Monothiol Glutaredoxins in Microbial Iron Metabolism: From Iron-Sulfur Cluster Trafficking to Iron-Dependent Regulation

Abstract

Iron-sulfur (Fe-S) clusters are essential cofactors that support a wide range of metabolic and regulatory processes across all domains of life. The assembly and distribution of these chemically labile cofactors require tightly coordinated biosynthetic and trafficking systems that respond dynamically to cellular iron availability and redox conditions. Monothiol CGFS-type glutaredoxins (Grxs) have emerged as central components of these networks. Once considered primarily thiol-disulfide oxidoreductases, these proteins are now recognized as versatile Fe-S cluster carriers that coordinate and exchange [2Fe-2S] clusters through glutathione (GSH)-dependent mechanisms. This review synthesizes current understanding of monothiol Grxs across bacteria, fungi, and protists, highlighting both conserved biochemical functions and lineage-specific adaptations. In all systems, CGFS Grxs function as intermediates in Fe-S cluster trafficking pathways, facilitating the transfer of clusters from assembly machineries to downstream targets. In fungi, these proteins have been further co-opted into regulatory circuits, where Grxs alone or in partnership with BolA proteins directly couple mitochondrial Fe-S cluster biogenesis to nuclear transcriptional control of iron homeostasis. In contrast, bacterial and protist systems exhibit more indirect or emerging regulatory roles, often integrating Fe-S metabolism with broader redox and stress-response networks. We propose a unifying model in which monothiol Grxs act as dynamic Fe-S “rheostats” that sense and redistribute labile clusters in response to cellular conditions, thereby linking iron metabolism to physiological adaptation. Understanding how this conserved molecular framework is differentially deployed across organisms provides new insight into microbial iron homeostasis and reveals potential targets for therapeutic intervention in pathogenic systems.

1. Introduction

1.1. Iron in Microbial Physiology

Since iron is the most abundant metal on Earth, it is not surprising that microorganisms have evolved to harness the versatile chemical properties of this ubiquitous element. As such, virtually all microorganisms rely on this redox-active metal for growth, survival, and virulence (with the exception of the Lyme disease pathogen Borrelia burgdorferi and the free-living soil bacterium Lactobacillus plantarum) (Archibald, 1983; Posey & Gherardini, 2000). Proteins and enzymes involved in essential functions such as anaerobic and aerobic respiration, photosynthesis, DNA replication and repair, nitrogen fixation, cofactor biosynthesis, oxidative stress defense, and gene regulation recruit iron in various forms as either a structural or functional cofactor. Protein-bound iron exists in different configurations, including heme and iron-sulfur (Fe-S) clusters, as well as mono- and di-nuclear iron centers. Despite its abundance in the environment, the acquisition of iron is complicated by the relative insolubility of this transition metal at neutral pH in oxygenated conditions. Furthermore, during infection, host organisms will strategically restrict access to iron stores to hamper the growth and survival of invading pathogens, in a process known as nutritional immunity (Murdoch & Skaar, 2022).

Microbes have adapted, in kind, with a diverse set of strategies to acquire iron from the environment or host, including ionic iron import systems, heme and hemoprotein uptake pathways, and siderophore (high affinity Fe³⁺ chelators) deployment and capture systems (Caza & Kronstad, 2013; Garg et al., 2024; Zhang et al., 2020). These systems are designed to ensure microbial survival under low iron bioavailability; however, dysregulation of iron metabolism can lead to the other extreme – iron overload – which creates another set of challenges. Elevated intracellular iron can catalyze formation of damaging reactive oxygen species and lead to mismetallation and subsequent inactivation of metalloproteins. Microorganisms must therefore tightly regulate iron uptake, storage, and utilization to balance the essential roles of iron with its reactive tendencies.

Importantly, intracellular iron does not primarily exist as a freely exchangeable pool but is rapidly incorporated into metalloproteins or sequestered in storage complexes (Foster et al., 2022; Helmann, 2025). Among iron-containing cofactors, Fe-S clusters occupy a central position in microbial physiology. These modular inorganic assemblies serve as catalytic centers in enzymes of the tricarboxylic acid cycle, electron transport chains, nitrogen fixation, nucleotide metabolism, and amino acid biosynthesis, and they function as regulatory ligands in transcription factors that respond to oxygen, nitric oxide, and iron availability. Consequently, a large proportion of cellular iron is committed to Fe-S cluster biogenesis and maintenance. The biogenesis of Fe-S clusters is itself a complex, highly regulated process. Dedicated assembly pathways synthesize clusters on scaffold proteins and deliver them to recipient apoproteins through a network of carrier and transfer factors. Because Fe-S clusters are chemically labile and susceptible to oxidative damage, their assembly must be coordinated with iron availability, sulfur mobilization, and cellular redox state. Perturbations in Fe-S cluster homeostasis frequently manifest as global defects in iron regulation, suggesting that the ability to assemble Fe-S clusters serves as an intracellular proxy for iron sufficiency.

This realization has reframed how iron sensing is conceptualized in microbes. Rather than responding directly to free iron concentrations, increasing evidence suggests that cells appear to gauge iron status through the assembly state of Fe-S-dependent regulatory proteins. Within this emerging paradigm, a specialized class of Fe-S-binding proteins, the monothiol CGFS-type Grxs, has come to the forefront. These proteins, once categorized primarily as redox enzymes, are now recognized as dynamic Fe-S cluster carriers and regulatory hubs that couple iron metabolism to transcriptional control and stress adaptation. This review provides an overview of monothiol Grxs and their roles in iron metabolism and physiological processes across microbial systems spanning prokaryotic and eukaryotic domains of life.

2. Evolutionary Distribution and Structural Conservation of CGFS Grxs

2.1. Grx Classes: From Classical Redox Enzymes to Iron-Sulfur Proteins

Grxs were first characterized as small thiol-disulfide oxidoreductases that belong to the thioredoxin (Trx) superfamily (Holmgren, 1976). The canonical Trx fold is characterized by a four or five-stranded β-sheet flanked by α-helices (Figure 1). Classical Grxs contain an active-site CP[Y/F]C motif and use the tripeptide glutathione (GSH) as a cofactor to catalyze reversible oxidation/reduction of protein disulfide bonds via S-glutathionylation and de-glutathionylation reactions (Holmgren et al., 2005) (Figure 1A). These dithiol Grxs, known as Class I Grxs, function in cellular redox buffering, protection against oxidative damage, and maintenance of thiol redox homeostasis. The emergence of a second major class of Grxs, known as Class II monothiol Grxs, represents a striking evolutionary departure from this canonical redox role. In these proteins, the active-site motif is altered from CXXC to Cys-Gly-Phe-Ser (CGFS) and two additional alpha helices are typically found at the N- and C-termini (Figure 1B). CGFS-type Grxs exhibit little to no classical oxidoreductase activity, but instead, function as Fe-S cluster carrier proteins (Couturier et al., 2015; Daniel et al., 2020; Mühlenhoff et al., 2020; Talib & Outten, 2021). Furthermore, unlike Class I Grxs which primarily function as monomeric enzymes, Class II Grxs reversibly bind [2Fe-2S] clusters in a dimeric configuration (Figure 1D) (Iwema et al., 2009; Johansson et al., 2011). Each monomer contributes its active-site Cys as a ligand, while two molecules of GSH complete the coordination sphere. This functional divergence stems from subtle differences in the GSH binding modes of Class I vs. Class II Grxs (Trnka et al., 2020). Members of both Grx classes accommodate GSH in a similar binding pocket, but the conserved active site residues in CGFS Grxs and the longer loop region adjacent to the N-terminal Cys disfavor formation of the mixed Grx-S-SG disulfide required for oxidoreductase activity. Instead, the thiol residue from GSH is positioned away from the active site in Class II Grxs to allow Fe-S cluster ligation (Figure 1D). This coordination mode is unusual because GSH becomes an integral structural ligand rather than serving as a redox cofactor.

