Deborah Grifagni

José Malanho Silva

Leonardo Querci

Michel Lepoivre

Cindy Vallières

Ricardo O Louro

Lucia Banci

Mario Piccioli
email: piccioli@cerm.unifi.it

Marie-Pierre Golinelli-Cohen
email: marie-pierre.golinelli@cnrs.fr

Francesca Cantini
email: francesca.cantini@unifi.it

Biochemical and cellular characterization of the CISD3 protein: molecular bases of cluster release and destabilizing effects of nitric oxide

Keywords: NEET protein, NMR, nitric oxide, iron-sulfur cluster, nitrosative stress

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

NEET family is a class of iron-sulfur proteins occurring in all domains of life (1). In humans, three genes encode for NEET proteins: CISD1 for mitoNEET, CISD2 for the nutrient deprivation autophagy factor-1 (NAF-1), also known as CISD2, and CISD3 for MiNT, also called CISD3. This family of proteins has a conserved domain, called CDGSH, consisting of the consensus sequence [C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H], and were initially annotated as zinc-finger proteins [START_REF] Wiley | MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity[END_REF].

Further investigation demonstrated that they actually bind a [Fe2S2] cluster [START_REF] Wiley | The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster[END_REF][START_REF] Valasatava | Metalpredator: a web server to predict iron-sulfur cluster binding proteomes[END_REF][START_REF] Andreini | The human iron-proteome[END_REF]. MitoNEET identified in 2004 (2) as a potential type II diabetes druggable target [START_REF] Song | Pioglitazone Inhibits Metal Cluster Transfer of mitoNEET by Stabilizing the Labile Fe-N Bond Revealed at Single-Bond Level[END_REF][START_REF] Takahashi | A novel MitoNEET ligand, TT01001, improves diabetes and ameliorates mitochondrial function in db/db mice[END_REF], is bound to the outer mitochondrial membrane via a N-terminal transmembrane domain with the main part of the protein, including the Fe-S cluster, which lies in the cytosol. Like mitoNEET, CISD2 is also a transmembrane protein with its cluster protruding into the cytosol but is anchored to the membrane of the endoplasmic reticulum (ER). MitoNEET and CISD2 proteins are structurally similar [START_REF] Inupakutika | Phylogenetic analysis of eukaryotic NEET proteins uncovers a link between a key gene duplication event and the evolution of vertebrates[END_REF], both are homodimeric proteins binding one cluster per monomer. Differently to both mitoNEET and CISD2, CISD3 is a soluble protein residing in the mitochondrial matrix. CISD3 is a monomer with two CDGSH domains, similar but not equivalent, each binding one [Fe2S2] cluster [START_REF] Lipper | Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells[END_REF]. Despite their different structure organization, the three human NEET proteins have similar three-dimensional structures [START_REF] Mittler | NEET Proteins: A New Link Between Iron Metabolism, Reactive Oxygen Species, and Cancer[END_REF].

A signature of the NEET proteins is the 3Cys:1His coordination of [Fe2S2] centers. This unique motif confers cluster lability [START_REF] Karmi | The unique fold and lability of the [2Fe-2S] clusters of NEET proteins mediate their key functions in health and disease[END_REF][START_REF] Valer | Histidine Ligated Iron-Sulfur Peptides[END_REF][START_REF] Gee | NRVS and DFT of MitoNEET: Understanding the Special Vibrational Structure of a [2Fe-2S] Cluster with (Cys)(3)(His)(1) Ligation[END_REF] and facilitates the in vitro cluster transfer from NEET proteins to an acceptor protein [START_REF] Karmi | A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics[END_REF]. CISD3 has 127 amino acids and, due to its significant cluster instability, it was necessary to mutate the coordinating histidine residue to a cysteine in order to crystallize it and to solve its X-ray structure [START_REF] Lipper | Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells[END_REF]. When the coordinating His is mutated to Cys, the protein ability to transfer the cluster is abolished, indicating that the Fe-bound His residue is crucial for cluster transfer. The coordinating His is exposed to protonation, suggesting a potential sensing function for this residue [START_REF] Tamir | Structure-function analysis of NEET proteins uncovers their role as key regulators of iron and ROS homeostasis in health and disease[END_REF]. In vitro, the NEET proteins behave as redox-sensors i.e., they are inert when in the reduced state, whereas in the oxidized state the two clusters can be transferred to apoacceptor proteins (16)[START_REF] Mons | The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein[END_REF][START_REF] Zuo | The two redox states of the human NEET proteins' [2Fe-2S] clusters[END_REF].

The interest in these proteins increased significantly when they were found to be potentially implicated in the regulation of reactive oxygen species (ROS), lipid and iron homeostasis, autophagy and apoptosis, and ageing [START_REF] Mittler | NEET Proteins: A New Link Between Iron Metabolism, Reactive Oxygen Species, and Cancer[END_REF][START_REF] Mons | The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein[END_REF][START_REF] Ferecatu | The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1[END_REF]. However, the exact functions of these proteins are still under discussion and their molecular mechanisms mostly unknown [START_REF] Salameh | New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET[END_REF]. The three human NEET proteins are overexpressed in various types of cancer [START_REF] Mittler | NEET Proteins: A New Link Between Iron Metabolism, Reactive Oxygen Species, and Cancer[END_REF]. In support of a possible role of CISD3 in cancer, changes in CpG methylation islands around the CISD3 gene have been observed and were associated with low expression levels in aggressive pediatric brain tumors [START_REF] Pérez-Ramírez | Genomics and epigenetics: A study of ependymomas in pediatric patients[END_REF]. Recently, CISD3 has been J o u r n a l P r e -p r o o f proposed as a regulatory factor in the process of ferroptosis, a non-apoptotic iron-dependent cell death [START_REF] Li | CISD3 inhibition drives cystine-deprivation induced ferroptosis[END_REF]. Inhibition of CISD3 cellular expression increases the sensitivity of the cell to inducers of ferroptosis. The use of ferroptosis as a weapon against tumors is of increasing interest, as tumor cells usually exhibit high endogenous oxidative stress levels [START_REF] Zhang | Ferroptosis in cancer therapy: a novel approach to reversing drug resistance[END_REF]. High intracellular iron concentrations can trigger ferroptosis by enhancing membrane lipid peroxidation, and this can be reverted using iron chelators [START_REF] Fanzani | Iron, Oxidative Damage and Ferroptosis in Rhabdomyosarcoma[END_REF]. CISD3 plays a role in mitochondrial function, morphology and dynamics, particularly in the regulation of the mitochondrial fission/fusion pathway [START_REF] Karmi | A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics[END_REF][START_REF] King | The mitochondrial localized CISD-3.1/CISD-3.2 proteins are required to maintain normal germline structure and function in Caenorhabditis elegans[END_REF]. A role of CISD3 in the biogenesis and function of complex I respiratory chain subunit NDUFV2 has also recently been proposed [START_REF] Marjault | CISD3 is required for Complex I function, mitochondrial integrity, and skeletal muscle maintenance[END_REF]. Finally, CISD3 [Fe2S2] clusters in their reduced [Fe2S2] + state, are capable of binding nitric oxide (NO) under anaerobic conditions in vitro (27,[START_REF] Cheng | Binding of Nitric Oxide in CDGSHtype [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2[END_REF]. NO is a small radical molecule that has a role in many physiological processes, such as angiogenesis, vasodilation, neurotransmission, inflammatory and immune responses [START_REF] Infante | Novel Insights Regarding Nitric Oxide and Cardiovascular Diseases[END_REF][START_REF] Ghasemi | Nitric Oxide and Mitochondrial Function in Neurological Diseases[END_REF][START_REF] Guzik | Nitric oxide and superoxide in inflammation and immune regulation[END_REF]. At the cellular level, NO is involved in the control of mitochondrial biogenesis and mitochondrial respiration, cellular signaling, cell proliferation and death [START_REF] Ghimire | Nitric oxide: what's new to NO?[END_REF]. In mammals, Nitric Oxide Synthase (NOS) enzymes synthesize NO by oxidizing Larginine to L-citrulline and NO [START_REF] Ghimire | Nitric oxide: what's new to NO?[END_REF]. The dysregulation of NO production generates a nitrosative stress, which has been implicated in several diseases [START_REF] Ghimire | Nitric oxide: what's new to NO?[END_REF][START_REF] Tuteja | Nitric Oxide as a Unique Bioactive Signaling Messenger in Physiology and Pathophysiology[END_REF][START_REF] Hirst | Nitric Oxide Physiology and Pathology[END_REF]. At the molecular level, reaction of NO with O2, O2 -and transition metal ions generates a variety of reactive nitrogen species (RNS) which induce lipid peroxidation, oxidative DNA damages, chemical modification of proteins and reactions with metal cofactors in metalloproteins [START_REF] Tuteja | Nitric Oxide as a Unique Bioactive Signaling Messenger in Physiology and Pathophysiology[END_REF][START_REF] Hirst | Nitric Oxide Physiology and Pathology[END_REF].

