# The ISC machinery assembles [2Fe–2S] clusters by formation and fusion of [1Fe–1S] precursors

Sylvain Gervason, Rafal Dutkiewicz, Kristian Want, Rania Benazza, Rémi Mor-Gautier, Aneta Grabinska-Rogala, Christina Sizun, Oscar Hernandez-Alba, Sarah Cianferani, Bruno Guigliarelli, et al.

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Sylvain Gervason, Rafal Dutkiewicz, Kristian Want, Rania Benazza, Rémi Mor-Gautier, et al. The ISC machinery assembles [2Fe–2S] clusters by formation and fusion of [1Fe–1S] precursors. Nature Chemical Biology, 2025, 21 (5), pp.767-778. 10.1038/s41589-024-01818-8. hal-05348439

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Submitted on 5 Nov 2025

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Biosynthesis of [2Fe2S] clusters via assembly and fusion of [1Fe1S]
precursors by the ISC machinery

Sylvain Gervason,<fnref n="1" />± Rafal Dutkiewicz,<fnref n="2" />± Kristian Want,<fnref n="1" />† Rania Benazza,<fnref n="3" />,<fnref n="4" />† Rémi Mor-
Gautier,<fnref n="1" />† Aneta Grabinska-Rogala,<fnref n="2" /> Christina Sizun,<fnref n="5" /> Oscar Hernandez-Alba,<fnref n="3" />,<fnref n="4" /> Sarah
Cianferani,<fnref n="3" />,<fnref n="4" /> Bruno Guigliarelli,<fnref n="6" /> Bénédicte Burlat,<fnref n="6" /> Benoit D'autréaux<fnref n="1" />*

<footnote n="1">¹ Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France</footnote>

<footnote n="2">² Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland</footnote>

<footnote n="3">³ Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France</footnote>

<footnote n="4">⁴ Infrastructure Nationale de Protéomique ProFI, FR2048 CNRS CEA, Strasbourg, France</footnote>

<footnote n="5">⁵ Institut de Chimie des Substances Naturelles, CNRS, Université Paris-Saclay, avenue de la terrasse, 91190 Gif-sur-Yvette, France</footnote>

<footnote n="6">⁶ Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines (BIP), 31 chemin Joseph Aiguier 13402, Marseille, France</footnote>

<footnote>±, † These authors equally contributed</footnote>
ABSTRACT

Iron-sulfur (Fe-S) clusters are essential metallo-cofactors biosynthesized by multi-protein machineries.
The core ISC machinery assembles [2Fe2S] clusters on the IscU scaffold protein using sulfur provided
in the form of a cysteine-bound persulfide by the IscS cysteine desulfurase and electrons delivered by
the Fdx-FdxR ferredoxin reductase system. Here we report a stepwise study of the Fe-S cluster
assembly process using an *in vitro* reconstituted *E. coli* ISC machinery and a combination of
biochemical and spectroscopic methods. Our data show that the assembly of [2Fe2S] clusters is
initiated by binding a ferrous iron to IscU followed by insertion of a persulfide in the vicinity of the
iron center by IscS, enabling the formation of a [1Fe1S] precursor upon cleavage of the persulfide
bond by Fdx. Then, IscU dissociates from IscS and dimerizes to generate a bridging [2Fe2S] cluster by
fusion of two [1Fe1S] precursors. The dimer dissociates into apo- and [2Fe2S]- monomeric forms.
These data provide a complete mechanistic description of the [2Fe2S] cluster assembly process by an
ISC machinery highlighting a concerted process.
INTRODUCTION

Fe-S clusters are among the oldest metallocofactors on Earth. One of the theories on the origin of life considers Fe-S clusters as central elements, likely generated nearby sulfide-emitting hydrothermal vents in the form of mixed Fe(Ni)-S minerals that catalyzed the formation of the first complex molecules.¹ Living organisms then evolved multi-protein machineries to control their biosynthesis. Fe-S clusters are now present in organisms from the three domains of life (archaea, bacteria and eukaryotes) where they fulfil essential biological roles. Fe-S clusters are present in many proteins and constitute the catalytic centers of a multitude of enzymes involved in many biological processes including respiration, the Krebs cycle, protein synthesis, cellular signalling and maintenance of genome integrity.² Fe-S clusters are made of iron and sulfide ions assembled in various structures. The most common forms in proteins are the [2Fe2S], [4Fe4S] and [3Fe4S] clusters, and more complex forms are found in specific enzymes such as nitrogenase, hybrid cluster protein and CO dehydrogenase.²⁻⁵ Due to their high versatility, Fe-S clusters cannot be acquired from food. They are biosynthesized *de novo* in each cell by multi-protein machineries.²,⁶,⁷ The Iron Sulfur Cluster assembly (ISC) machinery that is present in the mitochondria of eukaryotic organisms and in many bacteria biosynthesizes [2Fe2S] clusters that are the building blocks for the assembly of [4Fe4S] clusters.²,⁶ Although many proteins involved in Fe-S clusters biosynthesis and their insertion into recipient enzymes have been identified in the past two decades, several aspects of the mechanism of Fe-S clusters biosynthesis are still unclear.

The core bacterial ISC machinery encompasses four proteins that are essential for the assembly of [2Fe2S] clusters: IscU, the scaffold protein on which Fe-S clusters are assembled, IscS a cysteine desulfurase that provides sulfur to IscU in the form of a cysteine bound persulfide (Cys-SSH), and the [2Fe2S] ferredoxin Fdx that deliver electrons provided by a ferredoxin reductase (FdxR) from NADPH.²,⁸,⁹ Homologous proteins are present in eukaryotes, named ISCU2, NFS1, FDX2 and FDXR, respectively.²,⁶ Previous studies of the mouse and yeast ISC machineries revealed that the assembly of [2Fe2S] clusters is initiated by the insertion of a ferrous iron into the assembly site of ISCU2, followed by iron-dependent persulfide transfer from NFS1 to ISCU2 and then reduction of the persulfide into sulfide by the FDX2/FDXR system, ultimately leading to formation of a [2Fe2S] cluster.¹⁰⁻¹² While persulfide transfer and its reduction appear as key fundamental steps of the Fe-S cluster assembly process, it is still puzzling how two iron and two sulfide ions are gathered to generate a dinuclear [2Fe2S] cluster in ISCU2. Titration of iron and cysteine indicated that each ISCU2 monomer receives only one iron and one persulfide, suggesting that the synthesis of a [2Fe2S] cluster requires dimerization of ISCU2, which should lead to the formation of a bridging [2Fe2S] cluster.²,¹⁰ However, native MS showed that the main product of this process was an ISCU2 monomer holding one [2Fe2S] cluster, suggesting that the dimer with a bridging cluster is a transient species that is converted into a monomer via a yet undefined mechanism.¹⁰ In contrast to the mouse system, in the highly homologous
human system, the final product of the reaction was found to be an ISCU2 dimer holding [2Fe2S] clusters. Studies of an ISCU2 Y35A mutant showed that both formation of the ISCU2 dimer and Fe-S cluster assembly was abolished, suggesting that dimerization is a mandatory step during [2Fe2S] cluster assembly.¹³ However, evidence for the formation of a bridging [2Fe2S] cluster in this dimer is still lacking since the stoichiometry of [2Fe2S] clusters was not determined, and it is still unknown whether the persulfide in ISCU2 is reduced before or after dimerization of ISCU2.

The mechanism of [2Fe2S] cluster assembly has also been studied in the bacterial system. Similarly to the mouse ISCU2, the assembly site of *E. coli* (*Ec*) IscU binds a ferrous iron, which is required to allow subsequent persulfide insertion.¹¹,¹⁴ This metal-dependent persulfide transfer process has also been observed when zinc is in the assembly site instead of iron in both the bacterial and mouse systems.¹⁰,¹⁵ In contrast to the mouse system which receives a single iron and a single persulfide, insertion of two irons and several persulfides into bacterial IscU proteins has been reported.¹⁴⁻¹⁹ These data suggest a mechanism of [2Fe2S] cluster assembly by consecutive accumulation of iron and sulfides in a single monomer rather than dimerization. However, the biochemical conditions of these assays raise several questions on the functional relevance of the observed species. First, large excesses of iron were used to detect the binding of a second iron (up to 100 molar equivalents),¹⁴,¹⁵ which can promote non-specific iron binding.¹¹ Second, these reconstitutions used thiols such as GSH and DTT as reducing agents instead of Fdx.¹⁴,¹⁵,¹⁹ Thiols preferentially reduce the persulfide of the desulfurase in an iron-independent manner, leading to the release of sulfide and assembly of Fe-S clusters via a non-physiological process referred to as the thiol-based pathway.²,¹⁰,²⁰ Third, under these conditions, both [2Fe2S] and [4Fe4S] clusters were detected in IscU,¹⁹ while only [2Fe2S] clusters were formed in reconstitutions of the mouse system including the FDX2 ferredoxin and its reductase,¹⁰ consistent with *in vitro* and *in vivo* data²¹⁻²⁵ indicating that the assembly of [4Fe4S] clusters is not performed by IscU but by the IscA-type scaffolds. Altogether, these data raise questions on the validity of the mechanism of successive insertion of iron and persulfides into IscU.

Finally, *in vivo* data rather suggest a process based on the dimerization of IscU. In an *E. coli* strain strictly relying on Fe-S cluster biosynthesis for viability, the Y3A mutant, equivalent to the human Y35A ISCU2 mutant, was not viable.²⁶ The crystal structure of a holo form of the IscU protein from *Methanothrix thermoacetophila* revealed a dimeric structure in which Y7, equivalent to Y3 from *E. coli* IscU, was engaged in a π-π stacking interaction with the phenylalanine F61 from the other subunit, consistent with an essential role of this residue in dimerization.²⁷ Altogether, these data suggest that dimerization might be a critical step of the assembly process. However, the structure of the *Ec* IscS-IscU complex shows that the tyrosine Y3 of IscU is critical for the interaction with IscS, which could also explain the null-phenotype of the Y3A mutant. Moreover, in the holo form of dimeric *M. thermoacetophila* IscU, two adjacent [2Fe2S] clusters are present, one in each subunit, but
there is no bridging [2Fe2S] cluster. Therefore, it is still unclear whether [2Fe2S] cluster assembly
proceeds via a dimerization process involving formation of a bridging [2Fe2S] cluster or an
accumulation process via successive insertion of iron and sulfide ions into a single monomer.

Here we report a step-by-step analysis of the Fe-S cluster assembly process using a
reconstitution of the whole *Ec* ISC system, i.e. including the physiological Fdx/FdxR reducing system
instead of thiols. We show that *Ec* IscU acquires only one iron and one persulfide through a sequential
process where iron is inserted first then persulfide, followed by specific reduction of the persulfide in
IscU by Fdx. Using conditions in which IscS was present either under stoichiometric or catalytic
conditions, we isolated several intermediates that were characterized by EPR, NMR and native mass
spectrometry (nMS). Collectively, our data indicate that the assembly of [2Fe2S] clusters by the
bacterial ISC complex relies on the fusion of two [1Fe1S] precursors into a bridging [2Fe2S] cluster
upon dimerization of IscU.
# RESULTS

## Functional reconstitution of the *E. coli* ISC machinery

To investigate the mechanism of Fe-S cluster assembly by the *E. coli* ISC machinery, we reconstituted the complete system using purified IscU, IscS, Fdx and FdXR proteins overexpressed in *E. coli*. Since purified IscU proteins were shown to bind zinc in their assembly site, which could hinder iron insertion,¹⁰,¹¹ we first sought to assess the metal content of *Ec* IscU. We detected 0.3 - 0.6 zinc ions per IscU in the as-isolated protein. The *Ec* IscU protein was demetallated using DTPA and re-purified on size exclusion chromatography. The apo-IscU protein mainly eluted as a monomer with a minor proportion of aggregated forms which were discarded. The monomeric form was assayed in Fe-S cluster assembly assays containing 100 µM of apo-IscU incubated with 100 µM of Fe²⁺ ions (Mohr's salt), catalytic amounts of the IscS, Fdx and FdXR proteins (1 µM) in the presence of 200 µM of NADPH as a source of electrons for the Fdx/FdXR system and 100 µM of L-cysteine as a source of sulfur for IscS. The UV-visible spectrum of the mixture after 10 min of reaction (Fig. 1a, blue line) showed absorption peaks at 319, 417 and 456 nm, and a shoulder at 520 nm with extinction coefficients typical of [2Fe2S] clusters in other IscU proteins.¹⁰,¹⁹