Figure 1.

Apo and Fe-S-bridged structures of class I dithiol (Grx1) and class II monothiol (GrxD) Grxs and BolA proteins from E. coli shown in two different orientations. (A) NMR structures of apo-Grx1 (PDB entry 1EGR), (B) apo-GrxD (PDB entry 1YKA), (C) and apo-BolA (PDB entry 2DHM). The N- and C-termini are labeled when visible, in addition to conserved active site or metal-binding cysteine and histidine residues. (D) X-ray crystal structure of the [2Fe-2S]-GSH₂-bridged GrxD homodimer (PDB entry 2WCI). (E) Model of E. coli [2Fe-2S]-GSH-bridged GrxD-BolA heterodimer created using the structural model of the human GLRX5-BOLA1 heterodimer (Nasta et al., 2017). GSH is shown in pink.

2.2. Phylogenetic Distribution of CGFS Grxs

CGFS-type Grxs are widely conserved across bacteria and eukaryotic microbes, including ascomycete and basidiomycete fungi as well as numerous protists. Phylogenomic analyses indicate that a single ancestral Grx gene existed in the last common ancestor of the three kingdoms of life (archaea, bacteria, eukarya), giving rise to both CGFS- and CPYC-type families by duplication and divergence (Vilella et al., 2004). Across all organisms, monothiol CGFS Grxs tend to share higher sequence similarity through evolution than dithiol Grxs, suggesting a more specialized function (Alves et al., 2009). However, most archaea and several bacteria branches apparently lost CGFS Grxs during the early stages of their evolution. Within prokaryotic species, CGFS Grxs are only found in proteobacteria (except the epsilon group) and cyanobacteria (Vilella et al., 2004). Within archaea, CGFS Grxs are only found in Halobacteriales, presumably due to horizontal gene transfer from a proteobacteria ancestor (Alves et al., 2009).

CGFS Grxs are widespread in eukaryotic species from fungi and protists to plants and mammals. Unlike bacterial genomes which typically encode only one CGFS Grx, eukaryotes possess multiple CGFS Grxs that are targeted to different organelles (Couturier et al., 2009; Talib & Outten, 2021). Their subcellular localization often dictates specific functional partners and regulatory roles. Mitochondrial CGFS Grxs are single domain proteins similar to their prokaryotic ancestors. As discussed below, these isoforms became central to Fe-S cluster biogenesis within this organelle. Cytosolic CGFS Grxs are multidomain proteins with an N-terminal Trx-like domain fused to one or more CGFS Grx domains. These proteins are proposed to have evolved from fusion of a Trx-like domain with an ancestor CGFS Grx gene early during eukaryotic evolution after endosymbiosis (Alves et al., 2009; Couturier et al., 2009). Interestingly, the active site in the Trx-like domain of these Trx-Grx fusions is modified from the canonical redox-active WCGPC motif to a WAxxC motif that is not redox active, suggesting that this domain evolved for a separate function. Fe-S clusters only bind at the Grx domains in these multidomain proteins. Most microbial eukaryotes have one of these cytosolic Trx-Grx fusions, although the yeast Saccharomyces cerevisiae has two cytosolic CGFS Grxs (Grx3 and Grx4). These paralogs share close homology and likely originated from a more recent duplication event limited to the evolutionary line of budding yeast (Vilella et al., 2004). As described in more detail in section 4.3, cytosolic CGFS Grxs in fungi shuttle between the nucleus and cytosol and directly participate in iron regulation. The functional expansion of CGFS-type Grxs in fungi illustrates how a conserved Fe-S-binding scaffold can be incorporated into increasingly complex regulatory networks.

In addition to the cytosolic and mitochondrial isoforms, photosynthetic eukaryotes have 1 or 2 additional CGFS Grxs in the chloroplast (Couturier et al., 2009). These isoforms have been shown to interact with chloroplastic Fe-S cluster assembly proteins in vivo and in vitro and to bind [2Fe-2S] clusters in vitro; however, their specific roles in this organelle are unclear (Berndt et al., 2021; Talib & Outten, 2021). Virtually all major protist lineages encode one or more CGFS-type Grx homologs, including parasitic organisms with reduced mitochondria like Giardia intestinalis (Pena-Diaz & Lukes, 2018). The retention of CGFS-type Grxs, even when other metabolic pathways were streamlined, underscores their essentiality for Fe-S cluster metabolism and suggests that Fe-S trafficking roles preceded regulatory diversification. Overall, the conserved presence of CGFS Grxs in all kingdoms of life supports a scenario of early emergence and pervasive retention, with modifications (duplication, fusion, loss) in specific lineages (Alves et al., 2009; Couturier et al., 2009; Pena-Diaz & Lukes, 2018; Vilella et al., 2004).

2.3. Structural and Biochemical Properties of CGFS Grxs

Recombinant CGFS Grxs from bacteria, fungi, and some protists have been shown to incorporate [2Fe-2S]²⁺ clusters upon overexpression and purification from E. coli (Comini et al., 2008; Dlouhy et al., 2017; Dlouhy et al., 2016; Eap et al., 2025; Li et al., 2009; Mapolelo et al., 2013; Picciocchi et al., 2007; Shakamuri et al., 2012; Yeung et al., 2011; Zhang et al., 2013). However, several features distinguish CGFS Grxs from classical Fe-S proteins: the metal cofactor is surfaced exposed rather than deeply buried, dimerization is required for stable cluster coordination, and the GSH ligands are labile and exchangeable. Furthermore, CGFS Grx-bound Fe-S clusters tend to be both oxidatively and reductively labile, since they undergo degradation when exposed to oxygen or upon anaerobic reduction with dithionite. These characteristics make CGFS Grxs uniquely suited for dynamic cluster transfer or signaling rather than stable catalysis. Their Fe-S clusters are sufficiently stable to serve as regulatory modules but sufficiently labile to permit exchange.

In addition to [2Fe-2S]²⁺ clusters, spectroscopic evidence suggests that some CGFS Grxs can coordinate alternate Fe-S species, including cubane [4Fe-4S]²⁺ or linear [3Fe-4S]⁺ clusters (Eap et al., 2025; Zhang et al., 2013). For example, S. cerevisiae Grx5 was shown to bind a cubane [4Fe-4S]²⁺ cluster upon in vitro reconstitution in the absence of GSH, with the conserved Cys near the C-terminus serving as a possible cluster ligand in this conformation. However, in the presence of GSH, this single domain Grx primarily binds a linear [3Fe-4S]⁺ cluster at the dimer interface as evidenced by Mössbauer, electron paramagnetic resonance (EPR), and variable temperature magnetic circular dichroism spectroscopy (Zhang et al., 2014). More recently, the cytosolic CGFS glutaredoxin GrxD from the fungal pathogen Aspergillus fumigatus was shown to bind a mixture of [2Fe-2S]²⁺ and linear [3Fe-4S]⁺ clusters following semi-enzymatic Fe-S cluster reconstitution (Eap et al., 2025). Whether these alternate forms are an artefact of in vitro Fe-S cluster reconstitution or functionally relevant in vivo has yet to be determined. Furthermore, the three-dimensional structures of CGFS Grxs binding these alternate Fe-S clusters are currently not available.