Within this framework, we have delineated for the first time the behavior of CISD3 overexpressed in human HEK-293 cells submitted to oxidative or nitrosative stress or in irondepletion conditions. We also have analyzed the in vitro spectroscopic properties of purified CISD3 binding Fe-S clusters in both reduced and oxidized states and compared the results with mitoNEET and CISD2 proteins. Nuclear Magnetic Resonance (NMR) spectroscopy, in combination with UVvisible absorption and Electron Paramagnetic Resonance (EPR) spectroscopies and cyclic voltammetry, provided a full description of the redox states of the clusters of CISD3 as well as of the kinetics of the redox reaction and of the cluster release. The extended NMR assignment of the reduced state of CISD3 [START_REF] Grifagni | Relaxation-based NMR assignment: Spotlights on ligand binding sites in human CISD3[END_REF][START_REF] Silva | 1)H, (13)C and (15)N assignment of the human mitochondrial paramagnetic iron-sulfur protein CISD3[END_REF] opens the possibility to monitor the structural stability of both reduced and oxidized forms and to understand which residues are affected by the oxidation event and which residues are involved in the interaction with nitric oxide.

J o u r n a l P r e -p r o o f

Results

In cellulo stability of CISD3: cellular levels are affected by iron depletion

We investigated the half-life of CISD3 in HEK-293 cells using the protein synthesis inhibitor cycloheximide (CHX). First, a V5-tagged CISD3 protein (hereafter CISD3-V5) was overexpressed, as our efforts to detect endogenous or untagged CISD3 with commercially available antibodies were unsuccessful. Cells were then treated with 50 µM CHX to study the decay of CISD3-V5 over a 48hours treatment (Figure 1A-B). The protein level of CISD3-V5 decreased slowly with a half-life of 53.5 ± 2.4 h, indicating that the protein is highly stable. Then, we treated HEK-293 cells overexpressing CISD3-V5 with two different iron chelators, desferrioxamine (DFO) and salicylaldehyde isonicotinoyl hydrazone (SIH), to assess the stability of CISD3-V5 under iron scavengers. Results showed that the protein level of CISD3-V5 decreased by 53.7 ± 5.7 % after a 24hour treatment with 50 μM DFO (Figure 1C and1E). A slightly larger decrease (69.6 ± 6.2 %) was observed with 25 μM SIH (Figure 1D-E). We then considered the possibility that CISD3-V5 cellular level decreases due to mitophagy induction, which may happen under iron limiting conditions [START_REF] Hara | Iron loss triggers mitophagy through induction of mitochondrial ferritin[END_REF].

However, the presence of 3-methyladenine and chloroquine, respectively to block autophagosome formation and to inhibit lysosomal hydrolytic degradation [START_REF] Yang | Application and interpretation of current autophagy inhibitors and activators[END_REF], did not allow any recovery of CISD3-V5 protein level. Similarly, proteasome inhibitors such as MG-132 or bortezomib did not prevent the decrease in CISD3-V5 protein amounts induced by iron chelators (Supporting Information Figure S1). This indicates that neither mitophagy nor proteasome-dependent proteolysis are responsible for the decrease in CISD3-V5 protein level observed in response to cellular iron depletion.

In vitro stability studies of CISD3

CISD3 has only two redox states

The half-life of CISD3-V5 and its sensitivity to cellular iron deprivation led us to characterize CISD3 redox states and to investigate their role in protein stability via in vitro studies. We attempted to determine the reduction potentials of the two clusters using protein film voltammetry. We observed a single voltammetric wave, narrower than the 91mV expected [START_REF] Jeuken | Electron-Transfer Mechanisms through Biological Redox Chains in Multicenter Enzymes[END_REF], which gives a reduction potential of -33mV vs SHE at pH 7.8 (Figure 2). Sharp voltammetric signals, as shown in Figure 2, are an indication of positive cooperativity between the redox states of the two clusters [START_REF] Léger | Enzyme electrokinetics: using protein film voltammetry to investigate redox enzymes and their mechanisms[END_REF]. The apparent Hill coefficient n of the signal is 1.24 considering the average of anodic and cathodic waves, which is very significant for a protein with two redox clusters [START_REF] Dalgleish | Biophysical Chemistry: Part III 'The Behaviour of Biological Macromolecules: by CR Cantor and PR Schimmel[END_REF]. A positive cooperativity as observed here, requires a redox-associated conformational change in order to overcome the electrostatic repulsion that would normally arise between the two Fe-S clusters as they receive electrons upon reduction. This means that CISD3 is either fully reduced (hereafter 2[Fe2S2] + CISD3 or reduced J o u r n a l P r e -p r o o f CISD3) or fully oxidized (hereafter 2[Fe2S2] 2+ CISD3 or oxidized CISD3), and it does not have any mixed redox state in the present conditions. Moreover, the relatively high reduction potential is consistent with the in cellulo EPR (Figure S2), which indicates that at least a significant fraction of the protein in E. coli cells, and possibly in human cells, is in the reduced form.

Oxidized CISD3 is less stable than reduced CISD3

Oxidized and reduced CISD3 have different UV-visible absorption and 2D 1 H- 15 N HSQC spectra that can be used to characterize the stability of the holo protein as a function of the oxidation state of the cluster. As shown in Figure 3A, the UV-visible absorption spectrum of the oxidized form shows a peak at 460 nm and a shoulder at 550 nm, while the reduced form presents two peaks at 430 nm and at 550 nm respectively [START_REF] Lipper | Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells[END_REF]. The stability of the two oxidation states was measured by monitoring, at 25 °C, the time evolution of the absorbance bands at 460 nm (for the oxidized form) and at 550 nm (for the reduced form). Under anaerobic conditions, reduced CISD3 is very stable and less than 10% of cluster loss was observed in 24h, without pH-dependence (Figure 3B). On the contrary, the spectrum of the oxidized protein under anaerobic conditions changes with time (Figure 3C-D). Bands at 460 nm and 550 nm decrease in intensity, indicating a cluster loss. At pH 7.0. a shift of the band at 460 nm to lower wavelengths is also observed, revealing the reduction of the clusters. The latter process is less evident at acidic pH, where the decrease of the intensity of the observed bands is faster (Figure 3D). The reaction progress of the oxidized CISD3 under anaerobic conditions is reported, both cluster loss and cluster reduction are observed as a function of pH and time (Figure 3E).

