# When iron and sulfur met on an anoxic planet and eventually made clusters essential for life

Pierre Simon Garcia, Simonetta Gribaldo, Frédéric Barras

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Pierre Simon Garcia, Simonetta Gribaldo, Frédéric Barras. When iron and sulfur met on an anoxic planet and eventually made clusters essential for life. Biochimica et Biophysica Acta - Molecular Cell Research, 2024, 1871 (5), pp.119730. 10.1016/j.bbamcr.2024.119730. pasteur-05003086

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Review

# When iron and sulfur met on an anoxic planet and eventually made clusters essential for life

Pierre Simon Garcia<sup>a, b</sup>, Simonetta Gribaldo<sup>b</sup>, Frédéric Barras<sup>a, ∗</sup>

<figure><img src="image_2.png" /></figure>

A B S T R A C T

[Fe—S] clusters are co-factors that are essential for life and are synthesized by dedicated multiprotein cellular machineries. In this review, we present the current scenario for the emergence and the diversification of the [Fe—S] cluster biosynthesis machineries. In addition to well-known NIF, ISC and SUF machineries, two alternative minimal systems, SMS, and MIS, were recently identified. Taxonomic distribution and phylogeny analyses indicate that SMS and MIS were present in the Last Universal Common Ancestor (LUCA), well before the increase of oxygen on Earth. ISC, SUF and NIF systems emerged later in the history of life. The possible reasons for the emergence and diversification of these machineries are discussed.

**1. Introduction**

[Fe-S] clusters are ubiquitous and found in structurally and functionally diverse proteins [1,2]. A recent survey of *Escherichia coli* proteomes indicates that at least 181 protein species carry [Fe-S] clusters and are found in many unrelated biological pathways [3]. [Fe-S] clusters exist under different composition and geometry. The simplest and most frequently found are the [2Fe-2S] and [4Fe-4S], which are planar and cubic, respectively. A [3Fe-4S] cluster can be found as a decayed form, produced for instance under oxidative stress conditions [4]. In fact, clusters can exhibit different redox states, e.g. [4Fe-4S]⁰, [4Fe-4S]¹⁺, [4Fe-4S]²⁺, which might confer different biological properties to the hosting protein, as reported in seminal work by Beinert and colleagues on aconitase [5]. A previous bioinformatic study reported how [Fe-S] proteins tend to be selected against in organisms living under aerobiosis as compared with those living under anaerobiosis [6]. Presumably, this distribution is linked with the damaging effect of reactive oxygen species (ROS) on [Fe-S] clusters. One should underline that susceptibility to ROS is not a general rule that applies to any [Fe-S] of any protein. Most of the reported cases concern solvent exposed [Fe-S] clusters, as found for example in dehydratases [4]. Yet, this clearly points to specific relationships between a [Fe-S]-based life and environmental O₂.

Since the 90's and from the seminal work of Dean and collaborators on *Azotobacter vinelandii* nitrogenase, we know that *in vivo*, [Fe-S] clusters biogenesis can be described by two steps: assembly and distribution [7]. These steps and the associated components are the objects of

several chapters in this special issue, and we will here only briefly recall the basic principles and actors (Fig. 1). Sulfur is produced from L-cysteine thanks to the action of cysteine desulfurases. A dedicated source of iron has not been identified and it remains a possibility that intracellular iron can be provided by several molecules such as DNA, polyphosphate, or sugars, whereas extracellular iron was proposed to be provided by siderophores [8,9]. Then, a so-called scaffold protein captures sulfur as a persulfide form and iron under its ferrous form and provides the molecular environment allowing a cluster to assemble [10–12]. The cluster is then delivered to apo-targets via a network of [Fe-S] carriers, whose composition varies depending on the environmental conditions and the final targeted apo-protein [13,14].

**2. Diversity and distribution of [Fe-S] clusters biogenesis systems**

Several [Fe-S] clusters biogenesis systems have been described. Despite the fact they broadly obey the general construction plan depicted above, each presents significant specificity, both in terms of mechanisms and components. In a recent article, we made a survey of [Fe-S] clusters biogenesis systems in over 10,000 genomes of Bacteria and Archaea [15]. Notably, we analyzed homologues of all known associated components and depicted the homologue families they belong to by combining phylogenomics and genomic neighborhood, as this tends to be conserved (Figs. 2 & 3A). This allowed us to accurately identify each distinct [Fe-S] clusters biogenesis systems, which are described below.