2.4. Conservation of CGFS Grx-BolA Interactions

Comparative genomic analysis coupled with physical interaction experiments first revealed a conserved functional relationship between CGFS Grxs and another widespread protein family known as BolA proteins (Couturier et al., 2009; Huynen et al., 2005) (Figure 1C). BolA proteins are categorized into four main classes: BolA1, BolA2, BolA3, and BolA4 (Couturier et al., 2014). BolA1 proteins are found in all organisms except for archaea and cyanobacteria, while BolA2 and BolA3 are specific to eukaryotes. BolA4 is exclusively expressed in photosynthetic eukaryotes and prokaryotes, including archaea and cyanobacteria. In eukaryotes, BolA1, BolA3, and BolA4 proteins are localized in mitochondria and/or chloroplasts while BolA2 is found in the nucleus and cytoplasm. Gene neighborhood analyses of bacterial genomes frequently place CGFS-type Grx genes adjacent to BolA-like genes or Fe-S assembly operons, implying early functional coupling. Furthermore, these two protein families exhibit strong genome co-occurrence: almost all organisms with a CGFS Grx also possess a BolA-like protein, while organisms without CGFS Grxs also lack BolA proteins. This genomic association likely facilitated the co-evolution of a conserved Grx-BolA module that later diversified functionally. In vivo physical interaction experiments (e.g. yeast two-hybrid assays, bimolecular fluorescence complementation, affinity purification) have confirmed a functional interaction between CGFS Grxs and BolA proteins in a broad range of prokaryotic and eukaryotic species (Couturier et al., 2015; Daniel et al., 2020; Philpott et al., 2020; Talib & Outten, 2021).

Although there are no high-resolution structures of intact Grx-BolA complexes, converging evidence from spectroscopic studies, mutagenesis and biochemical reconstitutions, and comparative modeling have revealed that CGFS-type Grxs form [2Fe-2S]-bridged heterodimeric complexes with BolA proteins (Figure 1E). These complexes have been characterized in both bacterial systems (e.g., E. coli GrxD-BolA) (Dlouhy et al., 2016; Yeung et al., 2011) and fungal systems (e.g., S. cerevisiae Grx3/4-Fra2) (Encinar del Dedo et al., 2015; Hati et al., 2023; Li et al., 2011; Li et al., 2009) using biochemical and spectroscopic methods (e.g. analytical gel filtration chromatography, resonance Raman, EPR, electron-nuclear double resonance spectroscopy, extended X-ray absorption fine structure, Mössbauer, circular dichroism (CD), and UV-visible absorption spectroscopy) on WT and mutant versions of Grx-BolA proteins. These data collectively support a conserved heterodimeric architecture in which the [2Fe-2S] cluster is coordinated across the BolA-Grx interface by the Grx active-site cysteine and GSH on one side, and two residues from BolA on the other. One BolA Fe-S ligand is an invariant histidine located at the end of the 3^rd β-sheet and the other ligand is either a His or Cys residue (depending on the specific BolA class) located in the loop between the 1st and 2^nd β- sheet of BolA (Figure 1C,E). These heterodimers differ structurally and dynamically from Grx homodimers, often exhibiting altered cluster stability and coordination chemistry. Typically, [2Fe-2S]-bridged CGFS Grx-BolA heterodimers are less labile and more stable to reduction than [2Fe-2S]-bridged CGFS Grx homodimers, although there are a few exceptions (Talib & Outten, 2021). Apo forms of some microbial CGFS Grxs and BolA proteins have been also shown to interact in the absence of Fe-S cluster binding (Chi et al., 2018; Dlouhy et al., 2016; Roret et al., 2020; Yeung et al., 2011), suggesting a potential iron-independent function for these binding partners.

3. Integration of Monothiol Grxs in Fe-S Biogenesis Pathways

3.1. Monothiol Grxs in Bacterial Fe-S Cluster Biogenesis

The creation of Fe-S clusters in cells requires specialized biosynthetic systems to safely assemble and deliver these cofactors to their target proteins, while avoiding generation of toxic free Fe²⁺ and sulfide (S²⁻). In bacteria, three major systems have been characterized: the housekeeping ISC (iron-sulfur cluster) pathway, the stress-responsive SUF (sulfur mobilization factor) pathway, and the specialized NIF (nitrogen fixation) system required for nitrogenase maturation. These systems share a common logic in which sulfur is mobilized from cysteine by a desulfurase (IscS, SufS, or NifS), transferred to a scaffold protein (IscU, SufBCD complex, or NifU), and assembled into nascent [2Fe-2S] and [4Fe-4S] clusters prior to delivery to downstream carriers (Figure 2) (Blahut et al., 2020; Garcia et al., 2024; Sourice et al., 2024). Despite this conserved framework, the existence of multiple assembly systems reflects the chemical fragility of Fe-S clusters and the need to tailor their synthesis to environmental conditions. The ISC system functions under normal growth conditions but is sensitive to oxidative stress, whereas the SUF system is induced under iron limitation and reactive oxygen species stress and is more resistant to oxidative damage (Blahut et al., 2020; Esquilin-Lebron et al., 2021). The NIF system, in contrast, is highly specialized and operates under anaerobic conditions to assemble complex metalloclusters required for nitrogen fixation (Martin Del Campo et al., 2022). While the enzymology of cluster assembly has been extensively characterized, a central challenge remains: once formed, Fe-S clusters must be safely trafficked to a diverse array of target proteins. Because free Fe-S clusters are both unstable and potentially toxic, cells rely on a network of carrier and transfer proteins, including A-type proteins (IscA, SufA, ErpA), Nfu proteins, and Isa factors, to mediate cluster delivery. Within this network, monothiol Grxs function as Fe-S transfer intermediates that facilitate the exchange between assembly systems and client pathways.

Figure 2.

Schematic diagram for Fe-S cluster assembly via the SUF and ISC pathways in E. coli. Left, the SUF pathway, which functions under low iron or oxidative stress conditions, requires the SufS cysteine desulfurase to mobilize sulfur from cysteine, generating a persulfide (Cys-S-SH) intermediate. SufE serves as a sulfur shuttle that transfers the persulfide from SufS to the SufBC₂D scaffold complex. SufBC₂D binds an FADH₂ that is proposed to provide electrons to reduce the persulfide (S⁰) to sulfide (S²⁻) and/or reduce Fe³⁺ to Fe²⁺ on the scaffold complex. The specific iron donor for the SUF pathway and the molecular details of Fe-S cluster assembly on the SufBC₂D scaffold are unclear. The scaffold can assemble either [2Fe-2S]²⁺ or [4Fe-4S]²⁺ clusters and transfer these to the A-type carrier protein SufA or to [2Fe-2S]²⁺ or [4Fe-4S]²⁺ client proteins (Blahut et al., 2020). Genetic experiments suggest that GrxD functions with the SUF pathway but this has not been verified in vitro. Right, the housekeeping ISC pathway relies on the cysteine desulfurase IscS and ferredoxin(Fdx)/ferredoxin reductase(FdxR) to provide sulfur and electrons, respectively. A [2Fe-2S]²⁺ cluster is first assembled on the scaffold protein IscU, with iron atoms putatively provided by either CyaY (frataxin homolog) or IscX. The chaperone/co-chaperone pair HscA/HscB facilitate release of the nascent cluster from IscU and allow delivery to the Fe-S cluster trafficking proteins IscA or GrxD. GrxD binds a GSH-ligated [2Fe-2S]²⁺ cluster that can be delivered to ErpA or to [2Fe-2S] client proteins. IbaG/BolA are BolA proteins that form [2Fe-2S]²⁺-bridged heterodimers with GrxD and may promote or inhibit the [2Fe-2S] transfer activity of GrxD. SufA, IscA, and ErpA are all A-type carriers that can bind and transfer both [2Fe-2S]²⁺ and [4Fe-4S]²⁺ clusters to client proteins under different conditions. Genetic studies suggest they these carriers have overlapping and distinct target proteins that vary depending on O₂ tension and iron bioavailability (Esquilin-Lebron et al., 2021). Dotted arrows indicate putative pathways. Created in BioRender. Outten, C. (2026) https://BioRender.com/jl64tyv