Cluster loss also happens under aerobic conditions as the instability of the oxidized protein is much higher and was found to slightly increase at acidic pH values (Figure S3). Moreover, the in vitro stability of oxidized holo-CISD3 in the presence of oxygen is significantly lower than the other NEET proteins (Figure 4). At pH 7.0 the half-life time of oxidized CISD3 under aerobic condition is about five times shorter than CISD2, whereas mitoNEET is at least 10 times more stable than CISD3 [START_REF] Mons | The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein[END_REF][START_REF] Salameh | New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET[END_REF].

The behavior observed for oxidized protein under anaerobic conditions at pH 7.0 suggests the possibility that cluster reduction is coupled to cluster loss, as already observed in mitoNEET [START_REF] Mons | Chapter Four -Combined Biochemical, Biophysical, and Cellular Methods to Study Fe-S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET[END_REF].

This can be better monitored by NMR spectroscopy. The overlay of diamagnetic-type and paramagnetic-type 1 H- 15 N HSQC spectra of holo-CISD3 for both the oxidized and the reduced state are shown in Figure 5A. An extended resonance assignment is available for the reduced form [START_REF] Grifagni | Relaxation-based NMR assignment: Spotlights on ligand binding sites in human CISD3[END_REF] and, due to the small chemical shift differences between the spectra of the two oxidation states, it can be extended to the oxidized form by comparison. As already observed by electronic absorption J o u r n a l P r e -p r o o f spectroscopy, under anaerobic condition the 1 H-15 N-HSQC spectra of the reduced form are stable with time, while for the oxidized form, the resonances of the reduced CISD3 appear at the expenses of corresponding signals of the oxidized form (Figure 5B). In about 300 min, at pH 7, the signals of the oxidized form completely disappear (Figure 5C). About 40% of reduced form is obtained over time. The intensity of peaks whose chemical shift does not change with the oxidation states, such as A53 and G54, provides information on the amount of holo-forms, because the signals of the apo-form are in a different spectral region. Their time evolution shows that 60% of the holo-protein loses clusters and precipitates over time (Figure 5D). Therefore, NMR spectroscopy clearly demonstrate the intrinsically instability of the oxidized CISD3 which loses the clusters even under anaerobic conditions. This event induced the reduction of the remaining holo-protein leading to a mixture of apo-and reduced holo-CISD3. The apo-protein has, in solution, no secondary structure, is unstable, and undergoes protein precipitation. This behavior is also observed when holo-CISD3 is treated with 5 mM ferricyanide and 10 mM of EDTA i.e, the protein partly precipitated and the UV-visible absorption spectrum showed no absorption in the visible part indicating a total loss of the clusters bound to the protein (Figure S4A). In these conditions, the 1 H- 15 N HSQC spectrum shows that amide resonances collapse in the region typical of an unfolded protein (Figure S4B). The latter behavior demonstrates that the [Fe2S2] clusters are required for the global folding and stability of CISD3, as previously observed for mitoNEET [START_REF] Ferecatu | The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1[END_REF] and CISD2 [START_REF] Salameh | New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET[END_REF].

Atomic level characterization of reduced and oxidized CISD3

The NMR assignment of the amide backbone resonances allowed us to perform a per-residue analysis of chemical shift perturbations between the two redox states (Figure 6A). At variance with mitoNEET, where no assignments are available in the 20 aminoacids region encompassing the metal coordinating residues and the α helix region [START_REF] Camponeschi | The long-standing relationship between Paramagnetic NMR and Iron-Sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?[END_REF], in the case of CISD3, tailored NMR approaches and relaxation-based assignments allowed us to identify chemical shift perturbation (CSP) values also for residues belonging to the regions in proximity of the two clusters [START_REF] Grifagni | Relaxation-based NMR assignment: Spotlights on ligand binding sites in human CISD3[END_REF][START_REF] Silva | 1)H, (13)C and (15)N assignment of the human mitochondrial paramagnetic iron-sulfur protein CISD3[END_REF] J o u r n a l P r e -p r o o f showed a very similar shift difference (0.092 vs 0.088). The paramagnetism of the cluster impedes the identification of NMR signals of residues close to the [Fe2S2] clusters, even when tailored approaches are used [START_REF] Trindade | NMR of paramagnetic metalloproteins in solution: Ubi venire, quo vadis?[END_REF][START_REF] Trindade | Sequencespecific assignments in NMR spectra of paramagnetic systems: A non-systematic approach[END_REF], as a consequence we do not have enough data for a reliable comparison among the two cluster sites. However, Figure 6B shows that larger perturbations occur in the Cterminal CDGSH domain and, therefore, the two cluster sites might not be equivalent. To provide atomic-level insights into the proximity of the two clusters, we performed 1 H NMR spectra of the reduced and oxidized CISD3. Paramagnetically-shifted 1 H NMR signals constitute a very unique and sensitive fingerprint for the characterization of the first coordination sphere [START_REF] Trindade | NMR of paramagnetic metalloproteins in solution: Ubi venire, quo vadis?[END_REF][START_REF] Trindade | Sequencespecific assignments in NMR spectra of paramagnetic systems: A non-systematic approach[END_REF][START_REF] Banci | Correction to: The NMR contribution to protein-protein networking in Fe-S protein maturation[END_REF][START_REF] Hsueh | NMR investigations of the Rieske protein from Thermus thermophilus support a coupled proton and electron transfer mechanism[END_REF]. As shown in protein reduction, no paramagnetic spectrum can be observed. Again, the same behavior of 1 H paramagnetic NMR spectrum has been observed for mitoNEET [START_REF] Camponeschi | The long-standing relationship between Paramagnetic NMR and Iron-Sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?[END_REF], thus confirming that the similarity between mitoNEET and CISD3 in terms of coordination sphere and electronic properties holds for both oxidation states.

Effects of H 2 O 2 and nitric oxide on CISD3 protein stability

Decreased expression of the CISD3-V5 protein in cells exposed to a chemical NO donor

A recent study has proposed that CISD3 is involved in the control of mitochondrial ROS levels and cell resistance to ROS [START_REF] Karmi | A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics[END_REF]. Moreover, CISD3 clusters can bind NO in vitro and NO-bound clusters become resistant to redox transition [START_REF] Cheng | Binding of Nitric Oxide in CDGSHtype [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2[END_REF][START_REF] Tinberg | Characterization of Iron Dinitrosyl Species Formed in the Reaction of Nitric Oxide with a Biological Rieske Center[END_REF], but how NO affects CISD3 protein level in cellulo is unknown. Therefore, we investigated the effects of H2O2 and NO on the CISD3-V5 protein There is thus a specificity in CISD3 instability in the presence of NO compared to CISD2 under the same conditions in cells.

In vitro study of the effect of H2O2.

We monitored the stability of CISD3 clusters in the presence of a large excess of H2O2 (250 µM) at different pH values, under anaerobic conditions, by using UV-visible absorption spectroscopy (Figure 9A). Addition of H2O2 on reduced NEET proteins in vitro is known to oxidize the cluster and we might expect that the same occurs in cellulo [START_REF] Landry | Redox control of human mitochondrial outer membrane protein MitoNEET [2Fe-2S] clusters by biological thiols and hydrogen peroxide[END_REF]. Then, the in vitro studies were performed only on the oxidized protein. At pH lower than 6.7, CISD3 clusters were highly labile upon H2O2 treatment, with a half-life shorter than 15 min. At higher pH values, the destabilization of the clusters by H2O2 treatment was less efficient, with a t1/2 in the range of 100-200 min for pH values between 6.7 and 8.0 (Table 1). NMR has also followed the induced oxidative stress. When two equivalents of H2O2 are added to the oxidized CISD3 under anaerobic conditions, a significant decrease of peak intensity is observed for the signals of the oxidized form, together with the appearance of new peaks in the 6.5-8.5 ppm region, typical of unfolded polypeptide chains. When four equivalents of H2O2 are added, only the spectrum of the unfolded protein appeared (Fig. 9B). The oxidative stress induced by H2O2 results in a complete loss of the two clusters and of the secondary structure of CISD3 in vitro.