<footnote><sup>a</sup> Institut Pasteur, Université Paris Cité, CNRS UMR6047, Department of Microbiology, Unit Stress Adaptation and Metabolism in Enterobacteria, Paris, France</footnote>

<footnote><sup>b</sup> Institut Pasteur, Université Paris Cité, Department of Microbiology, Unit Evolutionary Biology of the Microbial Cell, Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: frederic.barras@pasteur.fr (F. Barras).</footnote>
## 2.1. The NIF system

The NIF (NItrogen Fixation) system was the first to be identified [7,16]. It is specific for maturation of the nitrogenase complex, which catalyzes the conversion of atmospheric N₂ into bioavailable ammonium and H₂ [17]. The best-studied molybdenum nitrogenase consists of two component proteins: the molybdenum iron (MoFe) protein, which contains a [8Fe–7S] cluster (P-cluster) and a [7Fe-Mo-9S-homocitrate-R] cluster (FeMo-cofactor), and the iron (Fe) protein, which contains a [4Fe–4S] cluster. The genes encoding the nitrogenase complex form a large gene cluster, i.e. *nifABDEFAHKLMNQSTUWXYZ*, which includes functions for both the synthesis of nitrogenase and its maturation [18,19].

The NIF system *per se* is composed of three proteins: a cysteine desulfurase NifS, a U-type scaffold NifU, and a A-type carrier IscA\_nif (Figs. 2 & 3A). NifU is composed of three distinct domains: the NifU domain (N-terminal, Pfam NifU_N PF01592, InterPro IPR002871) that assembles a [2Fe–2S] cluster, the ferredoxin domain (middle, Pfam Fer2_BFD PF04324, InterPro IPR007419) that provides electrons, and the Nfu domain (C-terminal, Pfam NifU PF01106, InterPro IPR001075) that binds a [4Fe–4S] cluster but whose role is still not clear. Finally, the IscA\_nif is an A-type carrier that is likely involved in the transfer of the cluster from NifU to NifKD and NifH [20].

The NIF complex has been characterized in several organisms, especially in *Azotobacter vinelandii* (*Gammaproteobacteria*) and in *Cyanobacteria* [21,22]. The precise taxonomic distribution [15] reveals that the NIF system is found exclusively in *Proteobacteria* (*Alphaproteobacteria*, *Betaproteobacteria*, *Gammaproteobacteria*, *Acidithiobacillia*) and *Cyanobacteria* but is patchily distributed within these groups (Fig. 3B).

## 2.2. The ISC system

The ISC system is the most studied [Fe-S] cluster biogenesis system in Bacteria and Eucaryotes where a mitochondrial version exists [10–12,23,24]. From studies in *E. coli*, it is considered as the housekeeping system, providing [Fe–S] clusters to all cellular proteins under normal conditions. It is composed of eight proteins, IscASUX, HscAB, Fdx and CyaY (Figs. 2 & 3A).

The scaffold IscU belongs to the same family as NifU and SufU (see below) but encompasses only a Pfam NifU_N domain. It has been proposed that IscU first binds the iron, and then receives the persulfur [25]. IscU interacts with the cysteine desulfurase IscS to allow the persulfide linked to IscS to be transferred to IscU. The ferredoxin Fdx intervenes to reduce the IscS-bound persulfur. Evidence tends to show that CyaY acts as a regulator of the IscU-IscS complex, potentially as an iron sensor [26]. The role of IscX remains unclear. Once assembled, the [Fe–S] cluster is transferred to the A-type carrier IscA with the help of the two chaperones HscA and HscB, belonging to the DnaK and DnaJ families, respectively [12,27]. Finally, the [Fe–S] cluster is delivered to the apo-

targets. A mechanistic model of delivery to apo-target has been proposed [12,27]. First, HscB binds to IscU causing the dissociation with IscS. HscB then recruits IscU to ATP-bound HscA. Finally, the ATP hydrolysis causes the release of HscB and the stabilization of the interaction between HscA and IscU. Then, the FeS cluster is delivered to IscA, which transfers it either directly to an apo-target or to another carrier, ErpA, that in turn transfers it to the apo-target.

Our recent survey uncovered the surprising fact that the ISC system is found in a very restrained set of phyla [15] (Fig. 3B). At first this contradicts the *E. coli* promoted vision of ISC as being the house keeping system. In fact, ISC is mainly found in *Proteobacteria* (*Alpha*-, *Beta*- and *Gammaproteobacteria*) and closely related phyla (*Nitrospinae*, *Nitrospirae*, *Rokubacteria*, *Acidobacteria*). Interestingly most of the *isc* genes are conserved in a same genomic cluster, except for *cyaY*. Some organisms possess a split genomic cluster, notably the *Chlorobi* and the *Bacteroidetes*.