<figure><img src="image_2.png" /><figcaption>**Figure 1: Reconstitution of the *E. coli* ISC machinery**</figcaption></figure>

(a) UV-visible spectrum of Fe-S cluster assembly assays containing 100 µM of apo-IscU, 1 µM of IscS, Fdx and FdXR (*catalytic conditions*), 100 µM of iron, L-cysteine and 200 µM NADPH after 30 min of reaction (blue line) and in assays lacking IscU (green line), IscS (red line), Fdx (purple line) or FdXR (orange line). The spectra were obtained by desalting of the reaction mixtures to remove remaining NADPH and subtraction of the spectrum of the initial mixture before addition of L-cysteine and iron. The data are displayed as extinction coefficients based on the initial concentration of iron. (b) Paramagnetic 1D ¹H NMR spectra of reaction mixtures containing 400 µM apo-IscU, Fe²⁺ and L-cysteine, 800 µM NADPH, and 10 µM of IscS, Fdx and FdXR (red line), and 10 µM of Fdx and FdXR with 800 µM NADPH (black line), both after exchange into 20 mM Tris pH 8.0, 100 mM NaCl D₂O buffer, measured at 600 MHz and 293K. Arrows indicate hyperfine shifted signals of IscU, and the star a signal of Fdx. (c) Temperature dependence of the chemical shifts of 8 hyperfine-shifted signals of IscU between 283 K and 298 K. (d) Kinetics of Fe-S clusters assembly monitored at 465 nm in reactions containing 100 µM of apo-IscU, 10 µM of IscS, Fdx and FdXR, 100 µM of iron, L-cysteine and 200 µM NADPH (+ Fdx) and without Fdx and FdXR (- Fdx). (e, f) Kinetics of Fe-S cluster assembly performed as in (b) but with various concentrations of L-cysteine from 25 to 200 µM, without Fdx and FdXR (- Fdx) (e) and with (+ Fdx) (f). All experiments were repeated at least 3 times.
To further characterize the Fe-S cluster in IscU, we acquired paramagnetic 1D ¹H NMR spectra of the ISC reconstitution mixture after exchange in D₂O buffer (Fig. 1b, red line). Eight hyperfine shifted signals were visible in the 10-55 ppm chemical shift range, where cysteine β-CH₂ signals bound to [2Fe2S] clusters are expected.²⁸,²⁹ The presence of several well-resolved dispersed signals, rather than a single broad signal as observed for ferredoxins, can be ascribed to a non-symmetric coordination, as reported for the (Cys)₃(His)₁ coordination of the [2Fe2S] cluster in mitoNEET.²⁸,³⁰ A dissymmetric coordination is consistent with the (Cys)₃(Asp)₁ coordination reported for the [2Fe2S] cluster in other ISCU proteins.¹⁰,²⁷ No signal indicative of other iron-containing species could be detected more downfield, even by increasing the transmitter frequency. A weak signal at 14 ppm was assigned to the [2Fe2S] cluster of Fdx, by comparison with a sample containing exclusively Fdx, FdxR and NADPH (Fig. 1b, black line). The eight hyperfine shifted signals assigned to IscU displayed an anti-Curie temperature dependence characteristic of an S = 0 ground state, indicating that the [2Fe2S] cluster formed onto IscU was in the oxidized state (Fig. 1c). The [2Fe2S] clusters assembled in IscU via a Fdx-based reaction by the mouse ISC machinery or a thiol-based reaction by the *Azotobacter vinelandii* ISC machinery were also in the 2+ oxidized state, suggesting that this is a common feature.¹⁰,¹⁹

The kinetics of Fe-S cluster assembly monitored at 465 nm showed that the absorbance reached a plateau after 5 min corresponding to ~50 µM of [2Fe2S] clusters with 100 µM of iron and L-cysteine, indicating that nearly 100% of iron and the sulfur from L-cysteine were incorporated as [2Fe2S] clusters (Fig. 1d, + Fdx trace). Reactions performed in the absence of IscS did not produce Fe-S clusters (Fig. 1a, red line), consistent with its essential role in the mobilization of sulfur. In the absence of IscU, weak and broad absorptions between 400 and 800 nm were observed (Fig. 1a, green line), that could be attributed to undefined “iron-sulfur” species, confirming the specific role of IscU as a scaffold protein. In the absence of Fdx or FdxR, [2Fe2S] clusters were still produced (Fig. 1a, purple and orange lines), but at a much slower rate than in the presence of all the ISC components (Fig. 1d, - Fdx trace). Under similar conditions with the mouse ISC machinery lacking the FDX2/FDXR system, L-cysteine was shown to provide the reducing equivalents for persulfide cleavage leading to Fe-S cluster assembly via a non-enzymatic process, referred to as the thiol-based reaction and considered as non-physiologically relevant.¹⁰,²⁰ To test this hypothesis, we assessed the effect of L-cysteine concentration on the rate of assembly, since the thiol-based reaction is cysteine-dependent in contrast to the enzymatically catalyzed Fdx-based reaction. In the absence of Fdx/FdxR, the rate of assembly increased with the concentration of L-cysteine (Fig. 1e), but not in its presence (Fig. 1f), indicating that the Fe-S cluster assembly process in the reaction including Fdx and FdxR proteins is enzymatically catalyzed. The quantity of [2Fe2S] clusters generated in the presence of Fdx and FdxR increased proportionally with the concentration of L-cysteine, indicating that at each concentration of L-cysteine the reaction was close to completion.
Quantification of iron and persulfide acquired by IscU

To elucidate the mechanism of the Fdx-based reaction, we performed a stepwise analysis of
the assembly process. We first sought to titrate iron in IscU using circular dichroism (CD). The
binding of Fe²⁺ to the assembly site of IscU is characterized by ligand-to-metal charge transfer
(LMCT) bands in the near UV region arising from the sulfur of the cysteine residues. The incubation
of apo-IscU with Fe²⁺ ions led to the appearance of absorption peaks at 270 nm and 310 nm typical of
Cys to Fe(II) transitions (Fig. 2a).¹⁰,¹¹ Titrations by Fe²⁺ showed that the signals reached a maximum
for one equivalent of Fe²⁺ ion relative to apo-IscU, indicating that IscU hosts a single Fe²⁺ ion.

We next examined persulfide transfer to *Ec* IscU. Mouse ISCU2 was shown to acquire a single persulfide when a ferrous iron is bound to its assembly site. In contrast, studies of reactions between *Ec* IscU and IscS by mass spectrometry reported that about 20 % of IscU was able to acquire a second persulfide in the presence of an excess of iron relative to IscU.¹⁴ We thus examined the persulfidation of IscU with one and two equivalents of iron and L-cysteine.

<figure><img src="image_3.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Figure 2: Quantification of iron and sulfur inserted into IscU</figcaption></figure>

(a) CD spectra of apo-IscU (100 µM) incubated with various molar equivalents of Fe²⁺ ions from 0.25 to 3.0. (b) ARBS assays of IscS incubated without (lane 1) or with (lane 2) one molar equivalent of L-cysteine. (c) ARBS assays on IscS (top panel) and IscU (bottom panel) in mixture of apo-IscU (20 µM) and IscS (20 µM) incubated for 3 min without iron and L-cysteine (lane 1), with one molar equivalent of L-cysteine and iron (lane 2) or without iron (lane 3) and analyzed under reducing conditions using β-mercaptoethanol to reveal persulfides (left panel, reducing) or under non-reducing conditions by omitting the reducing agent (right panel, non-reducing). The star indicates the presence of oxidized cysteines in IscU other than persulfidated cysteines. (d) Kinetic of persulfide transfer monitored by ARBS assays initiated by adding 40 µM of L-cysteine to a mixture containing 20 µM apo-IscU, Fe²⁺ and IscS. (e, f) ARBS assays on IscS (top panel) and IscU (bottom panel). (e) Reaction mixtures containing 20 µM of apo-IscU and IscS incubated with two molar equivalents of L-cysteine without iron (lane 1) or with either one molar equivalent of iron (lane 2) or two (lane 3). (f) Reaction mixtures containing 20 µM of apo-IscU and IscS incubated with one molar equivalent of iron without L-cysteine (lane 1) or with either one molar equivalent of L-cysteine (lane 2) or two (lane 3) for 10 min, then with 20 µM Fdx and FdxR, and 50 µM NADPH (stoichiometric conditions) for another 10 min (lanes 4 and 5, respectively). All experiments were repeated at least 3 times.
The persulfidation of IscU was analyzed using the alkylation-reduction band-shift (ARBS assay).<sup>20,31</sup> The assay is performed in two steps. First, the persulfidation reaction was quenched by adding SDS and a thiol-specific alkylating agent carrying a PEG tail (Mal-dPEG<sub>4</sub>-(m-dPEG<sub>12</sub>)<sub>3</sub>, MW = 2.36 kDa, referred to as an alkylation unit afterwards) to denature the proteins and alkylate both reduced (Cys-SH) and persulfidated cysteines (Cys-SSH). In a second step, the mixture was incubated with β-mercaptoethanol to specifically cleave the sulfur-sulfur bond of persulfides, which led to the loss of the PEG tail of alkylated persulfides and consequently decreased the molecular weight of the protein. Then, the alkylated forms were separated on SDS-PAGE. The presence of a persulfide in a protein is revealed as a faster migrating band separated from the unpersulfidated form by one alkylation unit (2.3 kDa). The position of the bands depends on the total number of reduced and persulfidated cysteines. *Ec IscU* (15 kDa) and *IscS* (45 kDa) each contain three cysteines. In the absence of L-cysteine, all three cysteines of *IscS* (**Fig. 2b**, lane 1, *IscS +3 alkylation units*) and *IscU* (**Fig. 2c**, lane 1, *IscU +3 alkylation units*) were alkylated, indicating the absence of persulfide. After incubation of *IscS* with L-cysteine (**Fig. 2b**, lane 2), the species corresponding to fully alkylated *IscS* (*IscS + 3*) was converted into a species lacking one alkylation unit (*IscS + 2*), indicating the presence of a persulfide on *IscS*. We then analyzed the transfer of persulfide from *IscS* to *IscU* by incubating apo-*IscU* with one molar equivalent of iron and then with *IscS* and L-cysteine for 3 min. After the addition of L-cysteine, *IscU* was almost completely converted into a species lacking one alkylation unit (**Fig. 2c**, reducing, lane 2, *IscU + 2*), indicating that one cysteine was persulfidated. In the absence of iron, the persulfide was not transferred to *IscU* but stayed on *IscS* (**Fig. 2c**, reducing, lane 3), indicating that persulfide transfer is an iron-dependent process. The persulfide was transferred to *IscU* in less than 2 min (**Fig. 2d**). Two minor species appearing later were also detected (**Fig. 2c-d**, *IscU*\*). These species were missing two and three alkylation units (*IscU + 1*, *IscU + 0*). To determine their nature, we analysed the reaction under non-reducing conditions (**Fig. 2c**, non-reducing), which allows identification of other cysteine modifications, such as disulfide bonds. Under non-reducing conditions, the major part of *IscU* was in the + 3 state and virtually no + 2 species was present (**Fig. 2c**, non-reducing, lane 2), which confirmed that the + 2 species detected under reducing conditions was a persulfidated form. In contrast, the + 1 and + 0 species were still present, indicating that the cysteines were oxidized to other forms. The +1 species migrated faster under non-reducing conditions and the + 0 species appeared fuzzy, which are indications of the presence of intramolecular disulfide bonds. In conclusion, a single cysteine of *IscU* was persulfidated by transfer from *IscS*. This persulfide slowly converted into a disulfide bond, but this species was not related to Fe-S cluster assembly given its rate of formation (**Fig. 2d**, 10% in 3 min) compared to Fe-S clusters (**Fig. 1d**, 70% in 3 min).

We next examined the effect of adding two equivalents of iron on the ability of IscU to acquire a second persulfide in the presence of two equivalents of L-cysteine. In the presence of either one or two equivalents of Fe<sup>2+</sup>, apo-IscU received a single persulfide despite the presence of another
persulfide on IscS (Fig. 2e, lanes 2 and 3). We concluded that IscU was not able to acquire a second persulfide, even in the presence of two equivalents of iron.