In vitro study of the effect of NO

The interaction of CISD3 with NO was followed in vitro by UV-visible absorption, NMR and EPR spectroscopies, upon addition of Spermine NONOate. Spermine NONOate is a molecule that releases two molecules of NO and has a half-life of 40 minutes at pH 7.4 and 310 K (50). The UV-visible absorption spectrum, reported in Figure 10A, shows that the addition of Spermine NONOate to reduced CISD3 under anaerobic conditions gives rise to an intermediate species, monitored by the increase of the absorption bands at 430 nm. When the intensity of this band is plotted vs time (Figure 10A inset), a plateau is observed after about 40 minutes, corresponding to a ratio 1:2 protein:NO. This indicates that NO binds to the reduced clusters in CISD3 as previously proposed [START_REF] Cheng | Binding of Nitric Oxide in CDGSHtype [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2[END_REF]. When the J o u r n a l P r e -p r o o f concentration of NO increases, the UV-visible absorption spectrum evolves. The band at 430 nm decreases and the band at 550 nm disappears with time, indicating a cluster loss. A new absorption band at 366 nm is observed. The time dependence of the three bands (Figure 10A inset) indicates that the intermediate species observed after 40 min eventually disrupts the [Fe2S2] cluster, giving rise to a different metal center. The appearance of the 366 nm band has been already observed for Rieske proteins [START_REF] Tinberg | Characterization of Iron Dinitrosyl Species Formed in the Reaction of Nitric Oxide with a Biological Rieske Center[END_REF] and interpreted as the formation of dinitrosyl iron complexes (DNIC). This behavior is consistent with EPR spectra acquired on reduced CISD3 in the presence of increasing amounts of NO (Figure S5).

The interaction with NO has also been studied on the oxidized form of CISD3. At variance with the reduced form, UV-Visible absorption spectroscopy shows no evidence of a transient intermediate species nor of DNIC complexes. As shown in Figure 10B, the band at 460 nm shifts to 430 nm indicating the reduction of the cluster, consistent with the behavior already observed for oxidized CISD3 independently on the addition of NO. Once the cluster is reduced we observe that NO binds to the protein as confirmed by the slightly increase of the band at 430 nm. In about 12 hours, the cluster is lost and the DNIC is observed, confirming the behavior of the reduced form.

The addition of spermine NONOate to reduced CISD3 was also followed by 1 H-15 N-HSQC spectra. indicating that about 35% of the cluster is lost during the reaction. Consistently, the intensity of 1 H- 15 N HSQC signals of residues that are not subjected to any change upon NO binding, decrease to ca 65% of their initial intensity, as shown in Figure 11B for Ala 53 and Lys 55. Remarkably, residues that are closer to the cluster than Leu 51 and Ser 125, such as Lys 48 and Arg 58 show a different behavior i.e., the disappearance of the NO-free reduced species is 100% and it is not accompanied by the appearance of a NO-bound signal (Figure 11B-C). This can only be due to a longer electronic correlation time of Fe ions in the NO-bound form that causes a larger blind sphere. Signals that in the NO-free reduced CISD3 are affected by paramagnetic relaxation but are still observable and are broadened beyond detection in the NO-bound form. This is a very strong evidence that, upon NO binding, the cluster is disassembled but the iron ions are still bound to the protein, thus providing the experimental evidence that the DNIC complex [START_REF] Tinberg | Characterization of Iron Dinitrosyl Species Formed in the Reaction of Nitric Oxide with a Biological Rieske Center[END_REF][START_REF] Butler | Non-heme iron nitrosyls in biology[END_REF] is still bound to the protein, as already proposed for [Fe4S4] containing proteins [START_REF] Foster | Chemistry of Nitric Oxide with Protein-Bound Iron Sulfur Centers. Insights on Physiological Reactivity[END_REF]. In our hands, 1 H- 15 N HSQC spectra account for a 65% DNIC species, which is higher than 5% previously observed by Ding et al [START_REF] Cheng | Binding of Nitric Oxide in CDGSHtype [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2[END_REF]

Discussion

The in cellulo and in vitro characterization of human CISD3 provides a better understanding of the mechanisms underlying its function and permits the comparison with the other human NEET proteins, mitoNEET and CISD2. Our in cellulo experiments showed that CISD3 half-life in 50 μM CHX treated cells is about 50 h. In a previous study in HeLa cells [START_REF] Salameh | New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET[END_REF], mitoNEET half-life was found to be less than 6 hours and CISD2 to be highly stable (half-life higher than 48 h), meaning that mitoNEET is particularly unstable compared to the two other members of the NEET family, CISD2 and CISD3.

The high stability of CISD3 in unstressed cells suggests a functional role in fundamental cellular processes, whereas the instability of mitoNEET may indicate that it plays a role in adaptive cellular responses, as already proposed [START_REF] Ferecatu | The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1[END_REF]. From a structural point of view, NMR data demonstrate that, once the cluster is removed in vitro, CISD3 completely loses its 3D structure. The generated apoform is largely unfolded, unstable and rapidly precipitates. Consistently, CISD3 levels in iron deficiency conditions (cell treated with DFO) decrease by 50% in about 24 h, reproducing a trend similar to mitoNEET, which was barely detectable after 16 h [START_REF] Salameh | New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET[END_REF]. On the contrary, CISD2 showed no sensitivity to cellular treatment with iron chelators [START_REF] Salameh | New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET[END_REF]. However, the higher cellular instability of CISD3 and mitoNEET under conditions of iron deficiency with respect to CISD2 cannot be explained based on structural properties of their apo-forms i.e., CISD2 stability is potentially a consequence of a greater stability of the apo-CISD2 protein in cells or such different stability is probably linked to their respective functions.

J o u r n a l P r e -p r o o f

The results obtained by cellular studies raised the question whether CISD3 stability properties are linked to the release of its Fe-S clusters upon cluster oxidation. Protein film voltammetry showed two oxidation states for CISD3: the fully oxidized 2[Fe2S2] 2+ and the fully reduced 2[Fe2S2] + forms.

We have observed that the stability of the cluster in oxidized holo-CISD3 in the presence of oxygen is almost independent of the pH, whereas in mitoNEET it is highly dependent. Oxidized CISD3 loses the clusters also in the presence of H2O2 under initial anaerobic conditions in vitro. However, the pHdependence of cluster stability in the presence of hydrogen peroxide is not as pronounced as in the case of CISD2 and mitoNEET, which are practically insensitive to hydrogen peroxide at pH 8 (Table 1) [START_REF] Mons | The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein[END_REF][START_REF] Salameh | New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET[END_REF]. Although CISD3 Fe-S clusters are very labile in the presence of H2O2 in vitro, we did not observe a decrease in CISD3 protein levels in H2O2-treated HEK-293 cells. An explanation could be that CISD3 is protected from the effects of H2O2 by a partner in the cell, as it has been demonstrated in the case of bacterial nitrogenase and thiol-based peroxidase [START_REF] Schlesier | A Conformational Switch Triggers Nitrogenase Protection from Oxygen Damage by Shethna Protein II (FeSII)[END_REF][START_REF] Fourquet | The dual functions of thiol-based peroxidases in H2O2 scavenging and signaling[END_REF].