## 2.3. The SUF system

The SUF system has been mostly studied in *E. coli* and *Salmonella*. SUF is considered as the backup system that these enterobacteria use under stressful conditions such as oxidative stress and iron starvation and is responsible for the maturation of most if not all cellular target proteins [11,23,28,29]. In *Enterobacteria*, the SUF system is made of six proteins, SufABCDSE (Figs. 2 & 3A). A slightly different version occurs in the Gram-positive model *Bacillus subtilis* and the pathogen *Mycobacterium tuberculosis*, where it is composed of SufBCDSTU [30–33].

The Cysteine desulfurase first converts a cysteine into an alanine and a persulfur. Contrary to IscS or NifS, the persulfur is not directly transmitted to the scaffold, but instead to an intermediate sulfurtransferase, SufE [34,35]. Many organisms lack SufE but possess SufU, a U-type scaffold family member [36]. The function of SufU remains to be established but it is unlikely to act as a scaffold. A role of sulfur-transferase like that of SufE would be consistent with the mutual exclusion observed in their taxonomic distribution [15,37]. This view is fully supported by biochemical studies (see Dussouchaud et al., this issue).

The scaffolding activity is carried out by the SufBCD complex [38]. SufB and SufD belong to the same family, and both form a long flattened right-handed beta-helix. SufC is an ABC ATPase exhibiting the typical Walker motives and D- and Q-loops. SufBCD associate to form a complex, but different tetramers have been observed [28,39]. The structure of a *E. coli* SufBC₂D complex has been resolved. Some mutagenesis experiments suggest that the cluster is assembled at the interface of SufB and SufD, but this remains to be fully demonstrated [40]. The ATPase activity of SufC is thought to drive energetically the assembly of the cluster through a cycle of structural changes, in open and released conformation. Thus, scaffolding activity evolved twice, as U-type and Suf-type scaffolds are structurally unrelated, yet in both cases, general

<figure><img src="image_3.png" /><figcaption>Fig. 1. General mechanism of [Fe-S] cluster biogenesis systems.</figcaption></figure>
cellular components, an ABC-type ATPase and a DnaKJ duo-like proteins, were recruited to provide energy for [Fe–S] cluster biogenesis.

The *E. coli* SUF type includes SufA, an A-type carrier (ATC), which helps delivering clusters to target apoproteins. SufA is predicted to have higher efficiency in delivering cluster under stress conditions [13,41], yet other ATC such as ErpA, NfuA or IscA can also receive SUF-produced clusters and deliver them. Note that SufA, like IscA, was proposed to act as iron donor [42].

The role of SufT, another component of the SUF system, remains elusive. It was proposed that SufT could be another carrier, and it has been shown that it is essential in high lipoic acid conditions or when Fe–S cluster demand is high [31,43–46].

SUF is widely present in Bacteria, less distributed in Archaea and is the unique system in most of organisms which code for it (Fig. 3B) [15]. It is found in major lineages such as *Firmicutes*, *Proteobacteria*, *Cyanobacteria*, *Actinobacteria*, *Bacteroidetes* or *Chlamydiae*. Importantly, it is mainly found in aerobic lineages. The core SufBCDS (Cysteine desulfurase and scaffold) is the most conserved set of proteins, while the distribution of SufE, SufU, SufT and SufA is patchier. The two widely studied bacterial models *E. coli* and *B. subtilis* typically represent these variations. Indeed, *E. coli* SUF harbors the sulfur relay SufE, while *B. subtilis* SUF contains SufU. Also, the genomic cluster of SUF in *B. subtilis* does not contain any SufA encoding gene, although an A-type carrier is present elsewhere in the genome but has been shown as non-essential for [Fe–S] cluster assembly [47]. Recently, by analyzing the distribution of SUF components in the two major supergroups into which Bacteria are divided, the Gracilicutes (including *E. coli*) and the Terrabacteria (Including *B. subtilis*), we showed that SufE is mostly present in Gracilicutes, while SufU is widespread in both Terrabacteria and Gracilicutes [15]. SufA is nearly exclusively found in Gracilicutes,

posing the question of the nature of the carrier in *B. subtilis* and all Terrabacteria. Finally, the distribution of SufT is larger than the SUF system, which might point to a cellular role wider than [Fe-S] biogenesis.