We next tested the ability of Fdx to reduce the persulfide in IscU. A reaction containing equimolar amounts of IscS, apo-IscU and iron was incubated with 1 equivalent of L-cysteine for 10 min, then with the Fdx/FdxR system and NADPH for another 10 min. The alkylation assay revealed that the persulfide carried by IscU was reduced by the Fdx/FdxR system (Fig. 2f, lanes 2 and 4). In contrast, the IscU + 1 and IscU + 0 species were not reduced by Fdx/FdxR. Although IscU was not able to accumulate two persulfides at the same time, we questioned if it could accept another persulfide after the reduction of the first one. In reactions containing two equivalents of L-cysteine, the ARBS assay showed that only the persulfide of IscU was reduced (Fig. 2f, lanes 3 and 5). This indicated that i) the Fdx/FdxR system is not able to reduce the persulfide carried by IscS and ii) after reduction of the persulfide in IscU, the transfer of a second one is not possible. The hindrance of successive persulfide transfer suggests that the receptor cysteine environment is modified after reduction of the persulfide. Altogether, these data established that Fdx specifically reduces the persulfide in IscU.

## Characterization of the product of the reduction by Fdx/FdxR

The ARBS assay showed that the persulfide of IscU was reduced by the Fdx/FdxR system, likely into sulfide, which leads to the formation of a [2Fe2S] cluster in the Fe-S cluster assembly assays (Fig. 1). However, only one iron and one sulfide ion are expected per IscU based on iron and ARBS assay titrations (Fig. 2a and 2e-f), suggesting that a mononuclear iron-sulfide intermediate is first generated after reduction of the persulfide by the Fdx/FdxR reductase system. To detect such intermediate, we sought to analyse reactions conducted with stoichiometric amounts of IscU and IscS as for the ARBS assays (stoichiometric conditions). The kinetics of Fe-S cluster assembly monitored by following the absorbance at 465 nm revealed that [2Fe2S] clusters were not formed in reaction mixtures containing stoichiometric amounts of IscS, IscU, iron and L-cysteine incubated with stoichiometric amounts of Fdx, FdxR and NADPH (Fig. 3a, red line), despite reduction of the persulfide into sulfide by Fdx/FdxR as attested by the ARBS assay (Fig. 2f). In contrast, reactions performed with catalytic amounts of IscS, Fdx and FdxR (catalytic conditions) proceeded to the assembly of [2Fe2S] clusters (Fig. 3a, blue line). The formation of [2Fe2S] clusters under stoichiometric conditions was only observed after the addition of extra IscU loaded with one equivalent of iron (thereafter referred to as Fe-IscU), as indicated by the change in absorbance at 465 nm (Fig. 3b). This suggests that after reduction by the Fdx/FdxR system, a precursor of the [2Fe2S] cluster was formed under stoichiometric conditions. To determine the nature of this precursor, we first asked whether it still contained sulfide, as sulfide ions may have diffused out of the complex.
To assess the amount of sulfide bound to IscU after the reduction of the persulfide by Fdx, we quantified the sulfide and iron contents in the reaction mixture after a desalting step. Reaction mixtures containing Fe-IscU, IscS, and Fdx in stoichiometric amounts with FdxR and NADPH were incubated with or without L-cysteine and then desalted. The iron and sulfide contents were measured using ferrozine and the methylene blue assays, respectively. The contribution from the [2Fe2S] cluster of Fdx was subtracted by measuring sulfide and iron after desalting a sample containing Fdx alone. Sulfide and iron titrations yielded about one iron and one sulfide per IscU monomer after reaction with cysteine and Fdx then desalting, indicating that the sulfide ion remained in the IscS-IscU complex under stoichiometric conditions (Table 1).

**Table 1: Iron and sulfide quantifications.**

The values are reported as molar ratio of iron and sulfide per protein with standard deviation from 3 repeats. a The quantity of iron and sulfide from Fdx has been subtracted.

<table><thead><tr><td>Sample name</td><td>Iron / (IscU or Fdx) ratio</td><td>Sulfide / (IscU or Fdx) ratio</td></tr></thead><tbody><tr><td>Fdx</td><td>1.70 ± 0.05</td><td>1.9 ± 0.1</td></tr><tr><td>IscS-Fe-IscU - L-cysteine</td><td>0.95 ± 0.05<sup>a</sup></td><td>0.1 ± 0.1<sup>a</sup></td></tr><tr><td>IscS-Fe-IscU + L-cysteine</td><td>0.86 ± 0.05<sup>a</sup></td><td>0.8 ± 0.1<sup>a</sup></td></tr></tbody></table>

To characterize the sulfide-containing precursor, we analyzed the iron center by EPR spectroscopy at each step of the reaction: Fe-IscU alone, in complex with IscS both before and after incubation with L-cysteine, after reduction of the persulfide by the Fdx/FdxR system and after addition of extra Fe-IscU to generate the [2Fe2S] cluster (Fig. 3c). IscU binds a $Fe^{2+}$ ion and previous CD, EPR, XAS and Mössbauer analysis of mouse Fe-ISCU showed that the Fe(II) center is high spin ($S = 2$) at the ground state.¹¹ Paramagnetic centers ($S \neq 0$) can be studied by EPR spectroscopy which allows for the detection of spin transitions that are highly sensitive to the redox and spin states of a metal center as well as to the nature and the geometry of its coordination sphere. High spin ($S = 2$) ferrous species are non-Kramers systems that are rarely detected by X-band EPR spectroscopy because the energy of the transition is generally higher than the energy of the radiation ($h\nu \sim 0.3 \text{ cm}^{-1}$), due to the zero-field splitting anisotropy (ZFS) and its distribution.³²,³³ When detectable, X-band spectra of $S = 2$ species are usually recorded in the parallel mode ($B_1$ // $B_0$, maximum probability transition for $\Delta Ms = 0$) and show a large resonance line at low magnetic fields with an inhomogeneous line shape corresponding to “Ms’ = $\pm 2$” intra-doublet transitions. EPR spectra are displayed as the first derivative of the absorption spectrum, the signal position is usually indicated at the minimum of the derivative line and by converting the magnetic field value into g-value. The X-band parallel mode EPR spectrum of Fe-IscU displayed a signal pointed at g = 14 at the trough of the derivative line (Fig. 3c, blue line). This g = 14 signature is very close to the g = 14.9 signal previously reported for mouse Fe-ISCU2,¹¹ and is characteristic of a high spin ($S = 2$) Fe(II) center in the assembly site of IscU.
<figure><img src="image_9.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 3: Characterization of the product of the reduction by Fdx/FdxC**</figcaption></figure>

(a) Kinetics of Fe-S cluster assembly monitored by UV-visible at 465nm under catalytic conditions (100 µM of apo-IscU, Fe²⁺, L-cysteine, 10 µM of IscS, Fdx, FdxC and 200 µM of NADPH,) and stoichiometric conditions (100 µM of apo-IscU, Fe²⁺, IscS, Fdx, FdxC, L-cysteine and 200 µM of NADPH). The reactions were initiated by adding L-cysteine. (b) Sequential Fe-S cluster assembly reaction monitored by UV-visible at 465nm. The reaction was initiated by mixing 100 µM of apo-IscU, Fe²⁺, IscS and L-cysteine. 10 min after, 100 µM of Fdx, FdxC and 200 µM of NADPH were added (+Fdx). 10 min after, 200 µM of apo-IscU and Fe²⁺ were added. (c) Parallel mode X-band EPR spectra at each step of the Fe-S cluster assembly process under stoichiometric conditions starting with Fe-IscU: 100 µM apo-IscU and Fe²⁺ (**blue line**) and after successive additions of IscS: 200 µM apo-IscU and IscS, 180 µM Fe²⁺ (**purple line**), L-cysteine 5 min later: 200 µM apo-IscU and IscS, 180 µM Fe²⁺, 400 µM L-Cys (**red line**), Fdx/FdxC/NADPH 10 min later: 200 µM apo-IscU, Fe²⁺, IscS, Fdx, FdxC, 250 µM L-Cys, 400 µM NADPH (**orange line**) and 2 eq. of extra Fe-IscU 10 min later: 100 µM apo-IscU, Fe²⁺, IscS, Fdx, FdxC, 200 µM L-Cys, NADPH + 200 µM apo-IscU and Fe²⁺ (**yellow line**) and EPR spectra of the reaction mixture after completion of the Fe-S cluster assembly under catalytic conditions as performed in (a) (**green line**). All spectra were normalized to the same initial Fe²⁺ concentration. Experimental recording conditions: temperature, 7 K; microwave frequency, 9.38 GHz; modulation amplitude, 0.96 mT; microwave power, 10 mW; 5 accumulations. (d) UV-visible difference spectrum of a reaction containing 100 µM of apo-IscU, Fe²⁺, IscS, Fdx, FdxC and NADPH incubated with 100 µM L-cysteine for 20 min then desalted, minus spectrum of a mixture containing 100 µM of apo-IscU, IscS, Fdx, FdxC and NADPH and desalted (**orange line**) and UV-visible difference spectrum of a mixture containing 100 µM of apo-IscU, Fe²⁺ and IscS minus spectrum of 100 µM of apo-IscU and IscS (**blue line**). (e) Kinetic of Fe-S cluster assembly monitored by UV-visible scanning and performed under catalytic conditions using 100 µM of apo-IscU, Fe²⁺, L-cysteine and NADPH, and 10 µM of IscS, Fdx and FdxC. The contribution of the initial reaction mixture containing 100 µM apo-IscU and NADPH and 10 µM of IscS, Fdx and FdxC was subtracted from each spectrum. All experiments were repeated at least 3 times.
After adding one equivalent of IscS to Fe-IscU to form the complex, the g = 14 signal almost completely disappeared (**Fig. 3c**, purple line). The Fe center remained in the +II redox state, as the perpendicular-mode X-band EPR spectrum of the IscS-IscU complex showed only a weak g = 4.3 signal typical of adventitious high spin (S = 5/2) ferric ion, which accounted for less than 5 % of total iron (**Fig. S1a**). The disappearance of the g = 14 EPR signal may thus reflect structural changes of the Fe(II) center in the assembly site, which could modify the ZFS parameters of the Fe(II) center in such a way that the energy of the transition of the intra-doublet ‘M<sub>s</sub> = ± 2’ becomes higher than the energy quantum in X-band EPR. For instance, a small increase in the *E* parameter of 0.1 cm<sup>-1</sup> would extinguish the g = 14 signal. Additionally, iron could become labile in the IscS-IscU complex, which could correspond to the weak signal at g = 8.8 akin to free iron (**Fig. S1b**). These structural changes of the assembly site might be induced by binding of the catalytic cysteine of IscS to the iron center of IscU, as observed in the crystal structure of the homologous human system with zinc in the assembly site instead of iron.<sup>34</sup> Furthermore, the assembly site is probably flexible, as described for mouse ISCU,<sup>11</sup> which is an inherent property to enable persulfide transfer and subsequent steps of the assembly process.

After incubation of the IscS-Fe-IscU complex with L-cysteine for 3 min to allow persulfide formation and transfer to IscU, a new signature was observed at g = 8.3, again characteristic of a high spin Fe(II) center (**Fig. 3c**, red line), and clearly distinct from a standard of Fe<sup>2+</sup> in the same buffer conditions (**Fig. S1b**), indicating that the ferrous iron was ligated by IscU in the IscS-IscU complex. The appearance of the g = 8.3 signal thus indicated that the structure of the Fe(II) center was modified by the presence of the persulfide in IscU, either nearby or directly bound to it. Upon incubation of the persulfidated IscS-Fe-IscU complex with stoichiometric amounts of Fdx, FdxC and NADPH to generate sulfide, the g = 8.3 signal disappeared, and a new signal at g = 18 was observed (**Fig. 3c**, orange line), still characteristic of a high spin Fe(II) center but with a modified structure, suggesting formation of a Fe(II)-SH species. However, it could not be excluded that the sulfide ion was retained near the Fe(II).

We next analyzed the product of the reaction after the addition of 2 equivalents of extra Fe-
IscU versus IscS to a sample containing the sulfide-containing g = 18 species, to generate the [2Fe2S]
cluster as described in **Fig. 3b**. The EPR spectrum showed almost complete disappearance of the
signal at g = 18 and reappearance of the signals at g = 14 and 8.3, which were assigned to free Fe-IscU
and persulfidated Fe-IscU in the IscS-Fe-IscU complex, respectively (Fig. 3c, yellow line and Fig.
S1d). The g = 18 species was absent of reaction performed with catalytic amounts of IscS and
Fdx/FdxC (Fig. 3c, green line), strengthening the conclusion that the g = 18 species is only present
when the assembly reaction is stopped one step before the formation of the [2Fe2S] cluster. In both
cases, no specific feature of the [2Fe2S] cluster in IscU could be detected, apart from the signature of
the reduced [2Fe2S]$^{1+}$ cluster of Fdx (Fig. S2). This was expected for a cluster in its [2Fe2S]$^{2+}$ oxidized form as determined by NMR (Fig. 1b-c), which is $S=0$ in the ground state, thus EPR-silent. Altogether, this set of data indicates that the $g=18$ species is a precursor of the [2Fe2S] cluster.

