between the reacting species, including the sovent [164]. Thus, it is favorable for mFtx and defavorable for thmFtx. However ionic interactions are also part of the enthalpy change [164]. Since the binding of iron is most likely electrostatic by nature, it is genuine to assume that the binding of two iron (II) could be enough to increase the enthalpy change and make it negative.

In both titrations, enthalpy changes are small when compared with the entropy changes (TS), indicating a process that is entropically driven. Entropy change reflects hydrophobic interactions and the relative degree of disorder between the free and bound systems [164]. For both binding events, they are positive evidencing an increase in the disorder.

77

Those data can be rationalized as follows: upon binding frataxin, the iron solvation sphere is perturbed to accommodate the new ligands, carboxylates and maybe nitrogen

(for the His6 tag protein). Similarly water molecules solvating the protein are perturbed and have to rearrange themselves to compensate for the iron interacting with the amino acid residues. The displacement of the water molecules comes with an entropy penalty since the iron binding is increasing their disorder. Also the metal binding may also introduce some structural changes that may result in exposition of hydrophobic residues to solvent.

3.3.2.2. Truncated frataxin

Afterwards, we looked at the binding abilities of truncated frataxin towards iron, zinc, calcium, and magnesium by isothermal titration calorimetry (ITC). Studies on yeast frataxin have used magnesium during the purification process and interestingly the frataxin to iron ratio was lower [165] than ours. Therefore we wanted to address whether magnesium could populate iron binding site.

Iron binding between the His6 tag and non His6 tag version of the truncated frataxin displayed similar thermodynamic parameters (Table 3.2) according to the ITC titrations results (Figure 3.6 & 3.7).

78

Time (min) 0 20 40 60 80 100 120 140 160

0.0

-0.5

-1.0

µcal/sec -1.5

-2.0

0.0 -0.1 -0.2 -0.3 -0.4 Data: default1_NDH -0.5 Model: OneSites Chi^2/DoF = 386.3 -0.6 N 5.60 ±0.20 K 8.76E3 ±1.0E3 -0.7 H -1038 ±51.5 S 14.6 kcal/moleof injectant -0.8

0 2 4 6 8 10 12 14 16 18 20 Molar Ratio

Figure 3.6: 60 M trFtx- Ø titrated with 5 mM iron ammonium sulfate.

79

Time (min) -20 0 20 40 60 80 100 120 140 160

0.0

-0.5

-1.0

-1.5 µcal/sec

-2.0

-2.5 0.0

-0.2

-0.4 Data: default3_NDH Model: OneSites Chi^2/DoF = 200.1 N 5.90 ±0.17 -0.6 K 9.16E3 ±7.9E2 H -996.7 ±39.0 S 14.8

kcal/mole of injectant -0.8

0 5 10 15 20 25 30 35 40 Molar Ratio

Figure 3.7: 60 M trFtx-NTH titrated with 10 mM iron amonium sulfate. The injection volume for the last 8 injections was changed to 20 L to ensure complete saturation.

Both stoichiometries were similar 5.6 and 5.9 with close overall binding affinities, about 110 M. Even enthalpy and entropy changes were comparable. Thus both recombinant proteins behave alike, and showed analogous results to the previously reported binding parameters.

Even though the iron binding is more exothermic with the truncated frataxin than with the mature frataxin, the reaction is still entropically driven. The electrostatic binding of the 5-6 iron and the favorable rearrangement of the hydrogen bond network cannot

80 compensate for the desorganization of the solvation shell around the ferrous iron and the protein. Since there is no more N-terminus tail that could interact with the anionic patch

(Figure 1.7) and prevent the interaction with iron, more iron atoms are binding and are solvent exposed. So besides carboxylates and maybe histines, iron will also coordinate with water. Since iron is charged, it will produce an electric field capable of orienting water molecules which in turn are less free or less disordered. This would explain the decrease in entropy change with respect to the values obtained with the mature frataxin.