SUF is also found in Archaea and particularly in aerobic lineages (*Thermoplasmatales*, *Haloarchaea*, *Thaumarchaea*, *Thalassarchaea*, *Ponarchaea*, ...). Archaeal SUF systems have likely been acquired by horizontal gene transfer (HGT) from bacteria [15] (see below). Interestingly, in Archaea, the SUF contains either SufE or SufU, likely depending on the bacterium at the origin of the HGT.

## 2.4. The SMS system

Phylogenomic analysis recently allowed us to identify a new minimal system, referred to as SMS (for SUF-like Minimal System [15]). It consists of two components, a homologue of SufB and a homologue of SufC (Figs. 2 & 3A). It had been previously observed in some archaeal genomes but wrongly annotated as SUF. We believe this to be misleading as SMS is phylogenetically very distant from SufBCD and always contains exclusively two components. The absence of associated cysteine desulfurase and carrier in the genomic clusters coding for SMS raises several issues regarding how this system makes and delivers clusters. In particular, the nature of the sulfur donor is critical. For example, L-cysteine is not the sulfur source in *Methanococcus maripaludis*, an archaeon that contains only SMS, and it is possible that exogenous sulfide is used directly to build the [Fe–S] clusters [48]. Regarding the delivery step, in the absence of any predicted associated carrier, we can tentatively propose that SMS cooperates with Mrp or SufT, which are widespread in Archaea (Fig. 4). Another possibility is that SMS could transfer the [Fe–S] clusters directly to its targets, which remains to be

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Schematic view of the phylogenies of protein families involved in [Fe-S] cluster biogenesis based on 15. Each schematic represents a protein family, with triangles corresponding to subfamilies. The lines correspond to long branches that separate the subfamilies. Each subfamily has been delineated according to the phylogeny and the genomic neighborhood. Colors correspond to the biochemical role shown in Fig. 1. Dark gray triangles correspond to other roles not mentioned in Fig. 1. Light gray triangles correspond to other paralogues not involved in [Fe-S] cluster biogenesis or that could not be classified.</figcaption></figure>
demonstrated.

SmsB andRINGMsC from *Methanocaldococcus jannaschii* and *Meth-
anosarcina acetivorans* can assemble and deliver clusters, as demon-
strated *in vitro* [15]. Yet, no defect in [Fe-S] protein activity was
observed upon genetic inactivation of the two SMS systems present in
*M. acetivorans* [49]. In fact, *M. acetivorans* also possesses three copies of
the MIS system (see below) and some masking effect cannot be ruled out.

The SMS system is found mostly in Archaea (Fig. 3B) and particularly
in methanogenic and anaerobic lineages (Methanosarcinales, Meth-
anomassiliicoccales, Methanobacteriales, Marsarchaea, ...). Its genomic
organization is extremely conserved and presents almost no variation.
Interestingly, SMS is also found in some bacterial lineages, mostly
anaerobic (Chlorobi, Dictyoglomi, Clostridia, sulfate reducing bacteria),
which likely acquired it from different archaeal sources [15]. It is also
found in the anaerobic parasite Blastocystis, which likely obtained it
from Archaea via HGT (see Dussouchaud et al., this issue).

2.5. *The MIS system*

Phylogenomic analysis also allowed us to identify a second minimal
system, referred to as MIS (for Minimal Iron-sulfur System [15]). The
MIS system is composed of a cysteine desulfurase MisS and a member of
the U-type scaffold family MisU (Figs. 2 & 3A). MIS had been previously
identified but misannotated as NIF or ISC [50,51]. However, both
phylogenetic distance and genomic organization demonstrate MIS is
distinct from NIF and ISC.

Genetic analysis has shown that both *misS* and *misU* are essential for *H. pylori* viability [52]. Purified MisU (former NifU) was found to bind a redox-active [2Fe—2S]²⁺ cluster while purified MisS (former NifS) exhibited typical pyridoxal phosphate dependent cysteine desulfurase activity [50]. Subsequent two-hybrid-based screens suggested the existence of partnerships between MisSU and a subset of predicted cellular [Fe—S] proteins, including a *nfu*-like gene. Its deletion was viable and

two-hybrid interactions suggested a restricted set of potential partners,
including MisSU, confirming that the Nfu-like protein might collaborate
for the maturation of some [Fe—S] proteins or under specific conditions.
However, nfu is genomically unlinked to the MIS system and cannot be
considered as one of its components. Interestingly, using a plasmid
replacement technique, the *H. pylori* misSU operon allowed viability to
an *E. coli* mutant lacking both the *isc* and *suf* operons [52,53].