3.1.1. Monothiol Grxs and Fe-S Cluster Biogenesis in E. coli

The function of CGFS Grxs in prokaryotes is perhaps best studied in the model bacterium Escherichia coli. In bacteria, the monothiol glutaredoxin GrxD (also referred to as Grx4 or Grx3 in some species) is typically the sole CGFS-type Grx found in the genome. Unlike classical ISC or SUF components, GrxD is not required for de novo cluster assembly but instead functions downstream of scaffold proteins to facilitate cluster distribution (Figure 2). Early genetic studies in E. coli established that ΔgrxD strains grow poorly under iron-limiting conditions and exhibit synthetic or aggravating genetic interactions with Fe-S biogenesis components, implicating GrxD in cluster maturation pathways (Boutigny et al., 2013; Butland et al., 2008; Yeung et al., 2011). These conclusions were supported by in vitro work establishing that bacterial monothiol Grxs mediate rapid and efficient cluster transfer from Fe-S scaffolding complexes to Fe-S carrier proteins and other protein targets via direct ligand exchange (Mapolelo et al., 2013; Vranish et al., 2016; Yeung et al., 2011). More recent work has refined this view, positioning GrxD as a conditionally essential factor that becomes particularly important when Fe-S cluster supply is limiting or when cells are subjected to environmental stress, which activates expression of the SUF system (Fisher et al., 2024). A major advance in understanding bacterial monothiol Grx function in E. coli has been the demonstration of a functional link between GrxD and the A-type carrier protein ErpA, which is essential for the maturation of a subset of Fe-S proteins required for aerobic growth. Global chemoproteomic and biochemical studies have shown that GrxD participates in cluster delivery to ErpA, especially under iron-limiting conditions. Depletion of GrxD results in reduced activity of ErpA-dependent Fe-S enzymes, supporting a model in which GrxD acts upstream of or in parallel with A-type carriers (Fisher et al., 2024). These findings suggest that GrxD functions as a late-stage transfer intermediate, receiving clusters from ISC or SUF-derived carriers and passing them to specific client pathways via ErpA. Importantly, this role appears to be context-dependent, becoming more pronounced when Fe-S cluster flux is constrained. Furthermore, although GrxD is among the most abundant Fe-S proteins under normal growth conditions, its cluster occupancy is sensitive to intracellular iron availability and decreases under iron limitation, consistent with the redistribution of Fe-S clusters to essential pathways. GrxD is thus hypothesized to serve as a reservoir of pre-made Fe-S clusters that can be rapidly mobilized as environmental conditions change (Fisher et al., 2024). This dynamic behavior suggests that GrxD does not simply function as a unidirectional carrier but instead participates in a reversible exchange network with both the ISC and SUF pathways, helping to equilibrate Fe-S cluster distribution across the proteome (Figure 2).

GrxD’s role in cluster trafficking vs. storage may be further refined by its interactions with BolA proteins. The E. coli genome encodes two BolA proteins, BolA and IbaG, that belong to the BolA1 and BolA4 families, respectively. GrxD interacts with either BolA or IbaG to form [2Fe-2S]-bridged heterodimers, which exhibit distinct biochemical and spectroscopic properties compared to [2Fe-2S]-bridged GrxD homodimers (Dlouhy et al., 2016; Yeung et al., 2011). The [2Fe-2S]-GrxD-BolA complex is more stable than [2Fe-2S]-GrxD homodimers and the cluster is less exchangeable. In contrast, the [2Fe-2S]-GrxD-IbaG complex is more dynamic and labile than either GrxD or GrxD-BolA complexes. BolA and IbaG may therefore differentially tune the functional state of GrxD, allowing the cell to balance Fe-S cluster flux with Fe-S cluster storage. In any case, the precise role of BolA proteins in modulating GrxD activity in E. coli remains less well defined than in eukaryotic systems.

3.1.2. Monothiol Grxs and Fe-S Cluster Biogenesis in Pathogenic Bacteria

Pathogenic bacteria encounter a dynamic and hostile environment within the host, characterized by restricted iron availability, oxidative and nitrosative stress, and fluctuations in redox homeostasis. These conditions place particular strain on Fe-S cluster-containing proteins, which are both essential for core metabolic processes and highly susceptible to oxidative damage. As a result, the ability to maintain Fe-S cluster homeostasis has emerged as a critical determinant of bacterial fitness and virulence. Within this context, CGFS-type Grxs represent an important but relatively underexplored component of pathogen physiology. While these proteins are well established as Fe-S cluster carriers and regulators in model organisms, only recently have studies begun to directly link their function to stress adaptation and virulence in pathogenic bacteria. These emerging findings suggest that monothiol Grxs serve as key mediators of Fe-S cluster maintenance under host-imposed stress conditions, thereby supporting both metabolic integrity and pathogenic potential.

The most direct evidence for a role of monothiol Grxs in Fe-S cluster metabolism of bacterial pathogens comes from recent studies in Pseudomonas aeruginosa, an opportunistic pathogen that must withstand both oxidative stress and iron limitation during infection. Deletion of grxD in P. aeruginosa results in pronounced sensitivity to oxidizing agents such as paraquat and cumene hydroperoxide, indicating a defect in oxidative stress resistance (Saninjuk et al., 2023). Notably, this phenotype is accompanied by impaired activity of peroxide detoxification systems, including the organic hydroperoxide resistance protein Ohr, suggesting that GrxD contributes indirectly to redox defense. Mechanistically, the oxidative stress sensitivity of the ΔgrxD mutant appears to be linked to defects in Fe-S cluster metabolism. P. aeruginosa relies solely on the ISC pathway for Fe-S cluster biogenesis and expresses several Fe-S carrier proteins, including ErpA, IscA, and NfuA. Expression of each of these Fe-S carrier proteins can partially rescue the stress-sensitive phenotype of the ΔgrxD mutant, supporting a model in which GrxD functions within the Fe-S trafficking network rather than acting directly on peroxide detoxification pathways. These findings are consistent with the idea that oxidative stress leads to damage or loss of labile Fe-S clusters, and that GrxD is required to maintain or restore cluster availability under these conditions. Furthermore, the P. aeruginosa ΔgrxD mutant grows poorly under iron limitation, mirroring the same phenotype observed for E. coli ΔgrxD mutants and underscoring a key role for GrxD in adapting to both oxidative and iron starvation stress. Importantly, loss of GrxD also results in attenuated virulence in an invertebrate infection model, providing direct evidence that Fe-S cluster maintenance contributes to pathogenic fitness (Saninjuk et al., 2023). This work establishes a functional link between monothiol Grxs, Fe-S metabolism, and virulence, and positions GrxD as a key factor in the adaptation of P. aeruginosa to the host environment.