To determine whether the sulfide ion in the $g=18$ species was bound to the iron center in the form of a Fe(II)-SH species or just retained nearby, we sought for specific features of iron-sulfide bonds by electronic absorption spectroscopy. Iron-sulfide bonds are well documented in Fe-S clusters, where they are characterized by LMCT transitions between 400 and 500 nm.<sup>35</sup> Additionally, an inorganic Fe(II)-SH complex has been documented in the literature, which displays an absorption peak at 402 nm attributed to an LMCT transition, thus possibly originating from the Fe-SH moiety.<sup>36</sup> Since mononuclear ferrous iron coordinated by His, Asp and Cys residues in protein binding-sites such as the assembly site of IscU exhibit LMCT transitions below 350 nm,<sup>37,38</sup> the appearance of absorption features in the 400 - 500 nm region would reveal the presence of an iron-sulfide bond in the $g=18$ species. We analyzed the product of a reaction containing stoichiometric amounts of IscU, IscS, iron, Fdx, FdxC and NADPH, incubated with L-cysteine for 10 min and then desalted. The contributions of IscS, Fdx and FdxC were withdrawn by subtracting a spectrum of the initial mixture containing IscU, IscS, Fdx, FdxC and NADPH, and then desalted. The difference spectrum displayed a shoulder at 330 nm and a peak with a maximum at 423 nm indicative of the presence of an iron-sulfide-containing species (Fig. 3d, orange line). The 423 nm feature was not present in the initial IscS-Fe-IscU complex (Fig. 3d, blue line). We next performed time course analysis by UV-visible scanning under catalytic conditions. The UV-visible spectra revealed the appearance of a peak at 423 nm at the earliest time point of the reaction (5s), pointing to the formation of an iron-sulfide-containing species, which was converted into a [2Fe2S] cluster in IscU with its characteristic absorption maxima at 417 and 456 nm (Fig. 3e). The negative feature near 340 nm was due to NADPH consumption.

Taken together, the sulfide and iron titrations, the EPR, UV-visible and kinetic data indicated that the reduction of the persulfide at the iron center of IscU by the Fdx/FdxC reductase system led to the formation of a transient mononuclear Fe(II)-SH intermediate during the assembly process which is a precursor of the [2Fe2S] cluster in IscU. Afterwards, we will refer to this Fe(II)-SH species as the [1Fe1S] precursor.

## NMR characterization of the complexes formed by IscU in the course of the reaction

We next examined the formation and dissociation of the complexes between IscU, IscS and Fdx along the Fe-S cluster assembly process by NMR (Fig. 4). This technique was previously used to study complex formation between IscU, IscS and Fdx based on line-broadening of the IscU NMR signals.<sup>39,40</sup> As molecular weight increases upon complex formation, molecular tumbling is slowed down and transverse relaxation increases, which results in broader NMR signals. Here we used <sup>15</sup>N-labelled *Ec*
IscU in combination with 2D ¹H-¹⁵N correlation experiments to selectively observe the signals of the ¹⁵N-labelled protein.

The ¹H-¹⁵N SOFAST-HMQC⁴¹ spectrum of ¹⁵N-labelled *Ec* apo-IscU displayed two sets of signals (**Fig. 4a**). A set of well dispersed sharp signals was indicative of a folded protein, whereas the broad overlapping signals in the central part of the spectrum pointed to the presence of a disordered state characterized by conformational exchange on a µs-ms timescale. These sets of signals were previously attributed to a structured (S) and a disordered (D) states of IscU, which slowly interconvert.¹⁰,⁴⁰,⁴² These two forms appear as two separate signals for the Nε-Hε side-chain of the sole tryptophan of IscU, W76, with a ~80:20 intensity ratio assigned to the S and D states of IscU, respectively (**Fig. 4a inset, Table 2**).⁴²

When apo-IscU was incubated with Fe²⁺, the broad signals almost completely disappeared leaving essentially signals of a structured state (**Fig. 4b**). Notably, less than 10 % of the W76 Nε-Hε signal of the apo form remained (**Table 2**), indicating that Fe²⁺ stabilizes the S-state as previously described for mouse IscU.¹¹ A similar behaviour was observed when *Ec* apo-IscU was incubated with Zn²⁺ (**Fig. S3a**), as previously reported.⁴³ Nearly all signals of Fe-IscU displayed spectral perturbations compared to Zn-IscU due to the paramagnetic effects of the iron center, including chemical shift perturbations likely due to pseudo-contact shift (PCS) and line broadening due to paramagnetic relaxation enhancement (PRE).⁴⁴⁻⁴⁶ As the PRE effect increases in the vicinity of the paramagnetic center, signals near the iron center were too broad to be detected. Fe-IscU NMR signals were tentatively assigned using proximal Zn-IscU signals (**Fig. S3a,b**). PCSs were analyzed using the FANTEN software,⁴⁷ and were compatible with a Fe²⁺ positioned at the metal binding site defined by the conserved residues Cys37, Asp39, Cys63 and His105 in the S-state (**Fig. S3c,d**). The signals that could be assigned with high confidence corresponded to residues more than 15 Å away from the metal center, which is higher than the 8 Å blind sphere reported for other high spin Fe(II) centers,²⁹,⁴⁸,⁴⁹ Low signal intensities, high PCS values, signal overlap, and incomplete attribution of Zn-IscU signals precluded assignment for the residues closer than 15 Å. The assigned signals were therefore not affected by PRE and only experienced PCSs, like W76 that is located ~19 Å from the metal center (**Fig. 4g**).

Upon addition of a stoichiometric amount of IscS to Fe-IscU nearly all the resonances disappeared due to the formation of a high molecular weight complex between Fe-IscU and the 90 kDa IscS dimer, except those of three highly mobile C-terminal residues (**Fig. 4c**). No changes were observed after addition of stoichiometric amounts of L-cysteine, Fdx, FdxC and NADPH to the IscS-Fe-IscU complex (**Fig. 4d**). Importantly, this indicates that IscU, which at this stage carried a [1Fe1S] precursor according to the EPR data, remained associated to IscS.
<figure><img src="image_14.png" /><figcaption>Figure 4: NMR analysis of complex formation between IscU and IscS and Fdx</figcaption></figure>

(a-e) ¹H-¹⁵N SOFAST-HMQC NMR spectra of ¹⁵N-labelled *Ec* apo-IscU (100 µM) alone (a) and after successive incubations with one equivalent of Fe²⁺ (b), one equivalent of IscS (c), one equivalent of L-cysteine, Fdx and FdxR with two equivalents of NADPH (200 µM) (d), and upon addition of unlabelled Fe-IscU (200 µM) (e). (f) ¹H-¹⁵N SOFAST-HMQC NMR spectrum of ¹⁵N-labelled *Ec* apo-IscU (100 µM) after Fe-S cluster reconstitution under catalytic conditions in a reaction containing Fe²⁺ (200 µM), L-cysteine (200 µM), IscS (10 µM), Fdx (10 µM), FdxR (10 µM), and NADPH (400 µM). All spectra were acquired at a temperature of 293 K and at 800 MHz ¹H resonance frequency. Spectra were acquired with 64 scans, except spectrum (e) with 512 scans. The upper right box in each spectrum highlights non-assigned Asn-Gln NH₂ amide side chain signals (δ¹⁵N ≈ 110 ppm and δ¹H 7.0-7.4 ppm) and the bottom left box the W76 Hε-Nε side chain signal (δ¹⁵N ≈ 129 ppm and δ¹H 10.0-10.4 ppm). In (a) the signals observed for W76 Hε-Nε, corresponding to structured (S) and disordered (D) IscU, are magnified in the upper left corner of the panel. (g-i) Close-up and superimposed views of the boxed region in spectra (a, b, e, f and Zn-IscU spectrum in Fig. S3a) for the signals of IscU S-state W76 Hε-Nε (g-h) and Asn-Gln NH₂ regions (i). Contour levels were adapted to display similar signal intensities. Colour codes are the same as those of each spectrum with Zn-IscU in turquoise, apo-IscU in green, Fe-IscU in blue and Fe-S reconstituted-IscU in red. All experiments were repeated at least 3 times.
Following the addition of unlabelled Fe-IscU (Fe-¹⁴N-IscU), ¹⁵N-IscU signals re-appeared (Fig. 4e), indicating that ¹⁵N-IscU was released from IscS. The W76 Nε-Hε region displayed two contiguous signals (Fig. 4e, Table 2). One signal was consistent with the S-state of apo-IscU (Fig. 4h). Comparison with the entire HMQC spectrum of apo-IscU showed that a subset of amide signals also coincided with apo-IscU signals (Fig. S4a, Table S1). We therefore assigned these signals to ¹⁵N-apo-IscU. The second W76 Nε-Hε signal was close to that of Fe-IscU (Table 2), but broad and distorted, suggesting the presence of more than one species. A subset of amide signals coincided with Fe-IscU signals, confirming the presence of ¹⁵N labelled Fe-IscU (Fig. S4b, Table S1). Analysis of amide signal intensities suggested that apo-IscU and Fe-IscU were present in comparable amounts (Table S1, Fig. S4c). Fe-IscU may originate from an exchange of Fe²⁺ between unlabelled Fe-IscU and ¹⁵N-apo-IscU, or from an incomplete reduction of persulfidated IscU by Fdx. However, it is not excluded that IscU-[1Fe1S] precursor is also present and that its signals overlap with Fe-IscU signals for residues that are distal from the metal binding site and therefore less affected by PCS. Several signals could neither be attributed to apo-IscU nor to Fe-IscU (Fig. S4a, b), indicating the presence of a third species. For example, the Asn-Gln NH₂ amide side chain signal at δ¹⁵N ≈ 110 ppm and δ¹H 7.4 ppm displayed three signals arising from apo-IscU, Fe-IscU and a third distinct species (Fig. 4i).

<table><caption>**Table 2: Analysis of NMR W76 ¹Hε⁻¹⁵Nε signals.**</caption><thead><tr><th>Sample name</th><th>Chemical shifts<br>δ<sub>H</sub> (ppm) / δ<sub>N</sub> (ppm)</th><th>Peak volume<br>a.u. (%)</th><th>Species</th></tr></thead><tbody><tr><td rowspan="2">apo-IscU</td><td>10.330 / 129.08</td><td>9.17 (81 %)</td><td>apo S-state</td></tr><tr><td>10.116 / 129.18</td><td>2.21 (19 %)</td><td>apo D-state</td></tr><tr><td rowspan="2">Fe-IscU</td><td>10.267 / 129.10</td><td>6.03 (90 %)</td><td>Fe S-state</td></tr><tr><td>10.116 / 129.23</td><td>0.66 (10 %)</td><td>apo D-state</td></tr><tr><td>IscU-[2Fe2S]</td><td>10.269 / 128.82</td><td>100 %</td><td>[2Fe2S] S-state</td></tr><tr><td rowspan="2">+ 2 eq Fe-¹⁴N-IscU</td><td>10.317 / 129.03</td><td>22.2 (50 %)</td><td>apo S-state</td></tr><tr><td>10.258 / 129.02</td><td>22.2 (50 %)</td><td>(Fe, [1Fe1S], [2Fe2S]) S-states</td></tr></tbody></table>

Since the addition of Fe-IscU led to [2Fe2S] cluster formation under these conditions (Fig. 3b), we suspected that the third species might be a [2Fe2S]-bound IscU form. To assess the presence of IscU-[2Fe2S], we carried out NMR measurements on a sample of *Ec* IscU reconstituted with a [2Fe2S] cluster under catalytic conditions. The reaction was performed with two equivalents of iron and L-cysteine to allow complete reconstitution of IscU with [2Fe2S] clusters. The NMR spectrum of this sample displayed a set of well-dispersed peaks (Fig. 4f). Several signals were missing or broadened as compared to apo-IscU, Zn-IscU or Fe-IscU. These effects might be ascribed to PRE induced by the [2Fe2S] cluster, exchange between multiple [2Fe2S] binding modes or oligomerization of IscU-[2Fe2S]. Several signals of IscU-[2Fe2S] were clearly distinct from those of apo-IscU and Fe-IscU and superimposed with those of the third species identified in the mixture (Fig. S4d), notably the signals from W76 Nε-Hε (Fig. 4h,
**Table 2)** and Asn-Gln NH₂ amide side chain (Fig. 4i). We thus attributed the third species to IscU-[2Fe2S]. Line broadening and partial signal overlap between IscU-[2Fe2S], apo-IscU and Fe-IscU, precluded a reliable quantitative analysis of IscU-[2Fe2S] in the reaction mixture.