*M. acetivorans* contains three *misSU* operons (former *iscSU*). Purified MisS2 (*IscS2*) showed pyridoxal 5'- phosphate-dependent cysteine desulfurase activity [51]. Formation of a [4Fe—4S] cluster on MisS2 was achieved upon mixing MisU2, MisS2, cysteine, and iron (Fe²⁺) and the resulting holo-MisU2 was able to transfer its cluster to apo-aconitase. A mutant lacking the *misSU2* gene had no growth phenotype. A possible masking effect of the two remaining *misSU* systems in *M. acetivorans* is likely and this requires further investigation.

The MIS system is widely present in both Archaea and Bacteria
(Fig. 3B). Most of archaea possessing MIS are methanogenic/anaerobic
archaea. In contrast, no pattern related to aerobia/anaerobia is found in
Bacteria. Interestingly, the domain composition of MisU is scattered.
Most of them have a NifU_N domain that is signature of the U-type
scaffolds, but several archaea MisU contain an extra FeS_assembly_P
domain (Pfam PF01883, InterPro IPR002744) of unknown function.
Also, some bacteria exhibit a MisU with domain composition like NifU, i.
e. additional NifU and Fer2 domains (Pfam PF00111, InterPro
IPR001041).

3. Evolution of [Fe-S] biogenesis systems

3.1. *An ancient origin of [Fe-S] biogenesis*

The so-called “Iron sulfur world” was postulated to have arisen in an
ancient anaerobic environment on mineral surfaces at high tempera-
tures [54]. Pyrite – also known as *fool’s gold* – is an iron sulfide (FeS₂),

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Overall genetic organization of the genes coding for components of [Fe-S] cluster biogenesis systems, based on 15. The organization of SUF from *E. coli* (top) and B. subtilis (bottom) are presented. Colors correspond to the biochemical role shown in Fig. 1. Dark gray corresponds to other roles not mentioned in Fig. 1. B. Venn diagrams corresponds to the abundance and overlap of the different systems in genomes across Bacteria and Archaea.</figcaption></figure>
Bacteria

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Taxonomic distribution of proven or potential [Fe-S] cluster carrier proteins based on 15. The presence/absence of proteins per clade are mapped on reference trees of Bacteria and Archaea. For each clade/phylum and for each protein, the size of the dot indicates the percentage of genomes among the clade/phylum possessing the protein.</figcaption></figure>

some types of which were used by the Romans to spark fires [55]. Pyrite might have been used as such by inserting into proto-polymers or as a source of Fe and S, which was eventually exploited by emerging life. Another possibility is that clusters could have facilitated the formation of pseudo-cellular compartments [56,57].

But how did the cellular machineries for [Fe—S] biogenesis emerge? Following the hypothesis that [Fe—S] could form spontaneously in an “iron sulfur world”, [Fe—S] biogenesis systems may have arisen later to assist an already well-developed [Fe-S] cluster-based biology. One of the challenges that [Fe—S]-based biology had to face during evolution is the Great Oxidation Event (GOE), which occurred around 2.4 By ago following the emergence of oxygenic photosynthetic by Cyanobacteria (Fig. 5). As ROS levels associated to atmospheric oxygen increased, this was a threat for some types of [Fe-S] proteins, as described above. Moreover, soluble bioavailable ferrous form was converted to its insoluble oxidized ferric form, leading to a drastic shortage in iron. Based upon these considerations, it has been largely assumed that [Fe—S] biogenesis systems arose during the GOE to maintain a Fe—S based life

Archaea

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

(Fig. 5). The role of these systems would have been to facilitate harvesting of limiting iron, to protect clusters from ROS insults, and to catalyze their formation and/or distribution. To test this hypothesis, it was necessary to demonstrate bioinformatically that all [Fe—S] systems arose after the GOE.

An early bioinformatics study inferred that the SUF system was present in the Last Universal Common Ancestor (LUCA), therefore it had arisen prior to the GOE [53]. The ISC and the NIF would have appeared later during the diversification of Bacteria. However, the conclusions were based on the analysis of taxonomic distribution of [Fe-S] assembly systems in a very narrow sampling of 22 bacteria and 7 archaea.

A second study suggested that SUF emerged first in Archaea and was subsequently horizontally transferred to Bacteria [58]. The archaeal SUF system (SMS) only contained SufB and SufC, and the transfer to Bacteria would have been concomitant with a complexification of the SUF system, with the addition of SufD and SufS. This event would have allowed adaptation to the increase in oxygen rate concomitant with the GOE. This study was conducted on 1094 bacterial and archaeal genomes but
<figure><img src="image_8.png" /><figcaption>Fig. 5. Geological timeline showing the schematical evolution in concentration of Oxygen and available Iron on Earth.</figcaption></figure>

was mainly based on a phylogeny of SufBC. Moreover, the authors used
as outgroup SufD and ill-defined ABC-ATPases other than SufC. Thus,
the evolutionary scenario was based on the assumption that SufD is at
least as old as SufB (and SmsB), and that the chosen ABC-ATPases have
the same evolutionary history as SufD.