Beyond Pseudomonas species, monothiol Grxs have also been characterized in other bacterial pathogens. In Leptospira interrogans, the causative agent of leptospirosis, both a monothiol CGFS Grx (Lin1CGrx) and a dithiol CPYC Grx (Lin2CGrx) have been identified and functionally analyzed (Sasoni et al., 2022). Notably, these organisms rely on distinct Fe-S cluster assembly strategies: whereas Pseudomonas aeruginosa encodes only the ISC pathway, L. interrogans depends exclusively on the SUF system for Fe-S cluster biogenesis. This reliance on a stress-adapted assembly pathway in L. interrogans is consistent with its pathogenic lifestyle and may place increased importance on downstream Fe-S trafficking factors such as monothiol Grxs. Heterologous expression of Lin1CGrx in the model yeast S. cerevisiae was shown to complement the function of Grx5, the mitochondrial CGFS-type Grx required for Fe-S cluster biogenesis (see section 3.3.1), suggesting a conserved function for the L. interrogans homolog. Consistent with this idea, as-purified, recombinant Lin1CGrx was found to coordinate an oxygen-sensitive, GSH-ligated Fe cofactor with spectroscopic properties indicative of a [2Fe-2S] cluster. In contrast, the dithiol Grx Lin2CGrx displayed classical GSH-dependent oxidoreductase activity towards multiple substrates and was able to rescue the H₂O₂ sensitivity of yeast strains lacking the dithiol glutaredoxins Grx1 or Grx2. Notably, the apo form of Lin1CGrx exhibited weak disulfide reductase activity; however, this activity was substantially lower than that observed for Lin2CGrx (Sasoni et al., 2022). Collectively, these findings support a primary role for the CGFS-type Grx in L. interrogans in Fe-S cluster metabolism, although direct evidence for its in vivo function in this organism remains limited.

3.1.3. Monothiol Grxs and Fe-S Biogenesis in Nitrogen-Fixing Bacteria

In addition to the ISC/SUF systems used for general Fe-S cluster assembly, bacterial diazotrophs require a dedicated cluster assembly system for synthesizing and inserting two complex metalloclusters (P-cluster [8Fe-7S] and M-cluster [7Fe-9S-Mo-C-homocitrate]) required for maturation of the nitrogen-fixing enzyme nitrogenase. As with the ISC and SUF systems, the NIF system includes a canonical cysteine desulfurase (NifS), scaffold (NifU), and A-type carrier protein (^NifIscA) that coordinate to generate and traffic simple [2Fe-2S]²⁺ and [4Fe-4S]²⁺ clusters used as building blocks for downstream assembly of the complex P- and M-clusters (Buren et al., 2020). Monothiol CGFS Grxs are present in the nif operons of several nitrogen-fixing bacteria (Buren et al., 2020; Rouhier et al., 2010), and have been shown to rapidly transport [2Fe-2S] clusters to A-type carriers within the NIF pathway in vitro (Mapolelo et al., 2013). Although direct mechanistic links between specific CGFS Grxs and Nif protein maturation in vivo remain limited, emerging evidence from proteobacterial systems supports a model in which these proteins act at the intersection of Fe-S cluster homeostasis and nitrogenase biogenesis, thereby enabling efficient nitrogen fixation.

The impact of monothiol Grxs on rhizobial growth and survival has been assessed by genetic experiments in a variety of bacterial diazotrophs. In the gram-negative bacterium Sinorhizobium meliloti, mutations in the only CGFS Grx in the genome (SmGRX2) significantly increased intracellular iron levels, decreased the growth rate of the free-living bacteria, impaired the activity of Fe-S cluster enzymes, hampered nodulation efficiency, and reduced symbiotic nitrogen-fixing efficiency. These phenotypes are attributed to a role for SmGRX2 in Fe-S cluster biogenesis, supported by the observation that recombinant SmGRX2 is purified with a labile Fe-S cluster (Benyamina et al., 2013). Deletion of the single monothiol glutaredoxin GrxB in Rhizobium leguminosarum produced similar phenotypes, and additionally increased the H₂O₂ sensitivity of the bacterium (Zou et al., 2021). The microsymbiont Azorhizobium caulinodans expresses two CGFS Grxs (GRX1 and GRX2) with apparently redundant functions since deletion of both was required to observe phenotypes. Compared to WT A. caulinodans strains, the Δgrx12 mutant exhibited a slower growth rate in the free-living state, sensitivity to H₂O₂, a decreased GSH/GSSG ratio, decreased intracellular iron, reduced colonization ability, and a significant reduction in nitrogen fixation efficiency (Cao et al., 2020). Together, these findings across multiple rhizobial species highlight a conserved role for monothiol Grxs in coordinating iron homeostasis, Fe-S cluster biogenesis, and redox balance, processes that are tightly coupled to both free-living growth and symbiotic performance. More broadly, they underscore that disruption of CGFS Grx function compromises not only bacterial physiology but also the efficiency of plant-microbe interactions, emphasizing their central importance in sustaining effective nitrogen-fixing symbiosis.

3.2. Monothiol Grxs and Fe-S Cluster Biogenesis in Archaea

Although Grxs are evolutionarily conserved across all domains of life, understanding of CGFS-type Grxs in archaea remains largely limited to sequence-based and comparative genomic analyses. As mentioned earlier, CGFS Grx homologs have only been identified in halobacterial genomes within archaea, which was inferred through conservation of the hallmark CGFS active-site motif and phylogenetic relationships to bacterial and eukaryotic counterparts (Alves et al., 2009; Couturier et al., 2009). However, in contrast to the extensive biochemical and functional characterization available in bacteria and eukaryotes, there are currently no detailed experimental studies defining the physiological roles, interaction partners, or Fe-S cluster-related functions of CGFS Grxs in archaeal systems. As a result, proposed roles for archaeal CGFS Grxs in redox regulation or iron metabolism can only be largely extrapolated from other domains of life. Interestingly, halobacteria rely primarily on γ-glutamylcysteine rather than GSH as their low-molecular-weight thiol (Fahey, 2013), a distinction that likely influences the biochemical properties of CGFS Grxs in these organisms. The paucity of functional data for CGFS Grxs in halobacteria highlights a significant gap in our understanding of redox-iron integration in archaea and underscores the need for targeted experimental investigation.

3.3. Monothiol Grxs and Fe-S Cluster Biogenesis in Fungi

Eukaryotic cells exhibit compartmentalized and hence more complex Fe-S requirements, with Fe-S proteins distributed across the mitochondria, cytosol, ER, and nucleus to support distinct biochemical processes, such as mitochondrial respiration, ribosome biogenesis, and nuclear DNA replication and repair. In contrast to many bacteria, which encode multiple Fe-S assembly pathways (ISC, SUF, and in some cases NIF), most fungi rely exclusively on the mitochondrial ISC machinery to generate Fe-S clusters for both mitochondrial and extra-mitochondrial proteins (Mühlenhoff et al., 2020). Cytosolic and nuclear [4Fe-4S] proteins are matured by the cytosolic iron-sulfur assembly (CIA) pathway, which depends on a sulfur-containing intermediate exported from mitochondria via the ABC transporter Atm1 (Dancis et al., 2024; Lill et al., 2015). Bridging these core assembly machineries, CGFS-type monothiol Grxs function as critical intermediates in Fe-S cluster trafficking and regulation. In budding yeast and other fungi, these proteins are represented by two functionally distinct classes: the mitochondrial glutaredoxin Grx5 and the cytosolic glutaredoxins Grx3 and Grx4. Together, these proteins link mitochondrial Fe-S cluster assembly to cytosolic cluster utilization and iron homeostasis, acting as intermediates in cluster trafficking and regulatory signaling.