Taken together, the NMR data indicate that the addition of extra Fe-IscU to the IscS-IscU complex carrying the [1Fe1S] species led to release of IscU from IscS and formation of a [2Fe2S] cluster concomitantly with appearance of apo-IscU. The presence of the apo form and a [2Fe2S]-bound form by the end of the reaction further indicates that a transfer of metal has occurred from one IscU to another, suggesting that [2Fe2S] clusters were formed by dimerization of IscU and fusion of two [1Fe1S] precursors, followed by migration of the [2Fe2S] cluster onto one of the two IscU subunits, thereby releasing apo-IscU.

**Time-resolved analysis by native mass spectrometry (nMS)**

To characterize the formation of an IscU dimer during the [2Fe2S] assembly process, the reaction was analyzed by nMS at different time-points. The reaction was performed under catalytic conditions using two equivalents of iron and L-cysteine, relative to IscU (200 µM), to allow complete conversion of apo-IscU into holo-IscU harbouring a [2Fe2S] cluster. The assembly process was initiated by the addition of L-cysteine and analyzed after 25 and 50 minutes, corresponding to the mid-point and end-point of the reaction, based on the kinetic profile (Fig. 5a). The MS spectra of the reaction mixture before the addition of L-cysteine revealed that IscU was mostly in its apo form with only a minor proportion detected with one iron bound to it (16049.8 ± 0.5 Da, **Fig. 5b**, t₀, **Table 3**). This is most likely due to oxidation of the Fe²⁺ center in IscU during MS analysis, leading to the formation of Fe³⁺, which is not stable in IscU and spontaneously dissociates. Two different charge state distributions of monomeric apo-IscU were observed within the mass spectrum, suggesting the presence of two different monomeric conformations. As the number of charge states correlates with the solvent accessible surface area,⁵⁰ and thus to the folding state of a protein, the nMS signals observed between 700 - 1500 m/z range were assigned to the D state, while the narrower charge state distribution appearing within the 1800 - 2500 m/z range was attributed to the S state of apo-IscU. A dimeric species of apo-IscU was also detected (32,045.1 ± 0.6, **Fig. 5b**, t₀, dimer region, **Table 3**), showing the ability of IscU to dimerize as previously reported for *T. maritima*, *A. vinelandii*, *S. pombe*, *C. thermophilum* and *D. melanogaster* IscU proteins.²
<figure><img src="image_15.png" /><figcaption>**Figure 5:** *Native mass spectrometry analysis of the assembly reaction*</figcaption></figure>

(a) Kinetic of Fe-S cluster assembly monitored by UV-visible at 465nm and performed under catalytic conditions at 20°C with 200 µM of apo-IscU, 400 µM of Fe²⁺, 5 µM of IscS, Fdx and FdxC, 600 µM of NADPH and initiated by addition of 400 µM of L-cysteine. Arrows indicate time points at which samples were analyzed by nMS. (b) nMS analysis of Fe-S cluster assembly reaction at the time point indicated in (a) before the addition of L-cysteine (t₀) and after 25 and 50 min. (c) Zoom on the 7+ and 11+ charged states of the monomeric and dimeric regions, respectively. The attribution of each peak is indicated on the figure with the symbols defined in the top panel. (d) Relative quantifications of the monomeric (top panel) and dimeric (bottom panel) species determined from nMS spectra of **Fig. S5**. The monomer and the dimer in grey gather apo-IscU and Fe-IscU as the monomeric species and (apo-IscU)₂, (apo-IscU)(Fe-IscU) and (Fe-IscU)₂ as the dimeric species. All experiments were repeated at least 3 times.

After 25 and 50 min of the reaction, the nMS peaks corresponding to the S and D states of apo-IscU progressively decreased concomitantly with increasing proportions of three other IscU species (Fig. 5b). The zooms on the monomeric and dimeric regions of two representative charged states showed the presence of a monomeric form containing one [2Fe2S] cluster (16,170.4 ± 0.7, Fig. 5c, Table 3) and a dimeric form with a mass adduct consistent with either two [2Fe2S] clusters or a [4Fe4S] cluster (32,339.9 ± 0.5, Fig. 5c, Table 3). After 50 min of reaction, monomeric IscU with [2Fe2S] cluster was clearly detected as the most intense species, while in the dimeric region only the
dimer with either two [2Fe2S] clusters or a [4Fe-4S] cluster was observed. Importantly, a dimer with a single [2Fe2S] cluster was detected at 25 min of reaction time, while there was no evidence of this intermediate form after 50 min of reaction, suggesting that it is a transient species (32,166.1 ± 0.6, Fig. 5c, Table 3). In order to better explore the evolution of the dimeric form of IscU with one [2Fe2S] cluster, the kinetic study was extended to intermediate time points at 0, 5, 10, 15, 20, 30, 40 and 50 min (Fig. S5, Fig. 5d). The inspection of the relative intensities of the different peaks showed that the dimer with a single [2Fe2S] cluster increased and reached a plateau then progressively decreased after 30 min of reaction until its complete disappearance at 50 min. This kinetic profile indicates that it is a transient species leading to the formation of the monomer with one [2Fe2S] cluster and the dimer with two [2Fe2S] clusters.

<table><caption>**Table 3:** Experimental mass of the species detected by native MS and attribution</caption><thead><tr><td>Experimental mass (Da)</td><td>Attribution</td><td>Exact mass (Da)</td></tr></thead><tbody><tr><td>15,995.3 ± 0.3</td><td>apo-IscU</td><td>15,995</td></tr><tr><td>16,049.8 ± 0.5</td><td>Fe-IscU</td><td>16,051</td></tr><tr><td>16,170.4 ± 0.7</td><td>IscU-[2Fe2S]</td><td>16,171</td></tr><tr><td>31,990.2 ± 0.6</td><td>(apo-IscU)<sub>2</sub></td><td>31,990</td></tr><tr><td>32,045.1 ± 0.6</td><td>(apo-IscU)(Fe-IscU)</td><td>32,046</td></tr><tr><td>32,166.1 ± 0.6</td><td>(IscU)<sub>2</sub>-[2Fe2S]</td><td>32,165</td></tr><tr><td>32,339.9 ± 0.5</td><td>(IscU-[2Fe2S])<sub>2</sub></td><td>32,342</td></tr></tbody></table>

## Assessing the assembly mechanism using physiologically relevant ratios of the ISC components

The hindrance of [2Fe2S] cluster formation when IscU and IscS are present at stoichiometric amounts raised the question of the ability of the ISC system to synthesize Fe-S cluster under physiological conditions. Based on the number of IscU and IscS copies determined in *E. coli* cells cultured in different media,⁵¹ the ratio of IscU to IscS would be in the range of 0.9 to 1.3 (Table S2), thus close to equimolar amounts. However, available IscS for IscU is most likely lower since IscS forms complexes with at least two other partners, TusA and ThiI, that could compete with IscU.⁵² Thereby, the effective ratio of IscU to IscS would be in the range of 1.1 to 1.9. We tested Fe-S cluster assembly reactions with a range of IscU/IscS ratios of 10, 4, 2, 1.5, 1.25 and 1.0 where IscU was kept constant at 100 µM. The estimated ratios of Fdx and FdxR relative to IscS are 0.3 to 0.8 and 0.05 to 0.10, respectively (Table S2). We used 20 µM Fdx and 7 µM FdxR corresponding to the highest ratios of these proteins as compared to IscS. The Fe-S cluster assembly reactions showed a decrease of the absorbance reached at the plateau, corresponding to the total quantity of [2Fe2S] clusters formed, as the concentration of IscS was raised (Fig. S6a). At physiologically relevant IscU/IscS ratios of 2.0 and 1.5 the assembly of Fe-S clusters still occurred, which validates the mechanism described here using a reconstituted system.
Interestingly, two steps are clearly observed for the reactions performed with IscU/IscS ratios lower than 2. The absorbance suddenly increased before reaching a plateau at a slower pace. The first step could correspond to the first cycle of the assembly process. We estimated the amount of [2Fe2S] clusters formed at the first step and compared it with the amount that would be generated at the first cycle of the reaction by release of the IscU-[1Fe1S] precursor generated on the IscS-IscU complex by available free Fe-IscU at the start of the reaction. This was done by considering that the amount of [2Fe2S] clusters would be half the amount of IscU bound to IscS if dimerization occurs. The comparison yielded a good correlation (Fig. S6b), consistent with a mechanism relying on the dimerization IscU-[1Fe1S]. We also compared the total amount of [2Fe2S] clusters generated by considering the absorbance reached at the plateau after 20 min of reaction and we compared it with the expected amount if all iron and cysteine would be consumed minus the amount of IscU-[1Fe1S] trapped on IscS. The comparison showed a good correlation (Fig. S6c), confirming that IscU-[1Fe1S] did not spontaneously dissociate, which further indicates that IscU-[2Fe2S] is not able to displace IscU-[1Fe1S] on IscS. Taken together, these data support the hypothesis of a mechanism proceeding by dimerization of IscU-[1Fe1S] and indicate that IscU-[2Fe2S] is not able to exchange with IscU-[1Fe1S] on IscS. Although the amount of Fe-S clusters generated with physiologically relevant IscU/IscS ratios (between 1 and 2) appears suboptimal compared to a reaction with higher IscU/IscS (10 to 4), it must be noted that under physiological conditions, the assembly is a dynamic process where the [2Fe2S] cluster of IscU is transferred to acceptors, thereby recycling IscU for further Fe-S cluster assembly cycles.

## Targeting the transient dimer by mutating Y3 and Y61

The crystal structure of a holo form of the IscU protein from *M. thermoacetophila* (*Mt*) has revealed a centrosymmetric head-to-head dimeric structure containing two adjacent [2Fe2S] clusters, one in each subunit.²⁷ We thought that this arrangement could also drive the formation of the transient (IscU)₂-[2Fe2S] dimer of *Ec* IscU observed by nMS to condensate the [1Fe1S] precursors into a bridging [2Fe2S] cluster. The dimeric interface of holo *Mt* (IscU-[2Fe2S])₂ shows two intermolecular π-π stacking interactions between the tyrosine Y7 of one subunit and the phenylalanine F61 of the other one, which might be critical for the formation of this dimer. Sequence alignments showed that Y7 and F61 of *Mt* IscU are equivalent to Y3 and Y61 in *Ec* IscU (**Extended Data Fig. 1a**). Moreover, the structure of the *Ec* IscS-IscU complex indicates that Y3 and Y61 of IscU are also involved in the interaction with IscS. Y3 is part of a multi-bond interaction pattern centered on glutamate 311 of IscS, which interacts with Y3 via a hydrogen bond alongside the lysine 103 of IscU via a salt-bridge (**Extended Data Fig. 1b**). Y61 interacts with the backbone amide carbonyl of valine 308 on IscS, and forms energetically favourable hydrophobic interactions with the isobutyl side chain of leucine 386 and the aliphatic region of glutamate 309 of IscS (**Extended Data Fig. 1c**). Therefore, the substitutions of Y3 and Y61 in *Ec* IscU could affect both persulfide transfer from IscS and formation
of a putative holo dimer. To preserve the interaction with IscS while disrupting potential π-π stacking
in the IscU dimer, we substituted Y3 and Y61 by glutamine, a polar non-aromatic amino acid, as both
single and double mutants. We also tested substitutions by alanine to totally disrupt any polar
interactions.