3D structures of the NEET proteins may explain the intrinsic pH-independent instability of oxidized CISD3. Although all members of the family share a common structural core, mitoNEET and CISD2 encompass key inter-subunit interactions, which are missing in CISD3. In mitoNEET, the interaction between the side chain of Lys 55 and His 87 has been proposed to be the driving factor controlling redox-dependent and pH dependent solvent accessibility and, as a consequence, the overall protein stability [START_REF] Gee | NRVS and DFT of MitoNEET: Understanding the Special Vibrational Structure of a [2Fe-2S] Cluster with (Cys)(3)(His)(1) Ligation[END_REF][START_REF] Bak | Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding[END_REF]. In monomeric CISD3, this intersubunit interaction is missing and not replaced, this causes a larger solvent exposure of the reducible iron ion with respect to mitoNEET and gives the molecular basis of the lower and pH independent protein stability of CISD3. In agreement, protein film voltammetry shows that the redox potential remains constant at an average value of ca. -31 mV vs SHE with a standard deviation of 6 mV in the pH range 6.5 to 8.3 (Table S1). On the contrary, pronounced pH-dependence has been found for both mitoNEET and CISD2, with redox potential values ranging from about 0 mV at pH 7 to about -50 mV at pH 8.5 [START_REF] Bak | Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding[END_REF][START_REF] Conlan | Crystal structure of Miner1: The redox-active 2Fe-2S protein causative in Wolfram Syndrome 2[END_REF].

Further insights into the molecular and structural aspects arise from the analysis of NMR data obtained on the two redox states. The distribution of chemical shift perturbations is odd as the largest perturbations are all localized on the β-strands close to the C-term cluster, while corresponding residues at the N-term are affected to a much lesser extent. In metalloproteins, redox shifts can be due either to the change in the electronic structure of the paramagnetic center [START_REF] Miao | Paramagnetic Chemical Probes for Studying Biological Macromolecules[END_REF][START_REF] Welegedara | Double-Arm Lanthanide Tags Deliver Narrow Gd3+-Gd3+ Distance Distributions in Double Electron-Electron Resonance (DEER) Measurements[END_REF][START_REF] Nitsche | Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags[END_REF][START_REF] Bertarello | Pico-meter resolution structure of the coordination sphere in the metal-binding site in a metalloprotein by NMR[END_REF][START_REF] Cerofolini | How Do Nuclei Couple to the Magnetic Moment of a Paramagnetic Center? A New Theory at the Gauntlet of the Experiments[END_REF] or to a conformational change associated to the redox process [START_REF] Messias | Solution structure of Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3: structural basis for functional cooperativity[END_REF][START_REF] Paquete | Molecular basis for directional electron transfer[END_REF]. Several studies indicate that redox shifts in Fe-S proteins are not related to the different paramagnetism between oxidized and reduced clusters, but they can be associated with conformational rearrangements [START_REF] Lehmann | Redox-related chemical shift perturbations on backbone nuclei of high-potential iron sulfur proteins[END_REF][START_REF] Pochapsky | Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin[END_REF][START_REF] Xia | Evidence for oxidation-state-dependent conformational changes in human ferredoxin from multinuclear, multidimensional NMR spectroscopy[END_REF]. In CISD3, a remarkably large redox shift is observed for Lys 48, which is close to the H-bonded residues Ile 47-Leu 97, the latter preceding the iron bound Cys 98 (Figure 6B). Both Ile 47 and Leu 97 are too close J o u r n a l P r e -p r o o f to the iron ions to be observed, however the perturbation observed on Lys 48 suggests that structural changes associated with the redox event may occur at the C-terminal site. Another protein fragment that might be concerned with redox driven structural rearrangements is the terminal α-helix of the CDGSH domain: this helix includes the cluster bound histidine (His 75/His 113) and, from the structural point of view, seems to be important to control the accessibility of the cluster. Also, for this fragment, the large perturbation observed for Ser 115 points out the possibility that structural rearrangements specifically occur at the C-terminal site of the protein (Figure 6B). Remarkably, the amino acids of the C-terminal site of CISD3 are conserved compared to mitoNEET, whereas the α helix at the N-terminal contains two hydrophobic residues (Phe 76 and Phe 77) that are not present in mitoNEET. It seems therefore that when the C2 symmetry of mitoNEET (and CISD2) is broken and the two clusters have similar but not identical environments, a conformational change around the C-terminal cluster gives rise to an "open" conformation for the oxidized state. This, redox driven, conformational switch is likely the molecular basis of the protein instability in the oxidized state i.e., the open conformation allows the release of the oxidized clusters. This finding is also completely consistent with the single reduction potential with a cooperative behavior of the two clusters observed by protein film voltammetry. The allosteric communication between the two clusters has been also

shown in silico in mitoNEET [START_REF] Zuo | The two redox states of the human NEET proteins' [2Fe-2S] clusters[END_REF]. Residues of mitoNEET that are suggested, from a co-evolutionary analysis [START_REF] Zuo | The two redox states of the human NEET proteins' [2Fe-2S] clusters[END_REF], to provide crosstalk between the two clusters are retained also in CISD3, explaining the single redox potential observed for two clusters with different environments.

The analysis of the 3D protein structure barely explains that CISD3 protein levels in cells are not affected by H2O2 whereas they are dramatically affected by NO. In mammals, nitric oxide is produced by three different NO synthase enzymes present in a large variety of cells and a plethora of biological effects have been attributed to NO under physiological or pathophysiological conditions [START_REF] Clementi | Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione[END_REF][START_REF] Drapier | Interplay between NO and [Fe-S] clusters: relevance to biological systems[END_REF]. A number of Fe-S cluster-containing proteins in prokaryotes and eukaryotes have been identified both as sensitive sensors of NO and effectors of a cellular adaptive response to damaging NO levels [START_REF] Mettert | Fe-S proteins that regulate gene expression[END_REF][START_REF] Vine | Nitrosative stress in Escherichia coli: reduction of nitric oxide[END_REF][START_REF] Spiro | Regulators of bacterial responses to nitric oxide[END_REF]. However, only a handful of studies have questioned the reactivity of NEET proteins towards NO and the effects of NO on NEET protein structure, function, stability and subcellular distribution.

Earliest studies have shown no binding of NO to mitoNEET in vitro and no mitoNEET protein instability in cells exposed to NO injury. Indeed, Ferecatu et al. suggested a protective role for mitoNEET in the repair of NO-mediated damaging of some Fe-S cytosolic proteins such as the IRP-1/cytosolic aconitase protein, a central regulator in cellular iron metabolism [START_REF] Ferecatu | The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1[END_REF]. However, the inability of NO to bind to mitoNEET has been questioned in a more recent study [START_REF] Fontenot | Nitric oxide reversibly binds the reduced [2Fe-2S] cluster in mitochondrial outer membrane protein mitoNEET and inhibits its electron transfer activity[END_REF] and, to date, mitoNEET sensitivity to NO is still an open question. By contrast, the work of Ding's group [START_REF] Cheng | Binding of Nitric Oxide in CDGSHtype [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2[END_REF] and

J o u r n a l P r e -p r o o f

this study conclusively demonstrated that NO affects CISD3 in several ways. In cells, we disclosed for the first time a complete degradation of CISD3 induced by prolonged exposure to DETA-NO, a compound mimicking high-throughput NO synthase 2 activity. CISD2 levels were not affected suggesting that NO is not able to interact with this NEET protein, as already proposed in vitro [START_REF] Cheng | Binding of Nitric Oxide in CDGSHtype [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2[END_REF].