3.3.1. Mitochondrial Grx5 Links the Early and Late ISC Machinery

Work in numerous fungal systems has firmly established that the mitochondrial CGFS-type glutaredoxin Grx5 functions downstream of the ISC assembly machinery and plays an important role in both mitochondrial and extramitochondrial Fe-S protein maturation (Mühlenhoff et al., 2020; Pandey et al., 2025; Talib & Outten, 2021). Seminal genetic studies performed in the budding yeast Saccharomyces cerevisiae and corroborated in the fission yeast Schizosaccharomyces pombe have shown that deletion of GRX5 leads to severe defects in mitochondrial Fe-S enzyme activity, respiratory deficiency, and intracellular iron accumulation, highlighting its central role in Fe-S metabolism (Kim et al., 2010; Rodriguez-Manzaneque et al., 2002). The precise positioning of Grx5 as an intermediate carrier within the mitochondrial Fe-S trafficking network has been confirmed by complementary in vitro and in vivo studies (recently reviewed in (Braymer et al., 2021; Dancis et al., 2024; Lill & Freibert, 2020)). According to the current model for mitochondrial Fe-S cluster assembly, initial assembly occurs on the scaffold proteins Isu1/Isu2, where sulfur is provided by the cysteine desulfurase Nfs1 and electrons are supplied via the ferredoxin/ferredoxin reductase (Yah1/Arh1) redox couple. The nascent [2Fe-2S] cluster is subsequently transferred from Isu1/2 to Grx5 in a process facilitated by the chaperone/co-chaperone pair, Jac1 (Hsp40) and Ssq1 (Hsp70). The grx5Δ phenotypes are thus consistent with a failure in cluster transfer rather than cluster assembly per se, as Fe-S clusters still accumulate on the scaffold protein Isu1 in vivo in the absence of Grx5 (Mühlenhoff et al., 2003). Grx5 then delivers [2Fe-2S] clusters to downstream carriers in the late-acting ISC machinery, including Isa1/Isa2 and Iba57. The Isa1-Isa2-Iba57 complex is proposed to catalyze the reductive coupling of two [2Fe-2S]²⁺ clusters to form a [4Fe-4S]²⁺ cluster, which is subsequently transferred to apo-target proteins by specialized maturation factors, including Nfu1, Ind1, Bol1, and Bol3, that facilitate delivery (Fig. 3).

Figure 3.

Schematic diagram for Fe-S cluster assembly via the mitochondrial ISC pathway in S. cerevisiae. In the initial phase, a [2Fe-2S] cluster is assembled de novo on the scaffold Isu1/2. Sulfur is mobilized from cysteine by the cysteine desulfurase Nfs1, generating a persulfide (Cys-S-SH) intermediate. Nfs1 requires additional binding partners, Isd11 and the acyl carrier protein Acp1, that both stabilize and regulate the enzyme’s function. The yeast frataxin homolog (Yfh1) promotes transfer of the persulfide to the scaffold protein. The persulfide (S⁰) is reduced to sulfide (S²⁻) with electrons provided by an NADPH-dependent ferredoxin (Yah1)/ferredoxin reductase (Arh1) system and ferrous ion (Fe²⁺) bound to the scaffold. Fe²⁺ may be acquired from labile iron pools or possibly Yfh1. Two ferric ions (Fe³⁺) and two sulfides (S²⁻) then combine to form the nascent [2Fe-2S]²⁺ cluster transiently bound to the scaffold protein. In the next phase, the nascent [2Fe-2S] cluster is transferred to Grx5 with the assistance of an ATP-dependent chaperone/co-chaperone system (Ssq1/Jac1) that drives release of the cluster from the scaffold protein. The [2Fe-2S] cluster may then be delivered to recipient apo-proteins or transferred to additional carrier/trafficking proteins for the synthesis of [4Fe-4S] clusters. In the final phase, two [2Fe-2S]²⁺ clusters are reductively coupled on a carrier/trafficking protein complex (Isa1/Isa1/Iba57) via electrons provided by Yah1/Arh1 to generate a [4Fe-4S]²⁺ cluster. Additional targeting factors (Bol1, Bol3, Nfu1, Ind1) facilitate trafficking and insertion of [4Fe-4S] clusters into recipient apo-proteins. Created in BioRender. Outten, C. (2026) https://BioRender.com/1xslzxr

3.3.2. Mitochondrial Grx5 and the Mitochondrial-Cytosolic Connection

A defining feature of fungal Fe-S metabolism is the dependence of cytosolic and nuclear Fe-S protein maturation on mitochondrial ISC activity (Figure 4). This intercompartmental coupling is mediated by the export of an iron- and sulfur-containing intermediate (Fe-S)_int via the ABC transporter Atm1 (Dancis et al., 2024; Kispal et al., 1999; Lill et al., 2014; Pandey et al., 2025). Although this dependency has been recognized for decades, the molecular nature of the exported species and the molecular mechanisms for its formation, export, and utilization in extramitochondrial Fe-S cluster biogenesis have remained major unresolved questions. Nevertheless, several studies have established that the early ISC machinery, Grx5, and the chaperone Ssq1 are essential for production of this intermediate, while the late-acting mitochondrial ISC machinery is dispensable (Dancis et al., 2024; Lill et al., 2014). Interestingly, the early ISC machinery also produces another sulfur-containing intermediate (X-S) that is exported by Atm1 and used specifically for tRNA thio-modifications. However, Grx5 is not required for production of this important metabolite (Figure 4) (Pandey et al., 2018; Pandey et al., 2019). Multiple lines of evidence suggest that (Fe-S)_int is GSH-coordinated, consistent with the central role of GSH in Fe-S metabolism (Bandyopadhyay & Outten, 2022; Dancis et al., 2024; Mühlenhoff et al., 2020). Firstly, depletion of GSH in cells phenocopies an atm1 deficient yeast strain, leading to defects in cytosolic/nuclear Fe-S cluster proteins and dysregulation of iron metabolism (Kumar et al., 2011; Ozer et al., 2015; Sipos et al., 2002). Secondly, the crystal structure of yeast Atm1 includes a GSH molecule in the substrate binding cavity (Lee et al., 2014; Srinivasan et al., 2014), suggesting that Atm1 transports GSH, oxidized glutathione (GSSG), or a GSH conjugate (Srinivasan et al., 2014). Thirdly, GSH, GSSG, GSH-metal conjugates, and/or Cys-containing peptides were shown to stimulate the ATPase activity of several Atm1 orthologs (Kuhnke et al., 2006; Lee et al., 2014; Schaedler et al., 2014). One proposed Atm1 substrate is a [2Fe-2S]²⁺ cluster ligated by four GSH molecules ([2Fe-2S] (GS)₄) (Li & Cowan, 2015; Qi et al., 2014). However, the existence of this [2Fe-2S] (GS)₄ complex under physiological conditions has not been firmly established. In any case, it is clear that Grx5 serves as a central integrator of intercompartmental Fe-S metabolism, coordinating cluster transfer within mitochondria with the generation of exportable intermediates that are used for cytosolic/nuclear Fe-S cluster biogenesis.

Figure 4.