We also analyzed the binding of two more ions, zinc (II) (Figure 3.8) and calcium

(II) (Figure 3.9). The binding parameters are gathered in table 3.2.

n Kd H TS G Metal -6 -1 -1 -1 No. of sites 10 M cal mol cal mol kcal mol HS-trFtx-Ø Fe2+ 5.6 110 -996.7 4410 -5.41 HS-trFtx-NTH Fe2+ 5.9 114 -1038 4351 -5.38 HS-trFtx-NTH Zn2+ 2.6 1.48 -3155 4798 -7.95 HS-trFtx-NTH Ca2+ N/A N/A N/A N/A N/A 2+ HS-trFtx-NTH Mg N/A N/A N/A N/A N/A

Table 3.2: ITC binding parameters from the HS-trFtx-NTH and HS-trFtx-Ø titrations with iron (II), zinc (II), calcium (II) and magnesium (II).

81

Time (min) -10 0 10 20 30 40 50 60 70

0 µcal/sec

-5

0

Data: default4_NDH -2 Model: OneSites Chi^2/DoF = 1601 N 2.60 ±0.011 K 6.76E5 ±8.6E4 H -3155 ±26.9

S 16.1 kcal/moleof injectant -4 0 1 2 3 4 5 6 7 8 9 Molar Ratio

Figure 3.8: 50 M trFtx-NTH titrated with 2.5 mM zinc sulfate.

82

Time (min) 0 10 20 30 40 50 0.14 0.12 0.10 0.08 0.06

0.04 µcal/sec 0.02 0.00 -0.02 0.10

0.09

0.08

0.07

0.06 kcal/moleof injectant 0.05 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Molar Ratio

Figure 3.9: 100 M trFtx-NTH titrated with 5 mM calcium chloride.

Zinc coordinates with the truncated frataxin with a ratio of 1:2.6. This represents about half of the iron binding sites. Zinc is smaller than iron and might be able to discriminate between strong and weak sites. Furthermore, Kd was about 1.5 M which is much lower than for iron. The enthalpy change was more negative and so the reaction was more exothermic. The entropy change is still dominating the reaction but to a lesser extent than with iron. This could be due to the strong electrostatic interactions between zinc and its ligands.

83

On the other hand calcium did not bind at all under these conditions. Calcium may be too big to fit in the iron binding sites. Finally, the titration of frataxin with magnesium showed that magnesium did not interact with frataxin under those conditions (Figure

3.10) signifying that magnesium is certainly not interfering with iron binding.

Time (min) 0 10 20 30 40 50 60 70 80 90 100 110 120 0.04

0.00 µcal/sec

-0.04

0.08

0.06

0.04

0.02

0.00 kcal/moleof injectant

0 2 4 6 8 10 12 Molar Ratio

Figure 3.10: 100 M trFtx-NTH titrated with 5 mM magnesium.

84

3.3.2.3 Conclusion

The two forms of frataxin, mature and truncated, bind iron with different stoichiometries and different affinities. So far no study has firmly established which forms of frataxin is physiologically relevant. One can assume that one form is more preponderant according the tissue specificity. For instance the the truncated form could be the relevant polypeptide in erythrocyte when there is a high demand in iron delivery for heme biosynthesis, whereas the mature form might be appropriate in cells with low iron requirements. Another possibility could depend on the iron concentration. Thus, under low iron conditions, mature frataxin could be the major type. Under high iron concentration, the N-terminus of mature frataxin would get cleaved and switch to the truncated form to prevent iron overload and keep the mitochondrion functioning.

3.3.3 Oximetry

Then, we tackled one of the most controversial properties of frataxin: its ferroxidase activity which is also a characteristic of ferritin. As explained in the first chapter, ferritin proteins are dedicated to iron storage. To accomplish their mission, they oxidize iron (II) to iron (III) extremely efficiently. When recombinant human H ferritin was incubated with ferrous iron at a ratio of 1:48, oxidation was 80% complete in 0.2 s and 99 % in 10 s

[32]. Furthermore kinetic parameters were determined (Table 3.3).

85

Table 3.3: Ferritin kinetic parameters for recombinant H-ferritin (rHF), ferritin (HLF) and horse spleen ferritin (HoSF). Conditions: 0.1 M NaCl, 50 mM Mops, pH 7.05, 20 °C. Adapted from [32].

It is also well known that any iron (III) complexing agent such as oxalate, phosphate, citrate, and EDTA, displays a ferroxidase activity. Harrison et al. measured the rates of oxidation of those aforementioned ligands by using a bathophenanthroline assay (Table 3.4) [166]. Interestingly, EDTA is able to oxidize in 10 s half of the initial amount of iron (II). This indicates that ferritin is even faster than EDTA.