Recently, we exhaustively deciphered the evolutionary history of all
five [Fe—S] systems described above [15] (Fig. 6). Using taxonomic
distribution and phylogenomic analysis in 1268 archaeal et 9596 bac-
terial genomes, we robustly inferred that SMS and MIS were already

present in the LUCA. These results suggest that from very early evolu-
tion, [Fe—S] proteins already required cellular machineries for their
maturation. In other words, we showed that enzymatically catalyzed
[Fe-S] biogenesis is an ancient process, and the occurrence of catalytic
systems emerged well before -and independently from- the GOE.

3.2. *The diversification of the [Fe—S] biogenesis systems*

Following their origin in the LUCA, the SMS and MIS systems

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Current scenario of the evolution of [Fe-S] cluster biogenesis systems based on 15. Circles represent proteins. Colors correspond to the biochemical role shown in Fig. 1. The hatched circles represent the mutual exclusion between SufE and SufU.</figcaption></figure>
diversified in different ways in Archaea and Bacteria. In Archaea, the two minimal systems remained identical, while in Bacteria, they underwent several major evolutionary events [15] (Fig. 6). On the other side, the SMS system was in fact made more complex by the addition of SufD, SufU and SufS to give rise to the SUF system, most likely in the ancestor of Bacteria. Then, SufB and SufD arose from a duplication of the ancestral SmsB. The origin of SufU and SufS is less clear because of the poor resolution of the phylogenetic trees of U-type scaffolds and cysteine desulfurase, respectively. Nevertheless, it is tempting to hypothesize that they emerged from MisU and MisS, likely by gene duplication events. SufE emerged later within the Gracilicutes and replaced SufU in several lineages. Then, several HGTs occurred between Archaea and Bacteria. Most aerobic archaeal lineages acquired the bacterial SUF system, while several anaerobic bacterial lineages acquired the archaeal SMS system. These HGTs were concomitant with the loss of the native system. Several intra-domain HGTs also occurred during the evolutionary history of SUF. This is for example the case of *Cyanobacteria* that likely replaced their native system by a SUF system of proteobacterial origin (Fig. 6). Another interesting case of HGT concerns SufS and SufE in *Gammaproteobacteria*. Indeed, some *Gammaproteobacteria* possess paralogues of SufS and SufE which are called CsdA and CsdE, respectively. Our results suggest that CsdA/CsdE are relics of the ancestral SUF system that was replaced by HGT.

We infer that the ISC system emerged later during the diversification of Bacteria, and especially in the ancestor of *Proteobacteria* and closely related phyla (*Nitrospinae*, *Nitrospirae*, *Rokubacteria*, *Acidobacteria*). It emerged from the tinkering of several components, some of which initially not related to [Fe-S] assembly (Fig. 6). This includes the recruitment of DnaK/DnaJ chaperones giving rise to HscA and HscB, respectively, the recruitment of a ferredoxin (Fdx), the emergence of frataxin CyaY and IscX, and the possible duplication of MisS and MisU giving rise to IscS and IscU, respectively. The ISC system was mostly vertically inherited in Bacteria and was lost in several lineages, mostly being replaced by SUF or MIS. Interestingly, the ISC of *Bacteroidetes* and *Chlorobi* was likely acquired by HGT.

We infer that the NIF system emerged in *Proteobacteria*, more precisely in the ancestor of *Alpha*-, *Beta*- and *Gammaproteobacteria*, likely by the integration of MIS genes in the NIF operon, giving rise to NifS and NifU. The NIF system was subsequently inherited vertically in *Proteobacteria* and transferred early in the diversification of *Cyanobacteria* (Fig. 6).

Finally, our updated taxonomic distribution shows that only a hand full of organisms lack any of the [Fe-S] cluster assembly system, confirming the critical role of these machineries for cellular life. These include *Lactobacillales*, which indeed lack some of the well-conserved and important [Fe-S] proteins (aconitase, fumarase, acetolactate synthase), indicating that these organisms may not use [Fe-S] proteins at all. Moreover, some intracellular bacteria (*Borrelia*, *Mollicutes*) also lack any known [Fe-S] system, likely leading to dependance on the host Fe-S cluster assembly machinery.