Our experiments by UV-visible absorption and EPR spectroscopy provide evidence for NO binding to reduced CISD3, with formation of DNIC, as previously published [START_REF] Cheng | Binding of Nitric Oxide in CDGSHtype [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2[END_REF]. Likewise, oxidized CISD3 did not react with NO. NMR indicates that [Fe2S2] + clusters are disrupted in NO-bound CISD3. This might explain the progressive breakdown of CISD3 in cells exposed to NO as the apo-protein is not stable in cells. Our experiments showing that iron deprivation decreases CISD3 stability also support this hypothesis.

Nitric oxide regulates several important functions and processes in mitochondria, where CISD3 is located. For instance, NO inhibits oxidative phosphorylation and regulates mitochondrial biogenesis and dynamics by different mechanisms including binding to metalloproteins and S-nitrosylation of key proteins [START_REF] López-Sánchez | Nitric oxide and tumor metabolic reprogramming[END_REF]. Dysregulated production of NO in human tumors has been correlated with the degree of malignancy and poor prognosis for survival [START_REF] Ekmekcioglu | Tumor iNOS predicts poor survival for stage III melanoma patients[END_REF][START_REF] Ko | Prognostic significance of three endothelial nitric oxide synthase (eNOS) polymorphisms and metabolic syndrome (MetS) in patients with colorectal cancer[END_REF]. NO contributes to metabolic reprogramming in tumors, in particular by stimulating mitochondrial biogenesis. It has been reported that CISD3 expression is largely elevated in various human cancers [START_REF] Li | CISD3 inhibition drives cystine-deprivation induced ferroptosis[END_REF] and it can exert a regulatory role in tumor progression [START_REF] Jiang | FGF4 improves hepatocytes ferroptosis in autoimmune hepatitis mice via activation of CISD3[END_REF]. Our study showing that CISD3 is a new target for NO in mitochondria warrants further investigations to determine to what extent NO binding to CISD3 may affect mitochondrial functions, especially in cancer cells exhibiting a dysregulated expression of NO synthase enzymes.

J o u r n a l P r e -p r o o f .

Experimental procedures

Cell line, culture and obtainment of stable CISD3-V5 expressing HEK-293

Human HEK-293 cells were routinely cultured in DMEM medium containing 4.5 g/L glucose, 2 mM L-glutamine, 10% Foetal Calf Serum and penicillin/streptomycin antibiotics. Cells were transfected using a Lipofectamine 2000 mediated transfection by a pcDNA3.1 plasmid expressing human CISD3 fused to a V5 tag (GKPIPNPLLGLDST) at its C-terminal end. Two days after transfection, cells were selected with G-418 at 1 mg/mL for two weeks. The resulting population was tested by immunofluorescence for V5 expression, which was localized exclusively in the mitochondria, and used for further experiments.

Cell Treatments with DFO, SIH, CHX, H 2 O 2 and DETA-NO

Stable CISD3-V5 expressing HEK-293 cells were seeded at 0.34 x 10 6 cells/cm 2 , incubated with either desferrioxamine (DFO) or salicylaldehyde isonicotinoyl hydrazone (SIH), or 50 μM of cycloheximide (CHX) for different times. Cell treatment with a protein synthesis inhibitor such as cycloheximide (CHX) is widely used to determine the half-life of a given protein [START_REF] Kao | Analysis of Protein Stability by the Cycloheximide Chase Assay[END_REF]. CHX inhibits translational elongation by interfering with the translocation step in protein synthesis. Treatment of the cells with CHX in a time-dependent decay experiment followed by western blotting of the cell lysates can provide an estimation of the protein half time. For NO and H2O2 challenge studies, CISD3-V5 overexpressing HEK-293 cells were seeded the day before and then exposed or not to DETA-NO or H2O2. The treatment was performed at increasing concentrations (0, 25, 50, 100 and 200 μM) for H2O2 and (0, 62,5, 125, 250 and 500 μM) for DETA-NONOate. Then we selected 100 μM and 250 μM concentrations for H2O2 and DETA-NONOate respectively to evaluate the half-life of CISD3-V5, quantifying protein amount at 0, 1, 5, 11 and 24 h. Autophagy inhibitors (3-methyladenine and chloroquine) were used with and without DFO 50 μM at 5 mM and 50 μM, respectively. Chemicals except DETA-NO (Cayman Chemical) were from Merck.

Immunoblot analysis of cell extracts

Total protein extracts from HEK-293 cells were obtained by harvesting cells in Laemmli buff er (0.06 M Tris HCl pH 6.8, 10% glycerol, 2% SDS) containing protease inhibitors (Sigma-Aldrich # P8340) and 2 mM Pefabloc (Calbiochem). Protein concentrations were determined using the DC protein assay (Bio-Rad). Equal amounts (30 μg) of proteins were separated on a 16% acrylamide SDS-PAGE and transferred on 0.45 µm of Polyvinylidene Difluoride (PVDF) membranes. The primary antibodies used were: anti-α-tubulin (Sigma-Aldrich #A5441) 1:1000, anti-CISD2 J o u r n a l P r e -p r o o f (Proteintech #13318-1-AP) 1:1000, anti-GADPH (Novusbio #NBP2-37563) 1:500, and anti-V5 (Novus Biologicals # NB600-381) 1:2000. Secondary antibodies were Alexa Fluor680-conjugated goat anti-mouse IgGs (A-21057, Life Technologies) 1:10000 and IRDye 800CW-conjugated goat anti-rabbit IgGs (926-32211, Li-Cor Biosciences) 1:15000.

Purification of holo-CISD3 for in vitro studies

The CISD3 ORF without the mitochondrial targeting signal peptide (amino acids 37 to 127) was inserted into a pET28a(+) plasmid. The plasmid was used to transform E. coli BL21 (DE3) GOLD competent cells. Cell growth was performed at 37 °C in either Luria-Bertani (LB), for nonlabelled protein, or in M9 minimal media supplemented with 1.2 g/L of ( 15 NH4)2SO4, 3 g/L of glucose, 4 mL of Q solution (metal mix solution: 50mM FeCl3, 20mM CaCl2, 10mM MnCl2, 10mM ZnSO4, 2mM of each of CoCl2, CuCl2, NiCl2, Na2MnO4, Na2SeO3, H3BO3). 500 μM Mohr's Salt was added when the cell culture reached an OD600 of 0.6. At an OD600 of 0.8-1, the temperature was lowered to 18°C and protein overexpression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were incubated at 18°C overnight and centrifuged.

Cells were resuspended in 80 mL of 20 mM Tris-HCl pH 7.5 buffer and lysed by adding CelLytic Reagent (0.8 g / 1 L culture) (Merck). After centrifugation, the supernatant was loaded on a 5 mL HiTrap SP FF cationic exchange column. The column was washed with a linear NaCl gradient (0-500 mM) until CISD3 protein solution eluted with 20 mM Tris-HCl pH 7.5 300 mM NaCl. The fractions containing the protein were collected, concentrated and injected in Superdex 200 Increase 16/60 75 pg previously equilibrated with Tris-HCl 50 mM NaCl 150 mM pH 7.5 buffer. All the buffers employed for purification were rendered oxygen-depleted by introducing a nitrogen flux into the solution. The iron content of the reduced CISD3, determined following a procedure reported in [START_REF] Nechushtai | Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism[END_REF][START_REF] Lipper | Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin, a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis[END_REF][START_REF] Banci | Anamorsin is a 2Fe2S cluster-containing substrate of the Mia40-dependent mitochondrial protein trapping machinery[END_REF][START_REF] Maione | CIAO3 protein forms a stable ternary complex with two key players of the human cytosolic iron-sulfur cluster assembly machinery[END_REF], is consistent with an 85% metallated protein (~1. 70 [Fe2S2] 2+ clusters per CISD3 protein).