Schematic diagram depicting the functions of CGFS Grxs in Fe-S cluster-dependent pathways in yeast. Left, Grx5 has a central role in mitochondrial Fe-S cluster assembly connecting the early and late ISC pathways. The core ISC machinery assembles an [2Fe-2S] cluster on Isu1/2 and provides a sulfur-containing compound that is exported by Atm1 for use in cytosolic tRNA thiolation. The newly assembled [2Fe-2S] cluster is transferred to Grx5 to form a GSH-ligated, [2Fe-2S]-bridged dimer. Grx5 transfers the cluster to the late ISC machinery or may deliver [2Fe-2S] clusters to recipient mitochondrial apo-proteins. The late ISC pathway assembles [4Fe-4S] clusters via reductive coupling of two [2Fe-2S] clusters and ensures specific delivery of [4Fe-4S] clusters to target apo-proteins. Middle, the early ISC pathway and Grx5 are also required to generate a sulfur- and iron-containing intermediate (Fe-S_int) that is exported by Atm1 to the cytosol. The maturation of most cytosolic/nuclear [2Fe-2S] proteins is achieved by an unknown mechanism that is dependent on (Fe-S_int) but independent of Grx3/4 and the CIA pathway. However, the CIA pathway is required for maturation of cytosolic/nuclear [4Fe-4S] proteins. (Fe-S_int) is required for [2Fe-2S] cluster loading on Grx3/4, which in turn impact Fe-S cluster loading on some CIA components. Since Grx3/4 cluster binding is labile, Grx3/4 cluster occupancy may be affected by changes in cytosolic GSH and/or Fe²⁺ pools. Right, cytosolic CGFS Grxs, often in partnership with Bol2/Fra2 proteins, interact with and modulate the activity of Fe-S binding transcriptional regulators in the nucleus that control iron homeostasis. Created in BioRender. Outten, C. (2026) https://BioRender.com/yd3wb81

3.3.3. Grx3/4 and Cytosolic Fe-S Cluster Biogenesis

Emerging evidence suggest that multiple pathways are required for synthesizing [2Fe-2S] and [4Fe-4S] clusters in the yeast cytosol, which are all strictly dependent on the mitochondria-generated, Atm1-exported intermediate (Fe-S)_int (Braymer et al., 2024; Dancis et al., 2024; Lill et al., 2015). The biogenesis of [4Fe-4S] clusters is specifically mediated by the CIA machinery. The CIA scaffold complex includes Cfd1 and Nbp35, two P-loop NTPases that transiently assemble and stabilize [4Fe-4S] clusters derived from the Atm1-exported intermediate. Electron transfer from the Fe-S binding protein Dre2 and the diflavin reductase Tah18 provides the reducing equivalents necessary for [4Fe-4S] cluster synthesis. Downstream targeting factors, including Nar1 and the CIA targeting complex (Cia1, Cia2, and Met18), facilitate the delivery and insertion of Fe-S clusters into specific client proteins such as cytosolic and nuclear enzymes involved in ribosome biogenesis, DNA replication, and DNA repair. Although the cytosolic CGFS glutaredoxins Grx3 and Grx4 are not strictly required for the core enzymatic steps of CIA-dependent cluster assembly, multiple lines of evidence support their function as accessory factors that facilitate cluster distribution and integration into cellular metabolism.

In S. cerevisiae, Grx3 and Grx4 are cytosolic CGFS Grx paralogs that possess an N-terminal Trx domain and a C-terminal Grx domain. The Trx domain is proposed to mediate protein-protein interactions while the Grx domain ligates a [2Fe-2S]²⁺ cluster via dimerization (Hoffmann et al., 2011; Li et al., 2009; Mühlenhoff et al., 2010; Picciocchi et al., 2007). Binding of [2Fe-2S]²⁺ clusters to Grx3/4 in vivo is independent of the CIA pathway, providing the first indication that multiple pathways exist for building cytosolic Fe-S clusters (Mühlenhoff et al., 2010). The dominant phenotype of grx3/4 mutant strains in all fungi studied to date is the dysregulation of iron metabolism (discussed in section 4.3), which leads to iron overload and sensitivity to oxidative stress in S. cerevisiae (Mühlenhoff et al., 2010; Ojeda et al., 2006; Pujol-Carrion et al., 2006). Consequently, grx3/4 deletion strains are either inviable or severely growth impaired, depending on the strain background (Mühlenhoff et al., 2010; Ojeda et al., 2006). Grx3/4-depleted cells also exhibit defects in cofactor insertion for mitochondrial and cytosolic Fe-S proteins, di-iron binding proteins, and heme (Li et al., 2017; Mühlenhoff et al., 2010; Zhang et al., 2014), suggesting a general role in intracellular iron trafficking. However, it was unclear whether these phenotypes were due to secondary effects of iron overaccumulation or a direct role for Grx3/4 in Fe-S cluster and iron trafficking.

Recent work has begun to clarify the role of Grx3/4 in cytosolic Fe-S protein maturation by examining the consequences of their depletion on [2Fe-2S] and [4Fe-4S] client proteins (Braymer et al., 2024). Notably, the severe growth defect of Grx3/4-depleted yeast can be rescued by preventing intracellular iron accumulation through deletion of the iron-responsive transcriptional activator Aft1. This finding indicates that Grx3/4 are not strictly essential for cytosolic Fe-S cluster biogenesis, an essential process for viability. However, even when iron levels are normalized, loss of Grx3/4 significantly impairs Fe-S cluster incorporation into a subset of cytosolic proteins, including [2Fe-2S] proteins such as Dre2 (a CIA component) and Apd1, and [4Fe-4S] proteins such as Rad3 (DNA repair) and Leu1 (leucine biosynthesis). Importantly, deletion of the cytosolic BolA protein Bol2, which is an established partner of Grx3/4 in iron regulation (see section 4.3), does not disrupt cytosolic Fe-S protein maturation, indicating that this function of Grx3/4 is independent of Bol2. Although Fe-S loading onto Grx3/4 themselves does not require the CIA machinery, cytosolic CGFS Grxs have been shown to physically and functionally interact with CIA components across multiple systems, including yeast, plants, and humans (Banci et al., 2015; Frey et al., 2016; Iñigo et al., 2016; Li et al., 2017; Michaelis et al., 2023; Saito et al., 2011; Zhang et al., 2011). Together, these findings support a model in which cytosolic CGFS Grxs do not function as dedicated Fe-S cluster carriers for specific client proteins within the CIA pathway. Instead, they likely act as dynamic buffering intermediates that help maintain and redistribute Fe-S cluster availability within the cytosol and nucleus (Figure 4). This buffering role may become particularly important under conditions where Fe-S cluster supply is limiting or fluctuating, which is supported by studies in E. coli, though not yet directly tested in yeast.