### 3.3. The complex history of Fe-S cluster carriers

Intriguingly, the ATC carriers are mostly present in the *Gracilicutes* and are not predicted to have been present in the LUCA [15] (Fig. 4). Unfortunately, retracing their history is hampered by very complex taxonomic distributions and phylogenies. Moreover, IscA and SufA, previously referred to as ATC-II subfamily, appear to have been the subject of multiple HGTs. This is consistent with the fact that in the *E. coli* model, they exhibit a high level of functional redundancy.

Mrp, presumably one of the carriers [46], presents a wide distribution both in Archaea and Bacteria, consistent with an ancient origin (Fig. 4). However, its phylogeny does not show a clear split between Archaea and Bacteria [15], precluding a clear inference of its presence in the LUCA.

Finally, SufT is likely to be a [Fe-S] delivery factor [46]. It is much

more widely distributed than SufBCDSU and is also found in genomes that do not have a SUF system (Fig. 4). This is observed both in Bacteria (e.g. *Aabyssubacteria*, *Aquificae*, *Desantisbacteria*, *Coprothermobacterota*, *Caldiserica*, *Dictyoglomi*) and in Archaea (e.g. *Hydrothermarchaea*, *Methanococcales*, *Methanobacteriales*, *Methanopyrales*). Moreover, the phylogeny of SufT is not congruent with the phylogenies of SufBCD, SufS and SufU, suggesting it was spread by multiple HGTs [15]. These results may point to a potential role of SufT beyond [Fe-S] cluster biogenesis, which remains to be demonstrated experimentally.

## 4. Considerations on the emergence and diversification of [Fe-S] cluster machineries

### 4.1. Life uses [Fe-S] biosynthesis machineries since the LUCA

Whether pyrite was a source of Fe and S or it was inserted as such in polypeptides to yield the first [Fe-S] protein is matter of speculation. What is certain however is that abundance of both elements in the early terrestrial crust and the lack of O₂ in the atmosphere certainly favored spontaneous formation of [Fe-S] clusters. For long, this has been the unique way to "reconstitute" [Fe-S] proteins *in vitro* using reducing conditions, iron, and sulfur sources. However, early studies on nitrogenase maturation -followed by a wide wave of interest from unrelated different fields of biology- showed that *in vivo* [Fe-S] cluster biogenesis depends on catalyzing systems instead of spontaneous assembly. When did [Fe-S] biogenesis systems appear during evolution and why were they selected have therefore been key pending questions. Given the enhanced difficulties of building and transferring clusters in iron limiting conditions or in the presence of ROS, it has been common sense to speculate that dedicated cellular [Fe-S] biogenesis systems evolved after the GOE to allow [Fe-S]-based biology. This was contradicted by our recent phylogenetic analysis along with preliminary analyses, which demonstrate that ancestral minimal [Fe-S] biosynthesis systems already existed in the LUCA, way before the GOE. This finding establishes that even in the most favorable early conditions, [Fe-S] cluster formation had to be catalyzed by dedicated machineries, thereby joining a long list of protein folding/maturation processes once thought to be spontaneous and eventually found to be catalyzed *in vivo* such as chaperone-assisted protein folding or catalyzed disulfide bond formation.

### 4.2. Driving forces for the diversification of [Fe-S] machineries

[Fe-S] biogenesis is essential for most living organisms and -as discussed above- it is ancient and ubiquitous [15]. It may be expected that all organisms would share a basic highly conserved molecular system. However, this is not the case as five distinct systems can be found in contemporary organisms, NIF, ISC, SUF, SMS, and MIS. While these systems share some components, each have their own specificities, the most distinct one being represented by having scaffolds of the U-type or of the SufBC-type, reflecting the independent origins of two different molecular devices to build clusters.

Only a few bacteria have been experimentally studied for their [Fe-S] biogenesis capacity. Yet, a very heteroclitic situation emerges already. Most bacteria have only one [Fe-S] system, yet some have two or even three different ones, such is the example of the phytopathogen enterobacterium *Erwinia chrysanthemi* (now *Dickeya dadantii*) that has ISC, SUF and NIF; or the archaeal methanogen *Methanosarcina acetivorans* that has three MIS and two SMS.