In Vitro CISD3 cluster loss

UV-Visible absorption spectroscopy was used to evaluate the kinetic of cluster loss of CISD3 cluster in both redox states, under aerobic and anaerobic conditions and at different pH. The purification of CISD3 was carried out in an aerobic environment, following the same protocol described above for the anaerobic purification. This ensures that the initial redox state of CISD3 is oxidized. After purification, the protein was concentrated and then put in an anaerobic environment. Cluster loss reactions were performed in degassed buffer (100 mM BisTris 100 mM NaCl for pH 5.8, 6.3 and pH 7 or 100 mM Tris-HCl 100 mM NaCl for pH 8). Reduced CISD3 was obtained by addition of 20 mM sodium dithionite. The anaerobic samples were sealed under an anaerobic atmosphere, whereas the J o u r n a l P r e -p r o o f aerobic samples were exposed to the oxygen in the atmosphere. Cluster loss reactions were followed by the acquisition of (240-900 nm) UV-visible absorption spectra over time by Cary 100 (Agilent, Santa Clara, CA, USA) spectrophotometer equipped with a temperature control apparatus set at 25°C. Cluster loss was measured for each time t using R(t) = (A(t)-Ainitial)/(Afinal-Ainitial) with R(t), the reaction progress at time t, A the absorbance at 460 nm for the oxidized cluster and 550 nm for the reduced cluster and Ainitial and Afinal, the absorbance at the beginning and at the end (when the cluster loss is complete) of the reaction, respectively.

NMR spectroscopy

A combination of NMR experiments was either recorded on Bruker AVANCE 500 MHz, 900 MHz, and 950 MHz spectrometers, on 0.3-0.5 mM 15 N-labelled samples in 20 mM Tris-HCl buffer, 150 mM NaCl, pH 7.5, 10 % (v/v) D2O, at 298 K. All NMR spectra were collected and processed using Bruker software (Topspin 3.6). 1 H- 15 N HSQC spectra were recorded on purified CISD3 protein in its reduced 2[Fe2S2] + and oxidized 2[Fe2S2] 2+ forms with the following parameters: 56 ms and 31 ms for direct (t2max) and indirect (t1max) acquisition times and a recycle delay of 1 s. A data-matrix of 1024x128 complex points, with 8 transients each increment, was collected. The auto-reduction process of 2[Fe2S2] 2+ CISD3 was monitored recording 1 H- 15 N HSQC experiments at 298 K on a 900 MHz spectrometer along a period of 10 h, with the parameter set described above. The paramagnetism tailored 1 H-15 N HSQC-AP experiments [START_REF] Ciofi-Baffoni | The IR-(1)(5)N-HSQC-AP experiment: a new tool for NMR spectroscopy of paramagnetic molecules[END_REF] were recorded using the following parameters: 27 ms and 31 ms for direct (t2max) and indirect (t1max) acquisition times and a recycle delay of 150 ms. A data-matrix of 1024x128 complex points, with 128 transients each increment, was collected. The interaction between NO and both oxidized and reduced forms of CISD3 was monitored at 11.7 and 21.1 T fields at 298 K, using a standard SOFAST-HMQC pulse sequence with the following parameters: 35 ms and 20 ms for direct (t2max) and indirect (t1max) acquisition times and a recycle delay of 200 ms. A data-matrix of 1024x128 complex points, with 16 transients each increment, was collected [START_REF] Schanda | SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds[END_REF]. 15 N-labeled samples were prepared to a final concentration of 0.5 mM (20 mM Tris-HCl buffer, 150 mM NaCl, pH 7.5, 10 % (v/v) D2O) and spermine NONOate was added up to a 1:2 protein:NO ratio. The NMR titration with H2O2 was performed on 0.6 mM 15 N-labelled CISD3 protein samples by adding 2 equivalents and 4 equivalents of H2O2, to a final concentration of 2.4 mM.

Protein film voltammetry

Protein film voltammetry was performed at 25ºC inside an anaerobic glove box (Coy) using a double walled vessel thermostated using an external water bath. A potentiostat (CH Instruments J o u r n a l P r e -p r o o f 1201A) was connected to a PGE working electrode, a graphite counter electrode, and an Ag/AgCl reference electrode (CHInstruments). The working electrode was treated for 5 minutes in 1 M nitric acid and thoroughly washed with double distilled water. It was then polished with Alumina (1 μm and 0.3 μm) washed thoroughly with double distilled water, and left to dry inside the glovebox. 10 μl of the 650 μM protein solution were deposited in the electrode and left to dry for at least 1 hour. The electrode was placed in the buffer solution (50 mM phosphate equilibrated at various pH values from 6.5 to 8.2) and three complete scans were collected at a scan rates of 50 and 100 mV/s. The pH of the solutions was checked after each experiment. Raw voltammograms were processed with SOAS [START_REF] Fourmond | SOAS: a free program to analyze electrochemical data and other one-dimensional[END_REF] to subtract the capacitive current and the position of the anodic and cathodic peaks was averaged and is reported vs SHE by adding 210 mV to the value obtained vs Ag/AgCl.

EPR spectroscopy

EPR spectra were acquired using a Spectrometer ELEXSYS E580 operating with at 9.8 GHz continuous wave X-band, with a power level of 0.1 mW, 100 kHz modulation frequency, 1.2 mT amplitude modulation, centered at 350 mT with a field sweep of 200 mT and at 20 K.

In-cell sample preparation E. coli BL21 (DE3) GOLD were transformed with the pET28a(+) plasmid containing CISD3 gene (amino acids 37 to 127). A 5 mL-preculture in LB medium was incubated overnight at 37°C. 1 mL of this preculture was inoculated into 100 mL of LB and cells were grown at 37°C up to OD600 of 0.8. Subsequently, protein overexpression was induced overnight at 18°C by addition of 0.1 mM IPTG. The cells were pelleted by centrifugation and washed twice with M9 medium to remove the culture medium containing waste products. The blank was performed following the same procedure with the only exception that protein overexpression was not induced. Sample cell numbers were normalized by measuring the OD600. Cells were resuspended in 300 μL of PBS containing 20% glucose and placed in the EPR tube which was frozen in liquid nitrogen.

In-vitro sample preparation

Protein samples were prepared in a buffered solution containing 50 mM phosphate, 150 mM NaCl, pH 7.5 at a concentration of 40 μM. To monitor the interaction of CISD3 protein with NO we added 1 or 2 equivalents of spermine-NONOate in a stock solution and let it react up to 24 hours. At The CSP shifts larger than 0.3 ppm are highlighted with spheres. In gray are shown the unassigned residues. The iron and sulfur atoms of the Fe2S2 cluster are shown as orange and yellow spheres respectively, the cysteine residues are labelled with their respective numeration.