The Fe-S cluster assembly assays showed that all the substitutions of Y3 and Y61 (Y3Q, Y3A, Y61Q, Y61A and Y3QY61Q) were active, with only slight effects on the rates of assembly (Extended Data Fig. 1d-e). The rates of assembly by the Y3Q and Y61Q mutants were strongly decreased in the absence of Fdx (Extended Data Fig. 1d-e), as with the WT (Fig. 1d-e). This indicates that in the assays including Fdx and FdxR, the reactions were proceeding via the Fdx-based mechanism. The mutants with substitutions of Y3 (Y3Q, Y3A and Y3QY61Q mutants) were those mostly affected, with a ~ 1.5-times lower rate of assembly than WT. The Y61Q mutant was similar to WT and the double mutant Y3QY61Q was similar to Y3Q and Y3A, suggesting that the Y3Q substitution was responsible for the defect of the double mutant. The Y61A mutant had a 1.3-times higher activity than WT. We next examined the effects of the Y3A, Y3Q, Y61A and Y61Q mutations on the rate of persulfide transfer. The kinetics of persulfide transfer monitored by ARBS assays showed that the Y3A and Y3Q mutants had 3-4 fold lower rates compared to WT, respectively, Y61Q was similar to WT and Y61A was slightly higher (Extended Data Fig. 1f-g). These data suggest that the effects of the Y3A, Y3Q and Y61A substitutions on Fe-S cluster assembly are due to interferences in persulfide transfer, consistent with the involvement of Y3 and Y61 in the interaction with IscS. Altogether, these data invalidate a mandatory role of Y3 and Y61 at a later step of Fe-S cluster assembly. Therefore, the formation of the transient (IscU)₂-[2Fe2S] dimer does not appear to rely on π-π stacking interactions between Y3 and Y61, suggesting that its dimeric interface differs from the interface of the *Mt* (IscU-[2Fe2S])₂ dimer.

Redox basis of the dimerization process

So far, our data indicate that two [1Fe1S] precursors merge into a [2Fe2S] cluster. UV-visible
and RMN data concluded that the [2Fe2S] cluster in IscU is in its oxidized state, denoted [2Fe2S]²⁺, in
which both iron ions are in the +3 state, as previously determined for the murine, yeast and *E. coli*
systems, resulting in a S = 0 ground state.<sup>10,12,19</sup> The EPR analysis also showed that the iron center in
the [1Fe1S] precursor is in the +2 redox state, denoted [1Fe1S]⁰ (**Fig. 3c, orange line**). Merging two
[1Fe1S]⁰ precursors would thus lead to a super-reduced cluster, noted [2Fe2S]⁰ (**Fig. 6a**, step 2) before
oxidation to the [2Fe2S]²⁺ state (**Fig. 6a**, step 3). Although unusual and unstable, the super-reduced
state has already been reached in proteins and inorganic complexes.<sup>53-56</sup>

To assess this hypothesis, we first tested whether the oxidized form of the [1Fe1S] precursor
could dimerize. To oxidize the [1Fe1S] precursor, we used 2,6-dichloro-phenol-indophenol (DCPIP),
a mild two-electrons oxidant (E°*pH*7 = + 0.217 V). We first tested the effect of DCPIP on the [1Fe1S]
precursor by EPR spectroscopy. The addition of DCPIP resulted in the complete disappearance of the g = 18 feature associated with the [1Fe1S] precursor, concomitant with the appearance of a new signal at g ≈ 4.26 (Fig. 6b), the intensity of which was enhanced in the perpendicular mode and characterized by several peaks at g = 4.19, 4.26, 4.35 and 9.5 (Fig. 6c). This signature is typical of a mononuclear ferric iron in the high spin state (S = 5/2) in a structured site with effective g-features indicative of high rhombicity (E/D ≈ 0.29). These data indicated that the [1Fe1S]⁰ was oxidized to the [1Fe1S]¹⁺ state.

<figure><img src="image_16.png" /><figcaption>Figure 6: Effect of DCPIP on the Fe-S cluster assembly process and NADPH titrations</figcaption></figure>

(a) Reaction scheme of persulfide reduction and formation of the [1Fe1S]⁰ precursor and the super reduced [2Fe2S]⁰ cluster. (b, c) X-band EPR spectra in the parallel (b) and perpendicular (c) modes of a reaction mixture containing 200 µM apo-IscU, Fe²⁺, IscS, Fdx, FdxR, 250 µM L-Cys and 400 µM NADPH (blue line, - DCPIP) and after reaction with 750 µM DCPIP for 10 min (orange line, + DCPIP). The upper orange line is a 20-fold amplification of the signal between 50 and 120 mT. (d) Effect of DCPIP on sequential Fe-S cluster assembly reactions monitored by UV-visible at 465 nm. Two reactions were initiated by mixing 30 µM of apo-IscU, Fe²⁺, IscS, L-cysteine for 5 min, then 30 µM of Fdx, FdxR with 60 µM of NADPH were added (Fdx). After 10 min of reaction, 30 µM of DCPIP was added to one reaction (DCPIP, orange line) and nothing to the other (blue line). After 10 min of reaction, 60 µM of extra Fe-IscU (Fe-IscU) were added to both reactions. (e) Fe-S cluster assembly reactions performed 100 µM of apo-IscU, 10 µM of IscS, Fdx and FdxR, 100 µM of iron and L-cysteine and either 50 µM (0.5 eq) or 100 µM (1.0 eq) of NADPH. All experiments were repeated at least 3 times.
We next tested the effect of DCPIP in sequential Fe-S cluster assembly reactions by monitoring the formation of the [2Fe2S] cluster by electronic absorption spectroscopy at 465 nm. Stoichiometric amounts of IscU, IscS and iron were incubated for 5 min with L-cysteine, then Fdx, FdxR and NADPH were added and the reactions were carried out for 10 min (Fig. 6d, orange and blue lines). After addition of DCPIP, the absorption at 465 nm immediately increased and progressively diminished over 10 min (Fig. 6d, orange line). The oxidized form of DCPIP absorbed in the visible region with a peak centered at 605 nm that strongly decreased upon reduction by ascorbic acid (Fig. S7). DCPIP also absorbed at 465 nm and the intensity decreases upon reduction. Therefore, the decrease in the absorption at 465 nm in the sample treated by DCPIP was an indication that oxidation of the [1Fe1S] precursor occurred. Upon addition of extra Fe-IscU, [2Fe2S] clusters were generated in the control reaction without DCPIP (Fig. 6d, blue line) whereas no cluster formation could be detected in the reaction incubated with DCPIP (Fig. 6d, orange line), indicating that the oxidized form of the [1Fe1S] precursor was not able to generate the [2Fe2S] cluster. Altogether, these data support a model in which only the reduced form of the [1Fe1S] precursor dimerizes, thereby leading to a super-reduced [2Fe2S]⁰ cluster (Fig. 6a, step 2).

To further assess this model, we examined the redox balance of this process by determining the minimal number of electrons required to generate the [2Fe2S]²⁺ cluster in *Ec* IscU. The reduction of a persulfide into sulfide to generate the [1Fe1S]⁰ precursor requires 2 electrons. The formation of two [1Fe1S]⁰ precursors thus requires a total of 4 electrons (Fig. 6a, step 1). The fusion of two [1Fe1S]⁰ precursors into the super-reduced [2Fe2S]⁰ cluster does not require additional electrons (Fig. 6a, step 2). In the last step, the oxidation of the [2Fe2S]⁰ cluster to the [2Fe2S]²⁺ state would release 2 electrons, which requires an electron acceptor (Fig. 6a, step 3). We hypothesized that Fdx could play that role, which would recycle electrons for further persulfide reduction. Therefore, a net number of 2 electrons would be needed if a [2Fe2S]⁰ cluster is formed and redox recycling operates. To test this hypothesis, we performed reactions with 2 and 4 electrons using 0.5 and 1.0 molar equivalent of NADPH per iron. The reaction was slower with 0.5 equivalent but reached completion as with 1.0 equivalent of NADPH, indicating that Fdx was recycled (Fig. 6e). The slower rate with 0.5 equivalent suggested that the reaction was rate-limited by Fdx recycling. These data support a model in which two [1Fe1S]⁰ precursors are fused to generate a super-reduced [2Fe2S]⁰ cluster with Fdx accepting electrons to reach the final [2Fe2S]²⁺ oxidized state.
DISCUSSION

Collectively, these data provide a step-by-step description of several key intermediates of the Fe-S cluster assembly process by the *E. coli* ISC machinery (**Extended Data Fig. 2a**). We report that the bacterial ISC machinery reconstituted *in vitro* with IscU, IscS, Fdx and FdxR, is functional for the assembly of [2Fe2S] clusters. Sequential analysis by CD and ARBS assays revealed that the reaction is initiated by the insertion of a ferrous iron into the assembly site of IscU. Upon binding of Fe-IscU binds to IscS, a cysteine-bound persulfide generated by IscS is transferred to IscU. This trans-persulfidation reaction requires iron, which compels to a sequential assembly process with iron coming first then sulfur, likely allowing to coordinate persulfide acquisition with the presence of iron in IscU. Once the persulfide is in IscU, it is reduced into sulfide by Fdx. These data provide the first evidence that the *E. coli* ferredoxin Fdx reduces the persulfide into IscU. The same mechanism was reported for the mouse ISC machinery with FDX2,¹⁰ indicating that the primary steps of Fe-S cluster assembly are conserved across species.

In the absence of the Fdx/FdxR system, formation of Fe-S clusters still occurs but relies on L-cysteine to reduce persulfides (referred to as the thiol-based reaction). In the murine system, L-cysteine was shown to reduce the persulfide of NFS1, an IscS homolog, leading to the release of free sulfide that combined with iron to generate Fe-S clusters in a process akin to chemical reconstitutions.<sup>10,20</sup> The thiol-based reaction is considered as a non-physiological process for Fe-S cluster biogenesis, only occurring *in vitro*, in contrast to the Fdx-based reaction that describes the biological process.<sup>2,10</sup> These data strengthen a general concept in Fe-S cluster assembly where sulfide has to be generated in the vicinity of the iron center of IscU to be trapped as it would otherwise diffuse out of the protein.

Next, to determine how two sulfides are generated and assembled with two irons into a dinuclear [2Fe2S] cluster, we titrated the number of iron and sulfide that IscU could accumulate at the first steps and we characterize the changes occurring at the iron center during the assembly process by spectroscopic methods. We found that IscU initially binds a single iron and acquires a single persulfide from IscS. This reaction was highly efficient with nearly all IscU converted into a monopersulfidated form, and despite the presence of two equivalents of iron and L-cysteine, IscU was not able to acquire more than one persulfide. Moreover, after the reduction of the persulfide by Fdx, IscU could not acquire a second persulfide. These data show that before a [2Fe2S] cluster is formed, IscU initially contained one iron and one sulfide. Strikingly, when IscU and IscS were present in equal amounts in the reaction mixture, no [2Fe2S] cluster was formed despite the generation of sulfide. The combination of sulfide titration, EPR and UV-visible analysis consistently concluded that after reduction by Fdx/FdxR, the sulfide ion was bound to the iron center and the iron center remained mononuclear. The EPR analysis showed that the iron was still in the high spin (S = 2) Fe(II) state. We thus concluded that a Fe(II)-S(H) species was formed, which we referred to as the [1Fe1S]<sup>0</sup> species.
Time course analysis by UV-visible further showed that the [1Fe1S]⁰ species was progressively converted into a [2Fe2S] cluster, indicating that it is a precursor of the [2Fe2S] cluster. These data provide the first proof of the existence of a [1Fe1S] precursor in the course of the Fe-S cluster assembly reaction.

The kinetics of Fe-S cluster assembly showed that formation of the [2Fe2S] cluster only occurred when IscU was in excess relative to IscS, and NMR data revealed that free Fe-IscU exchanged with IscU carrying the [1Fe1S] precursor on IscS. The analysis of the course of the reaction by nMS showed that a dimer with a single [2Fe2S] cluster is transiently formed and evolved into a monomer carrying one [2Fe2S] cluster and a dimer carrying two [2Fe2S] clusters. We conclude from these data that IscU carrying the [1Fe1S] precursor is released from IscS by exchange with free IscU, then dimerizes to merge two [1Fe1S] precursors into a bridging [2Fe2S] cluster. The transient nature of the (IscU)₂-[2Fe2S] dimer and the identification of a monomer with a single [2Fe2S] cluster as one of the main products suggest that the bridging [2Fe2S] cluster migrates onto one of the two subunits, which would trigger dissociation of the dimer and formation of the monomer carrying a single [2Fe2S] cluster, leaving the other subunit empty. This dissymmetric dissociation is supported by the NMR data showing that the product of the reaction performed with one equivalent of iron and L-cysteine relative to IscU, contains both apo-IscU and IscU-[2Fe2S]. Finally, the monomer with a single [2Fe2S] cluster dimerizes to generate the dimer with two [2Fe2S] clusters. At this stage, the reaction can engage a new cycle with the apo-IscU released from the dissociation of the dimer, until all the iron, cysteine or NADPH are consumed.