Ligand [Ligand] / [Fe] t1/2 (s) Initial pH Final pH First order Temperature (°C) None - 230 7.45 - - None - 2700 7.03 6.83 Yes 23 Oxalate 3.08 580 7.03 6.89 Yes 23 Phosphate 2.97 210 7.03 6.90 No 23 Citrate 3.84 60 7.03 6.99 No 23 Citrate 2.03 55 7.03 7.02 No 23 Nitrilotriacetate 2.01 12 7.03 6.90 No 23 EDTA 0.99 10 7.03 6.95 No 23

Table 3.4: The effects of ligands on the rate of oxidation of iron (II) by atmospheric oxygen. Adapted from [166].

86

Once oxidized, iron is stored as a mineral. The other function of ferritin is to detoxify the cell from hydrogen peroxide by the so-called detoxification reaction (Figure

1.4). As mentioned earlier it easily crossed membranes and exert its deleterious effects on more targets. This reaction has not been detected in mitochondrial ferritin. The latter is able to scavenge iron to a point where even cytosolic ferritin stores are completely depleted [80].

In 2005, O’Neill et al. reported a ferroxidase activity for the mature frataxin in human [74]. They measured the oxygen consumption upon incubation of about 100 M frataxin with 25 M to 10 mM iron, at 25 and 30 °C. Obviously those concentrations of iron are not physiologically relevant [4]. Furthermore a thorough analysis of the initial rates of oxidation and determination of kinetic parameters for the reaction were missing.

However from the displayed data, about 20 minutes are needed to deplete by half the initial concentration of iron. This makes frataxin activity comparable to oxalate (Table

3.2) or iron itself at pH 7.5.

We measured the ferroxidase activity of our mature frataxin and its truncated form both without His6 tag (Figure 3.11 & 3.12).

87

240

230

220

210

200

] (mmol/L) ] Buffer 2 Buffer + 25 M Fe2+ [O 190 Buffer + 50 M Fe2+ Buffer + 100 M Fe2+ Buffer + 200 M Fe2+ 180 Buffer + 100 M thmFtx Buffer + 25 M Fe2+ + 100 M thmFtx 2+ 170 Buffer + 50 M Fe + 100 M thmFtx Buffer + 100 M Fe2+ + 100 M thmFtx Buffer + 200 M Fe2+ + 100 M thmFtx 160 0 2 4 6 8 10 12 14 16 Time

Figure 3.11: Measurement of dioxygen consumption for thrombin cleaved HS-mFtx- NTH.

88

240

230

220 mol/L)

 210

] ( ] Buffer 2 2+

Buffer + 25 M Fe [O 2+ Buffer + 50 M Fe 200 2+ Buffer + 150 M Fe 2+ Buffer + 25 M Fe + 50 M trFtx 2+ Buffer + 50 M Fe + 50 M trFtx 2+ 190 Buffer + 150 M Fe + 50 M trFtx

180 0 2 4 6 8 10 12 14 16 Time (min)

Figure 3.12: Measurement of dioxygen consumption for truncated frataxin. WT indicates the presence of protein.

According to our data, there is almost no change in the iron oxidation rate. But comparison between the initial rates of the different proteins indicates that the ferroxidase of frataxin if any, contrasts to the reference, namely ferritin:

-Human ferritin: ~1.25 s-1 [167]

-Human mFtx: ~0.00007 s-1 [74]

-Human Ftx: ~0.00016 s-1 (This study)

-Yfh1p Ftx : ~0.0003 s-1 (yeast frataxin homolog [168])

It was reported that frataxin expression was independent of iron concentration

[162], it does not aggregate even at high concentration [169], the presence of iron do

89 promote self-assembly of frataxin [169], and iron deposits observed in FDRA cells are made of mitochondrial ferritin [170]. Our study completed those observations by measuring the rate of oxidation of iron in presence of frataxin. The latter is very slow compared to cytosolic ferritin. All those findings tend to infirm an iron storage role for frataxin as detected in ferritin. More cellular biology studies are needed to further investigate this function in vivo.

3.4 Conclusion

We were able to confirm the possible involvement of carboxylates as ligands for iron (II) in frataxin with the iron and zinc titrations. We also demonstrated that the ferroxidase activity of frataxin was not significant and was most likely physiologically irrelevant especially in the light of the presence of a mitochondrial ferritin.

90

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