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

  . Major differences are observed for residues Lys 48 and Ser 115, both next to the C-terminus Fe-S cluster (Ser 115 is two residues after the histidine ligand of the C-terminal cluster, part of the terminal α-helix). Their corresponding residues in the N-terminal part are Lys 86 and Phe 77, respectively. No assignment is available for Phe 77 while Lys 86 experiences a very small perturbation (0.013 ppm) below the threshold level (ca. 0.03 ppm). Differences above 0.1 ppm for residues close to the N-terminal Fe-S cluster are observed only for Lys 66 and Thr 80, whose corresponding residues in the C-terminal, respectively Gln 104 and Val 118, are not assigned. Among the other residues showing CSP above the threshold level, Phe 70 (N-terminal domain) and the corresponding Tyr 108 (C-terminal domain)

Figure 7 ,

 7 Figure 7, oxidized CISD3 shows five hyperfine shifted signals spread between ca. 60 and 25 ppm. The spectrum is very similar to that obtained for oxidized mitoNEET, both in terms of number of signals and of their linewidths (43). The spread of proton signals in mitoNEET and CISD3 is larger (15-60 ppm) than in any of the previously investigated [Fe2S2] 2+ proteins, indicating that the unusual 3Cys-1 His coordination provides distinctive and unique features. Signals of the oxidized forms decrease in intensity with time and almost completely disappear in about 16 hours, as observed with the other techniques. The large hyperfine shifts indicate that the observed signals belong to the residues that are directly bound to the iron ions, i.e. Cys βCH2/αCH and/or His imidazole. The similarity with the spectrum of oxidized mitoNEET suggests that, albeit sequence inequivalence between the two CDGSH domains occurring in CISD3, the geometry and the electronic structure of the ligands of the C-terminal and N-terminal clusters are spectroscopically indistinguishable. Upon

  overexpressed in HEK-293 cells. Cells were treated with increasing concentrations of H2O2 and DETA-NO, a long-lived nitric oxide donor mimicking inducible nitric oxide synthase 2 activity (t1/2 = 20 h at 37°C and pH 7.4), for 24 hours (see Material and Methods for details). At the highest concentrations tested (250 and 500 µM), steady-state concentrations of NO released by DETA-NO J o u r n a l P r e -p r o o f are in the micromolar range. Kinetics experiments were also performed using 100 µM H2O2 and 250 µM DETA-NO. CISD3-V5 protein levels were quantified by immunoblotting, together with CISD2 for comparison. As reported in Figure 8A-B, H2O2 treatments did not affect CISD3 and CISD2 protein levels. By contrast, nitric oxide induced a dramatic and dose-dependent reduction in CISD3 levels, leading to an almost complete loss of the protein after a 24-hour treatment with 250 µM DETA-NO (Figure 8C-D). CISD2 was much more resistant than CISD3 to nitric oxide and underwent only a 25% decrease in its level after 24h in the presence of 250 or 500 µM DETA-NO.

For 1 :

 1 2 protein:NO ratio, a few residues (Gly 36, Leu 51, Thr 56, Ala 89, Met 94, Gln 119 and Ser 125) showed a second NMR signal that appears in slow exchange with the reduced form, as shown in Figure 11A for residues Leu 51 and Ser 125. After about 300 min, the signals of the reduced CISD3 form vanished and the intensity of the NO bound form is about 65% of the initial intensity,

  using EPR. However, J o u r n a l P r e -p r o o f the quantification of the detected DNIC species is affected by several factors, i.e. NO-donor half-life, [Fe2S2] clusters and NO concentrations. In agreement with what was observed by UV-Visible absorption spectroscopy, the 1 H-15 N HSQC experiments also show that oxidized CISD3 does not bind NO. The protein undergoes reduction and precipitation over time (Figure S6), as already observed for the oxidized protein without NO. In conclusion, UV-Visible absorption, EPR and NMR spectroscopies provide evidence that NO binds only the reduced form of the [Fe2S2] clusters of CISD3, forming an intermediate which evolves in thiolate-ligated DNIC species. The blind sphere observed in the NMR spectra of the NO bound species indicates that DNIC species are still bound to iron ions within the protein frame. NO does not interact with oxidized CISD3; instead, the protein undergoes in part cluster loss and reduction of the still bound cluster as observed in the absence of NO.

5 ,

 5 210 and 1440 minutes from the addition of the molecule, 200 μL were collected in an EPR tube for data acquisition.

Figures LegendsFigure 1 .

 1 Figures Legends

Figure 2 .Figure 3 .

 23 Figure 2. Protein film voltammetry of CISD3 adsorbed on a PGE electrode at pH 8.0 and at a scan rate of 50 mV/s. The raw voltammogram in grey is reported versus the right-hand vertical axis and the data obtained upon subtraction of the capacitive current is reported in black versus the left-hand vertical axis.

Figure 4 :

 4 Figure 4: Comparison between [Fe2S2] 2+ clusters half-time of CISD1 mitoNEET (mNT, blue) CISD2 (black) and CISD3 (red) at different pH values exposed to air. The half-time corresponds to the time necessary to lose half of the cluster. Data have been obtained by measuring the 460 nm absorption band using UV-visible absorption spectroscopy.

Figure 5 .Figure 6 .

 56 Figure 5. Stability of the oxidized and reduced CISD3 monitored by NMR (A) diamagnetic 1 H-15 N-HSQC (left) and paramagnetic 1 H-15 N-HSQC (right) of reduced (red) and oxidized (blue) CISD3. (B) Insets of the Ala 121, Val 49 and Lys 120 in the 1 H-15 N-HSQC spectra of the oxidized CISD3 at different time points: blue (0 minute), red (200 minutes) and black (360 minutes). (C) Normalized peak areas of residues Lys 120 (red), Val 49 (blue) and Ala 121 (green) in slow exchange. (D) Normalized peak areas of residues Ala 53 (red) and Gly 54 (blue) that only experience a decay in peak area.

Figure 7 :Figure 8 .

 78 Figure 7 : 1 H unidimensional spectra of reduced (top) and oxidized (bottom) CISD3. A-E letters correspond to the Hβ protons of cluster coordinating cysteines.

Figure 9 . 8 (Figure 10 .Figure 11 .

 981011 Figure 9. CISD3 sensitivity to H2O2 in vitro. (A) Cluster loss reactions with 20 µM protein and 250 µM H2O2 were performed at 25 °C under anaerobic conditions and followed by UV-visible absorption spectroscopy. The intensity of the peak at 460 nm was monitored as a function of the time at various pH values. The reaction buffer (50 mM Tris HCl or Bis-Tris with 100 mM NaCl) was at pH 5.8 (blue) 6.2 (red), 6.7 (green), 7.0 (purple) and 8.0 (black), respectively. (B) 1 H-15 N-HSQC spectrum of CISD3 240 minutes after the addition of four equivalents of hydrogen peroxide (H2O2). Protein concentration was 600 µM in 20 mM Tris-HCl, 150 mM NaCl buffer at pH 7.5.

Acknowledgements

The authors are grateful to Inês B. Trindade for helpful discussions on the electrochemical data.

Funding and additional information

We acknowledge financial support from the Agence Nationale de la Recherche (ANR-21-CE44-0016) and from the European Union -NextGenerationEU -National Recovery and Resilience Plan, Mission 4 Component 2 -Investment 1.5 -THE -Tuscany Health Ecosystem -ECS00000017 -CUP B83C22003920001. This work was funded also by national funds through FCT-Fundação para a Ciência e a Tecnologia, I.P. (FCT), Project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020, and LS4FUTURE Associated Laboratory (LA/P/0087/2020). Cindy Vallières was supported by Marie Skłodowska-Curie Actions MSCA-IF-2020 (FungiFeS/ID 101030584). Deborah Grifagni was supported by European Molecular Biology Organization (EMBO) through Grant Application Number 9373 and by PHC Galileo PROJECT N° 48929TH. L.B.,F.C. and M.P. acknowledges the support by the Italian Ministry for University and Research (FOE funding) to the CERM/CIRMMP Italian Centre of Instruct-ERIC, a ESFRI Landmark.

Data availability

All the data described in the manuscript are contained within it.

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Authorship contributions

DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

J o u r n a l P r e -p r o o f