The assembly reactions performed with higher amounts of IscS compared to IscU, in which a larger fraction of IscU remained bound to IscS, revealed that the reaction proceeds in two distinct kinetic steps. The first step consumes half of the free IscU initially present, consistent with the formation of a dimer, which strengthens the dimerization model. The second step, slower than the first one, probably corresponds to the recycling of apo-IscU for further cycles. These data also showed that IscU-[2Fe2S], once formed, cannot displace IscU-[1Fe1S] on IscS, thus that IscU-[2Fe2S] remains free. Keeping IscU-[2Fe2S] in a free form might be an important feature to facilitate the interaction with downstream partners involved in the transfer of the [2Fe2S] cluster to recipient proteins. Strikingly, the physiological IscU/IscS ratios in *E. coli* cells are estimated between 2 to 1, suggesting that between 50% to 100% of IscU is permanently bound to IscS, retaining its [1Fe1S] precursor, and conversely a small fraction of IscU is free, which should significantly limit the amount of [2Fe2S] clusters generated. However, under physiological conditions, the presence of acceptors of the [2Fe2S] cluster in IscU will regenerate apo-IscU, thereby triggering the release of more IscU-[1Fe1S] precursors to generate [2Fe2S] clusters. These results suggest that the assembly of [2Fe2S] clusters is subordinated to the availability of acceptors.
The formation of a transient IscU dimer raised the question of its structure since only one structure of an IscU dimer has been documented so far, for *Mt* IscU.<sup>27</sup> The protein was crystalized in a holo-form in a head-to-head arrangement with each subunit harbouring a [2Fe2S] cluster. This holo-form may rather correspond to the (IscU-[2Fe2S])<sub>2</sub> dimer generated by the *Ec* reconstituted system. However, we questioned whether the transient dimer could also adopt this head-to-head arrangement to enable the fusion of the [1Fe1S] precursors. The dimerization interface of the holo *Mt* IscU dimer involves π-π stacking interactions between the tyrosine Y7 and the phenylalanine F61 of the other subunit.<sup>27</sup> Since the tyrosine equivalent to Y7 in *Ec* IscU (Y3) and human ISCU2 (Y35), were shown to be essential *in vivo*<sup>26</sup> and critical for dimerization,<sup>13</sup> respectively, we asked whether the formation of the transient (IscU)<sub>2</sub>-[2Fe2S] dimer could also rely on π-π stacking interactions between Y3 and Y61 of *Ec* IscU, equivalent to Y3 and F61 in *Mt* IscU. However, we found that mutations of Y3 in *Ec* IscU had only weak effects on Fe-S cluster assembly that correlate with alterations in the rate of persulfide transfer, likely due to the engagement of Y3 in the interaction with IscS. The mutations of Y61 in *Ec* IscU had only marginal effects, if any. Therefore, the structure of the transient (IscU)<sub>2</sub>-[2Fe2S] dimer in *Ec* IscU does not seem to rely on π-π stacking interactions between Y3 and Y61 but other undefined structural elements. The essential role of Y3 *in vivo* in *E. coli*<sup>26</sup> is probably due to the impairment of persulfide transfer or a later step after formation of the transient dimer.

Finally, the redox states of the intermediates and of the [2Fe2S] cluster produced by the ISC system raised the question of the redox basis of the mechanism. EPR showed that the iron center in the [1Fe1S] precursor is in the reduced state, denoted [1Fe1S]<sup>0</sup>, suggesting that when two of these precursors are fused by dimerization of IscU, a super-reduced [2Fe2S]<sup>0</sup> cluster is formed. The super-reduced state of [2Fe2S] clusters has only been reached using strong reductants.<sup>53-56</sup> A reaction scheme involving such a highly reactive species is thus surprising. An alternative pathway would involve the oxidized form of the precursor, [1Fe1S]<sup>+</sup>, which could directly generate the final [2Fe2S]<sup>2+</sup> form by dimerization of IscU. However, oxidation of the [1Fe1S]<sup>0</sup> precursor to the [1Fe1S]<sup>1+</sup> state by DCPIP precluded formation of [2Fe2S] clusters and NADPH titrations were consistent with formation of a super-reduced [2Fe2S]<sup>0</sup> cluster which donates two electrons to Fdx, thereby reaching the final [2Fe2S]<sup>2+</sup> oxidized state. These data therefore suggest that two [1Fe1S]<sup>0</sup> precursors are fused into a super-reduced [2Fe2S]<sup>0</sup> cluster upon dimerization of IscU (Extended Data Fig. 2b). The fusion of two [1Fe1S]<sup>0</sup> precursors, in which the net charge is zero, may be favoured over the dimerization of two positively charged [1Fe1S]<sup>+</sup> precursors due to charge repulsion effects. Such a reductive coupling is reminiscent of the formation of [4Fe4S]<sup>2+</sup> clusters by fusion of two [2Fe2S] clusters, which requires the reduction of the clusters to their [2Fe2S]<sup>1+</sup> state.<sup>19,24,27</sup> The conversion of the super-reduced [2Fe2S]<sup>0</sup> state to the final oxidized [2Fe2S]<sup>2+</sup> state requires an electron acceptor. In our assays, Fdx recycles these electrons for persulfide reduction, but it is unclear whether Fdx would be the natural acceptor *in vivo*, since other as-yet undefined electron acceptors could also play this role.
In conclusion, this stepwise study suggests that two [1Fe1S] precursors are fused into a
[2Fe2S] cluster via dimerization of IscU in the *E. coli* ISC system. This mechanism may also apply to
the eukaryotic systems since iron and persulfide titrations in the mouse ISC system pointed to
formation of a [1Fe1S] precursor and the analysis of the human Y35A ISCU2 mutant concluded that
dimerization is a mandatory step.¹⁰,¹³ Moreover, nMS analysis showed that the main product of the
reaction is a monomer coordinating a single [2Fe2S] cluster for both, the mouse and *E. coli* systems,
suggesting a similar mechanism.¹⁰ However, size exclusion chromatography analysis of the human
ISC system showed that the main product of the reaction was a dimer.¹³ Second, while the tyrosine
Y35 is critical for the dimerization of human ISCU2,¹³ it is dispensable for *E. coli* IscU, suggesting
that the structure of the dimer driving the fusion of the two [1Fe1S] precursors might be different.
Further investigations are needed to understand these subtle differences.

These data also provide characterisations of an iron-sulfido species for which only few
examples exist and exclusively as small inorganic complexes. To the best of our knowledge, only two
examples of non-heme iron-sulfido species have been reported in the literature and a single one with
iron in the +2 redox state as in the [1Fe1S] species.³⁶,⁵⁷ The data herein thus open new perspectives to
characterize iron-sulfido species and to unravel the mechanisms of [2Fe2S] cluster biosynthesis in
both the prokaryotic and eukaryotic systems. These data also lay down the basis to design new
biomimetic routes to control Fe-S clusters synthesis in inorganic reactions.
## Chemicals and materials

Sodium phosphate dibasic ($Na_2HPO_4$), tris-(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), sodium chloride ($NaCl$), ascorbate, sodium dodecyl sulfate (SDS), sodium sulfide ($Na_2S$), N,N-Dimethyl-p-phenylenediamine (DMPD), dithiothreitol (DTT), hydrochloric acid (HCl), L-cysteine, protease inhibitor cocktail (sigmafast), $His_6$-thrombin, diethylene triamine pentaacetic acid (DTPA), iron(III) chloride ($FeCl_3$), ferrous ammonium sulfate ($Fe-(NH_4)_2(SO_4)_2$), 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate (ferrozine), imidazole, zinc sulfate ($ZnSO_4$), $\beta$-mercaptoethanol, acrylamide: bisacrylamide (19:1), Mal-dPEG$_4$-(m-dPEG$_{12}$)$_3$ (2.36 kDa), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), pyridoxal phosphate (PLP), 2,6-dichloro-phenol-indophenol (DCPIP) and ascorbic acid were from Sigma-Aldrich; isopropyl $\beta$-D-1-thiogalactopyranoside (IPTG) was from Euromedex; HisBind Resin was from Novagen; PD10 column, Superdex, Q-Sepharose, and Phenyl Sepharose columns were from Cytiva; Amicon Ultra-15 Centrifugal Filter Units were from Millipore; Bio-Spin column from BioRad.

## Protein purification

The IscU, IscS, Fdx and FdxR proteins were produced following previously reported procedures.¹¹ To prepare the apo-IscU proteins WT and mutants (Y3A, Y3Q, Y61A, Y61Q, Y3QY61Q), the as-purified IscU proteins were incubated with 50 molar equivalents of DTT and 100 equivalents of DTPA during one hour at room temperature in order to reduce all cysteine residues and to remove the zinc ion. The IscU proteins were then loaded on a HiLoad 26/600 Superdex 75 prep grade column pre-equilibrated with 20 mM Tris, 100 mM NaCl, 10 mM DTT at pH 8.0. The monomeric forms were collected and concentrated on Amicon 15 mL (10 kDa cut-off).

All the assays (Fe-S cluster assembly, ARBS, sulfide and iron titrations) and preparation of the samples for CD, UV-visible, EPR, NMR, nMS were performed under anaerobic conditions in glove boxes (Jacomex, MBraun, $O_2$ < 2 ppm). The proteins were desalted on a NAP-5 column pre-equilibrated with 20 mM Tris, 100 mM NaCl at pH 8.0 inside the glove box, concentrated at desired concentration on Amicon 0.5 mL (10 kDa cut-off) and further let to deoxygenate for one hour at 10°C inside the anaerobic chamber. The proteins were aliquoted and flash frozen with 10% glycerol in liquid nitrogen before storage in a liquid nitrogen tank. Iron loaded IscU was prepared by mixing apo-IscU with one molar equivalent of ferrous ammonium sulfate prepared in water. Iron binding was routinely evaluated by circular dichroism on a Jasco J-815 CD spectrometer.

## Fe-S cluster assembly assays

For catalytic conditions, apo-IscU (100 µM) was incubated with one molar equivalent of ferrous ammonium sulfate (100 µM), IscS (10 µM), Fdx (5 µM), FdxR (1 µM) and NADPH (200 µM). For stoichiometric conditions, apo-IscU (100 µM) was incubated with one molar equivalent of ferrous
ammonium sulfate (100 µM), IscS (100 µM), Fdx (100 µM), FdxC (100 µM) and NADPH (200 µM). The mixtures were transferred into a septum sealed cuvette and the reactions were initiated by adding L-cysteine (100 µM) via a Hamilton syringe. The kinetics of Fe-S cluster assembly were monitored by UV-visible at 465nm at 25°C using a Secomam UVIKON XL spectrometer equipped with a refrigerated cell connected to a circulating water bath. For cysteine titrations, the reactions were initiated with 25, 50, 100 and 200 µM of L-cysteine. Sequential Fe-S cluster assembly reactions were performed under stoichiometric conditions by mixing apo-IscU (100 µM), iron (100 µM) and IscS (100 µM) then L-cysteine (100 µM). After 10 min of reaction, Fdx (100 µM), FdxC (100 µM) and NADPH (200 µM) were added. After another 10 min of reaction, extra Fe-IscU (200 µM) was added.

## Persulfide quantification by ARBS assays

The alkylation assays to quantify persulfides were conducted as previously described using Mal-dPEG2K as an alkylating agent.³¹ The reactions were carried-out under stoichiometric conditions using apo-IscU (20 µM) and IscS (20 µM) and various amounts of iron and L-cysteine from 1 to 2 molar equivalents in phosphate buffer containing 50 mM Na₂HPO₄, 100 mM NaCl, pH 8.0. The reactions were initiated by adding L-cysteine and incubated for 3 min. To assay the reduction by Fdx, the mixture containing apo-IscU (20 µM), IscS (20 µM) and iron (20 to 40 µM) were incubated with L-cysteine for 10 min then a mixture containing Fdx (20 µM), FdxC (20 µM) and NADPH (40 µM) was added and the mixture was incubated for 10 min. To monitor the kinetics of persulfide transfer, the reactions were sampled at different time and quenched by adding Mal-dPEG2K under denaturing conditions. All the reactions were quenched using a 2-fold excess of Mal-dPEG2K compared to the total amount of thiols in the reaction mixture. Typically, 15 µL of the reaction mixture were mixed to a solution containing Mal-dPEG2K, 1 mM EDTA and 2% SDS prepared in water. The reactions were incubated during 30 minutes at room temperature. Then, 10 µL of a Laemmli solution containing 10% SDS, 50% glycerol, 0.05% bromophenol blue, 0.1M Tris buffer at pH 6.8, with 0.5 mM β-mercaptoethanol (reducing conditions) or without (non-reducing conditions). The reaction was analysed by SDS-PAGE 14% and the proteins were revealed by Coomassie staining.