A classic rationale for selection of different and functionally redundant [Fe-S] systems is that the hosting organism exploits redundancy to keep up with fluctuating environmental conditions. This is perfectly illustrated with the *E. coli* model, which differentially uses ISC and SUF to maintain [Fe-S] biosynthesis steady throughout a diverse array of favorable and unfavorable conditions. The related pathogens *Salmonella typhimurium* or *Yersinia pseudotuberculosis* also have both ISC and SUF,
and they might use each system in different host compartments during their infection cycle [59,60]. However, this view is contradicted by the example of *Pseudomonas aeruginosa*, which exists as a free-living or opportunistic pathogen and thrives through different environmental conditions including oxidative stress and iron limitation, yet, has only one system, ISC [61,62]. This is also true for *Vibrio cholerae*. It will be interesting to test whether the ISC systems of *P. aeruginosa* and *V. cholerae* can keep up with the challenges that host-released ROS and iron limitation impose on their [Fe—S] biosynthesis capacities or if the bacteria have developed additional systems to solve these threats. This is even more intriguing because the SUF system -and not ISC- is predicted to be a better choice for a pathogen exposed to host-released ROS or fighting against iron limitation. This is in fact observed in *Mycobacterium tuberculosis*, a human pathogen exposed to macrophage-released ROS, which has only SUF [33].

Another selective force exerted on *E. coli* might stem from its capacity to grow under both anaerobic and aerobic conditions in the gut. Yet, this view falls short in the case of *Myxococcus xanthus*, which is strictly aerobic but also contains both ISC and SUF systems [63]. Thus, the reason for enterobacteria like *E. coli*, *S. typhimurium* or *Y. pseudotuberculosis* to have both ISC and SUF cannot be rationalized solely by considering niche constraint. Moreover, it should be noted that not all *E. coli* strains have two systems, as the laboratory strain BL21 derived from *E. coli* B has lost a functional SUF [64]. This last case opens the possibility that evolution is pushing also enterobacteria towards keeping only one system, like most bacteria?

Besides the environmental niche, another driving force possibly involved in the diversity of [Fe-S] biosynthesis systems could be the type and number of [Fe-S] proteins they synthesize, in other words, the nature and composition of the [Fe—S] proteome in each organism. One possibility is that selection of a specific [Fe-S] biogenesis system is dependent on the type of [Fe-S] apo-client present. If so, this would argue for some substrate specificity to occur between the different systems. Unfortunately, all experimental evidence points against the idea that [Fe—S] biosynthesis systems have substrate specificity: (i) *E. coli* ISC and SUF can mature the same set of cellular proteins, although possibly with different efficiencies [11,65,66], (ii) the *Helicobacter pylori* MIS system was shown to rescue a *ΔiscΔsuf* strain of *E. coli*, hence to mature essential *E. coli* [Fe-S] proteins [52,53], and (iii) NadA maturation in *E. coli* was achieved by using taxonomically unrelated SUF systems, while -conversely- taxonomically unrelated NadA could be matured by the ISC system of *E. coli*, showing that heterologous maturation of [Fe-S] protein followed phylogenetic proximity and not the type of system present [67]. Thus, recognition and maturation of the target protein does not seem to impose a strong constraint on the selection of which system is retained by an organism. Still, a driving force might reside in the importance, both quantitative (how many [Fe-S] proteins are to be matured?) and qualitative (how stable are the clusters of these proteins?), of an organism [Fe—S] proteome. This could lead to species-specific selection of the different types of [Fe-S] systems.

## 5. Conclusions

The NIF, SUF and ISC [Fe-S] systems have been widely characterized experimentally. However, how SMS and MIS build and transfer Fe—S cluster remains to be established. These two systems are present in non-model organisms, and new genetic tools in these lineages would be needed to investigate the molecular mechanism and physiological role of SMS and MIS. The main questions regarding these ancestral systems involve identifying the source of sulfur in SMS and the nature of the delivery factors cooperating with SMS and/or MIS. Also, although several major steps have recently led to a better understanding of the origin and evolution of [Fe-S] systems, the driving forces that led to the diversification from SMS and MIS and the emergence of SUF, ISC and NIF remain key open questions to be addressed in future investigations.

## CRediT authorship contribution statement

**Pierre Simon Garcia:** Writing – review & editing, Writing – original draft, Conceptualization. **Simonetta Gribaldo:** Writing – review & editing. **Frédéric Barras:** Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization.

## Declaration of competing interest

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

## Data availability

No data was used for the research described in the article.

## Acknowledgements

We wish to thank the SAMe unit for discussion. This work was supported by the European Union’s Horizon 2020 research and innovation program under grant 722361 (salary of PSG), the ANR-10-LABX-62-IBEID and the ANR-22-CE44-0043-First-FeS, the Institut Pasteur and the CNRS.

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