## Iron and sulfide quantification

The protein samples were desalted on a Biospin column. The iron content in protein samples was determined using a colorimetric assay with ferrozine. The protein samples were incubated with 100 µL of a solution containing ferrozine (2 mM) and ascorbate (10 mM) at room temperature for 1 hour. The reaction mixture was transferred in quartz cuvettes and the absorbance at 565 nm was measured. The iron content was determined using an extinction coefficient of 30,800 M⁻¹cm⁻¹ at 565 nm determined from a calibration curve with a solution of Fe(NH₄)₂(SO₄)₂ in the same buffer. The sulfide content in protein samples was determined using the methylene blue assay. The protein samples (125 µL) were incubated with 10 µL of NaOH 30% solution at room temperature during 15 minutes. The reaction
mixtures were then incubated with 125 µL of DMPD (N,N-dimethyl-1,4-phenylene-diamine, 0.1% in 1.5 M HCl) and 50 µL of ferric iron solution (11.5 mM in 1.5 M HCl) during 2 hours at 25°C. After 10 minutes, the reaction mixture was centrifuged at 13 000 rpm, the supernatant was diluted with 500 µL of H₂O and the absorbance of methylene blue was measured at 670 nm. The sulfide concentration was determined using an extinction coefficient of 25,500 M⁻¹cm⁻¹ at 670 nm previously determined by a calibration curve with a Na₂S solution in water (0-100 µM).

## EPR spectroscopy

The EPR samples (between 100 and 200 µM in IscU, final volume of 180 µL) were prepared under anaerobic conditions in the glove box in 20 mM Tris buffer pH 8 containing 100 mM NaCl and 10% of glycerol. The samples were transferred into calibrated quartz EPR tubes, sealed with a septum, precooled in cold ethanol still under inert atmosphere before freezing in liquid nitrogen outside the glovebox. Despite extensive care taken during transport in septum-sealed tubes and storage in liquid nitrogen, we found that, for some samples, O₂ could diffuse within cracks formed at the surface of the frozen sample in the EPR tube. The paramagnetic ground state (S = 1) of O₂ gives a complex EPR signature at 9 GHz in the parallel mode that partially overlays with the EPR signature of the S = 2 Fe(II) species (Fig. S1c). O₂ could be successfully removed by several cycles of vacuum/nitrogen flush on the frozen EPR sample prior to each EPR experiment. Alternatively, the EPR samples were prepared anaerobically and flame-sealed once the samples were frozen, which prevented O₂ contamination during liquid nitrogen storage and/or handling during EPR experiments (Fig. S1c). EPR spectroscopy experiments were performed on a Bruker Elexsys E500 spectrometer fitted with an Oxford Instruments ESR-900 helium-gas flow for cryogenic temperature studies and mounted with a Bruker dual mode resonator (ER 4116DM).

## NMR spectroscopy

¹⁵N-labeled IscU was produced in minimum medium complemented with 1 g L⁻¹ ¹⁵NH₄Cl and 20 µM ZnCl₂. The purification of ¹⁵N-IscU was carried as described for unlabelled IscU. Protein samples containing 100 µM ¹⁵N-IscU were prepared in 20 mM Tris pH 8.0, 100 mM NaCl buffer complemented with 7.5 % ²H₂O to lock the spectrometer frequency. ¹H-¹⁵N correlation spectra were acquired on a Bruker NMR spectrometer operating at 800 MHz ¹H frequency, equipped with an Avance III console and a TXI cryoprobe, using a SOFAST-HMQC pulse sequence⁴¹ at a temperature of 293 K, with a recycling delay of 0.2 s and 64 or 512 scans. NMR data were analysed with CcpNmr AnalysisAssign V3.1.1 software.⁵⁸

Samples for paramagnetic ¹H NMR were prepared in 20 mM Tris pH 8.0, 100 mM NaCl, 99.9% D₂O buffer by desalting on micro Bio-spin columns. The ISC reconstitution mixture contained 400 µM IscU, 1 molar equivalent of Fe²⁺ and L-cysteine, 2 molar equivalents of NADPH, and 10 µM of IscS, Fdx and FdxR. Control tubes were prepared with the mixture without IscU, and with buffer only.
Measurements were carried out on a Bruker Avance III 600 MHz spectrometer equipped with a TCI
cryoprobe, using a super-WEFT pulse sequence d1-180°-τ-90°-acq.⁵⁹ The 90° pulse length was 6.3-6.5
µs, the relaxation delay d1 35 ms, the interpulse delay τ 20 ms, and the acquisition time acq 21 ms.
The spectral window was set to 192 kHz (320 ppm), and the transmitter was centered at 20 ppm.
Spectra were acquired at 293 K, with 1k dummy scans to reach a steady state and 4k scans (sample
with IscU) or 16k cans (sample without IscU). The temperature was varied from 283 K to 298 K to
measure the temperature dependence of the chemical shifts of hyperfine-shifted signals. The
transmitter frequency was subsequently shifted in 20-50 ppm steps up to 300 ppm to account for
limited excitation bandwidth. Spectra were processed with squared sine bell apodization. The baseline
was corrected by subtracting spectra acquired on buffer under identical acquisition conditions.

All samples were handled inside a glove box, and samples were placed in a valved 3 mm NMR tube (Norell). ¹H chemical shifts were referenced to DSS. NMR data were processed with TopSpin 4.0 software (Bruker).

Pseudo-contact shifts (PCS) were measured as chemical shift differences in the ¹H and ¹⁵N dimensions between Fe-IscU and Zn-IscU amide signals. They were analyzed with the FANTEN software⁴⁷ accessed through the FANTEN web portal (http://abs.cerm.unifi.it:8080/). The crystallographic structure of *Ec* IscU in the IscS-IscU complex (PDB 3lvl) was used as an input, after adding hydrogens through access to the Protein Repair & Analysis Server (https://www.protein-science.com/).⁶⁰

nMS

Prior to nMS analysis, IscU was buffer-exchanged into 10 mM ammonium acetate (AcONH4) using a
Biospin column in a glove box under anaerobic conditions to avoid protein oxidation. Subsequently,
the proteins were diluted to 10 µM in 10 mM AcONH4 (pH 7.0). NMS measurements were performed
in positive ion mode on a Q-TOF Synapt G2 HDMS mass spectrometer (Water, Manchester, UK)
hyphenated to an automated chip-based NanoESI source (Triversa Nanomate, Advion Biosciences).
The instrument was calibrated and tuned using a 2 g/L solution of cesium iodide in 2-propanol/water
(50/50 v/v), and then operated in sensitivity mode using a + 1.5 kV capillary voltage. The backing
pressure (Pi) and the sample cone voltage (Vc) were set at 6 mbar and 60 V, respectively. In these
conditions, the non-covalent interactions were preserved to allow the detection of protein/iron-sulfur
clusters. The acquisitions were recorded in the 1000-5,000 m/z range with a 4 s scan time. Each
analysis was performed in triplicate under similar experimental and instrumental conditions. MS data
processing was performed using Mass Lynx V4.1 (Waters, Manchester, UK).
EXTENDED DATA

a

<table><tr><td>M. thermoacetophila</td><td>MIQQTGYSKKVMEHFMNPRNVGVIDDP---DGYGKVGNPVCGDLMEIFIKVGDEK-IEDI</td><td>56</td></tr><tr><td>E. coli</td><td>---MAYSEKVIDHYENPRNVGSFDNNDENVGSGMVGAPACGDVMKLQIKVNDEGIIEDA</td><td>56</td></tr><tr><td colspan="2">**:**:**:**:**: ******** :*: * * * *:**:**:**: ****.** ***</td><td></td></tr><tr><td>M. thermoacetophila</td><td>KFRTEGCGAAIATSSMITEMARGKSLEEAMRITRNDVADALDGLPPQKMHCSNLAADALH</td><td>116</td></tr><tr><td>E. coli</td><td>RFKTYGCGSAIASSLVTEWVKGKSLDEAQAIKNTDIAEEL-ELPPVKIHCSILAEDAIK</td><td>115</td></tr><tr><td colspan="2">*:*:*:*:*:*:*:*: .******:** *...*:*: * *** *:*:*: **:**:**:</td><td></td></tr><tr><td>M. thermoacetophila</td><td>AAINDYLSKKQKH</td><td>129</td></tr><tr><td>E. coli</td><td>AAIADYKSKREAK</td><td>128</td></tr><tr><td colspan="2">*** ** **:**:</td><td></td></tr></table>

b

<figure><img src="image_17.png" /></figure>

c

<figure><img src="image_18.png" /></figure>

d

<figure><img src="image_19.png" /></figure>

e

<figure><img src="image_20.png" /></figure>

f

<figure><img src="image_21.png" /></figure>

g

<figure><img src="image_22.png" /><figcaption>Extended Data Figure 1: Effects of Y3 and Y61 IscU mutants on Fe-S cluster assembly and persulfide transfer1</figcaption></figure>

(a) Sequence alignment of *Mt* and *Ec* IscU proteins. The position corresponding to Y7 and F61 of *Mt* IscU are highlighted in bold with a red dot. (b, c) Zoom on the *Ec* IscS-IscU binding interface (PDB 3LVL) centered on Y3 (b) and Y61 (c) with salt bridges, hydrogen bonds and hydrophobic interactions indicated as green, blue and red lines, respectively.⁶¹ (d) Kinetics of Fe-S cluster assembly monitored by UV-visible at 465nm and performed under catalytic conditions at 20°C with 100 µM of apo-IscU WT, Y3A, Y3Q, Y61A, Y61Q and Y3QY61Q, 100 µM of Fe²⁺, 5 µM of IscS, Fdx and FdxR, 600 µM of NADPH and initiated by addition of 100 µM of L-cysteine.

(e) Plot of rate constants of Fe-S cluster assembly assays, as performed in *d*, for IscU WT, Y3A, Y3Q, Y61A, Y61Q and Y3QY61Q representing mean values of 3 repeats. (f) Persulfide transfer assays from IscS to IscU WT, Y3A, Y3Q, Y61A and Y61Q analyzed by ARBS assays. The reactions were performed with 20 µM of apo-IscU WT, Y3A, Y3Q, Y61A and Y61Q, 40 µM of Fe²⁺, 20 µM of IscS and initiated with 40 µM of L-cysteine.

(g) Plot of rate constants of persulfide transfer assays, as performed in *f*, for IscU WT, Y3A, Y3Q, Y61A, Y61Q and Y3QY61Q representing mean values of 3 experiments.
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

**Extended Data Figure 2: Fe-S cluster assembly reaction schemes**

(a) Scheme of Fe-S cluster assembly: (1) a Fe²⁺ ion binds to the assembly site of IscU (Fe-IscU species), (2) two Fe-IscU bind to the cysteine desulfurase dimeric complex IscS, (3) persulfides are generated by IscS, (4) a persulfide is transferred to IscU via a cysteine residue, (5) Fdx reduces the persulfide in IscU into a sulfide ion which leads to formation of a [1Fe1S] precursor, (6) IscU carrying the [1Fe1S] precursor is released from IscS by exchange with Fe-IscU, (7) two IscU carrying the [1Fe1S] precursor dimerize and the [1Fe1S] precursors merge to generate a [2Fe2S] cluster bridging two IscU, (8) the [2Fe2S] cluster migrates onto one subunit of the dimer and the dimer dissociates into a monomer carrying the [2Fe2S] cluster and a monomer in its apo-form, (9) two monomers carrying a [2Fe2S] cluster dimerize, which generates a dimer carrying two [2Fe2S] clusters. (b) Redox state of the intermediates along the assembly reaction scheme.
## Acknowledgements

This work was supported by the "Agence Nationale de la Recherche" (Frataxur ANR-17-CE11-0021, FeSAM ANR-22-CE11-0006-04 awarded to B.D.A.), the Polish National Science Center Grant OPUS 21 UMO-2021/41/B/NZ1/00449, the CNRS, the University of Strasbourg, the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03), the EPR-MRS facility of the French Research Infrastructure INFRANALYTICS (FR2054) and the Aix-Marseille University EPR center. S.G. is grateful to the "Fondation pour la Recherche Médicale" for funding of his 4th year PhD (FDT202012010541), R. B. acknowledges the European Union's Horizon 2020 Research and Innovation Program Marie Skłodowska Curie Action ITN for funding their PhDs under Grant Agreement No. 859458. We are grateful to Emilien Etienne and Guillaume Gerbaud for their technical support and fruitful discussions, and Frédérique Berger, Service commun de soufflage de verre, Fédération des Sciences Chimiques de Marseille, Aix-Marseille University, for the sealing of the EPR tubes.
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