3.3.4. Monothiol Grxs and Fe-S Cluster Biogenesis in Fungal Pathogens

Given that both mitochondrial and cytosolic CGFS Grxs are highly conserved across fungal species, these proteins are expected to play key roles in Fe-S cluster metabolism in fungal pathogens. Indeed, several recent studies have clearly established that cytosolic monothiol Grxs (known as Grx3, Grx4, or GrxD depending on the species) serve as essential regulators of iron homeostasis and virulence in the human pathogens Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, and the plant pathogens Ustilago maydis and Fusarium graminearum (see section 4.3.3) (Alkafeef et al., 2020; Attarian et al., 2018; McCotter et al., 2023; Misslinger et al., 2019; Zhang et al., 2017). However, direct experimental interrogation of cytosolic and mitochondrial CGFS Grxs within the ISC and CIA Fe-S cluster assembly pathways remains comparatively limited in pathogenic fungi. As a result, current understanding of mitochondrial CGFS Grx function in these organisms is largely inferred from model yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe (Sections 3.3.1–3.3.2). Since other core components of the mitochondrial Fe-S cluster biogenesis pathway in fungal pathogens are indispensable for cellular viability and physiology (Dong et al., 2017; Misslinger et al., 2018; Ror & Panwar, 2019; Santos et al., 2004), the same is likely true for the mitochondrial CGFS Grx (usually named Grx5). In pathogenic fungi, mitochondrial metabolism is tightly linked to virulence, particularly during infection stages that require adaptation to nutrient limitation and host-imposed stresses, conditions that place high demand on mitochondrial function (Garcia-Santamarina et al., 2017; Horianopoulos & Kronstad, 2019; Ma et al., 2025). By maintaining the functionality of Fe-S-dependent enzymes in the tricarboxylic acid cycle and electron transport chain, the mitochondrial ISC pathway indirectly supports energy production, metabolic flexibility, and ultimately pathogenic fitness. As discussed in section 3.3.2, cellular Fe-S cluster metabolism requires coordination between mitochondrial and cytosolic pathways. This intercompartmental connection is mediated by the conserved ABC transporter Atm1, which exports a sulfur-containing intermediate generated by Grx5 and required for cytosolic Fe-S protein maturation. The functional importance of the ABC transporter Atm1 in linking ISC and CIA pathways, which is in turn is essential for virulence, has been confirmed in multiple fungal pathogens (Do et al., 2018; Garcia-Santamarina et al., 2017; Huang et al., 2025; Wang et al., 2019), further supporting the idea that Grx5 contributes not only to mitochondrial Fe-S assembly but also indirectly to cytosolic Fe-S cluster biogenesis.

While mitochondrial Grx5 plays a central role within the ISC machinery, cytosolic monothiol Grxs function at the interface between iron homeostasis and cytosolic Fe-S cluster biogenesis. As discussed above, Grx3/4 in model yeasts contribute to the delivery or regulation of Fe-S clusters destined for the CIA pathway. However, in fungal pathogens, direct evidence supporting a mechanistic role for Grx3/4 orthologs in cytosolic Fe-S cluster maturation remains sparse. To date, the most concrete evidence comes from the identification of physical interactions between cytosolic CGFS Grxs and components of the CIA machinery (e.g. Dre2, Cia1, Met18) (Alkafeef et al., 2020; Misslinger et al., 2019), suggesting a potential functional link between these Grxs and cytosolic Fe-S protein assembly. Although the precise role of these interactions has not yet been fully defined, they support a model in which cytosolic monothiol Grxs in fungal pathogens may act as intermediates in Fe-S cluster trafficking or as regulatory factors that coordinate iron availability with cytosolic Fe-S cluster biogenesis. Further work will be required to determine whether these interactions reflect direct cluster transfer activity or a more indirect role in pathway regulation.

Recent work in A. fumigatus provides additional, albeit unexpected, insight into the potential versatility of cytosolic CGFS Grxs in Fe-S cluster trafficking. In addition to interactions with CIA machinery, the cytosolic monothiol Grx (known as GrxD) was found to associate with the putative copper metallothionein CmtA, with this interaction being more pronounced under iron-replete conditions (Misslinger et al., 2019). However, earlier studies established that CmtA is not a classical copper-responsive metallothionein but is instead transcriptionally regulated by iron (Wiemann et al., 2017; Yap et al., 2022). More recently, biochemical and spectroscopic analyses of the recombinant protein revealed that CmtA is a novel [2Fe-2S]-binding metallothionein that can also coordinate copper and zinc. In vitro Fe-S cluster transfer assays monitored by CD spectroscopy demonstrated that GrxD can transfer a [2Fe-2S] cluster to CmtA in a transient 1:1 interaction (Eap et al., 2025). Notably, CmtA homologs are predominantly found in fungal pathogens, suggesting that this protein may confer a selective advantage in host-associated environments. This distribution raises the possibility that the GrxD-CmtA axis represents a pathogen-adapted mechanism for managing metal and Fe-S cluster homeostasis under the fluctuating and often restrictive conditions encountered during infection. Consistent with this idea, CmtA may function as a transient reservoir or buffering system for Fe-S clusters and/or metals in the cytosol. In this model, GrxD could facilitate the transfer of clusters not only to canonical CIA components but also to storage proteins such as CmtA, which may sequester Fe-S clusters under iron-replete conditions and mobilize them when demand increases or during stress. Such a mechanism would provide an additional layer of flexibility in coordinating iron utilization, Fe-S cluster availability, and metal homeostasis. Together, these observations expand the potential functional landscape of cytosolic monothiol Grxs in fungal pathogens. Beyond their proposed role in supplying or regulating Fe-S clusters for the CIA pathway, GrxD may participate in broader metal trafficking networks that integrate Fe-S cluster biogenesis with cellular metal storage and distribution. However, the physiological significance of the GrxD-CmtA interaction, and its relationship to canonical CIA-dependent Fe-S protein maturation, remains to be determined.

3.4. Monothiol Grxs and Fe-S Cluster Biogenesis in Protists

Protozoan parasites that include members of the order Kinetoplastida (e.g., Trypanosoma and Leishmania) and Apicomplexa (e.g. Plasmodium and Neospora), encode monothiol Grxs (1-C-Grxs) that function as analogs of CGFS-type Grxs found in other eukaryotes but are adapted to distinct cellular architectures and redox systems (Comini et al., 2013). These organisms experience highly dynamic host environments characterized by fluctuating iron availability and oxidative stress, necessitating robust mechanisms for Fe-S cluster biogenesis and maintenance.

In kinetoplastids, monothiol Grxs are integrated into canonical mitochondrial ISC and cytosolic CIA pathways, which are linked by the ABC transporter Atm1, as in fungi (Lukes & Basu, 2015). However, unlike fungi and most other eukaryotes that rely on GSH, these parasites utilize the GSH derivative bis(glutathionyl)spermidine, also known as trypanothione [T(SH)₂] as their principal low-molecular-weight thiol. Consistent with this adaptation, their monothiol Grxs coordinate [2Fe-2S] clusters using either GSH or T(SH)₂, highlighting biochemical flexibility in cluster ligation (Comini et al., 2013; Manta et al., 2013). Trypanosomatids typically encode multiple monothiol Grxs with distinct localizations and functions. In Trypanosoma brucei, the monothiol Grxs 1-C-Grx1 and 1-C-Grx2 are single domain proteins that localize to the mitochondrion, while 1-C-Grx3 is a cytosolic multidomain protein with a fused Trx-Grx architecture (Comini et al., 2013). Although all three possess monothiol Grx active site motifs that deviate from the canonical CGFS motif (CA[Y/F]S, C[G/R]FT, and CGFT, respectively), each has been shown to bind an Fe-S cluster in vitro (Comini et al., 2013; Manta et al., 2013). However, functional studies of the mitochondrial 1-C-Grxs in Trypanosoma brucei highlight a clear divergence among these paralogs (Comini et al., 2008; Filser et al., 2008). Notably, mitochondrial 1-C-Grx1, but not 1-C-Grx2, partially complements the Fe-S defects of a grx5Δ S. cerevisiae strain, indicating both evolutionary divergence from fungal Grx5 and non-redundant roles among trypanosomal Grxs. The enrichment of 1-C-Grx1 in non-dividing or stationary-phase parasites further suggests a role in stabilizing or storing Fe-S clusters during periods of reduced metabolic activity, thereby preparing cells for subsequent growth (Comini et al., 2008). Structural and biochemical studies underscore additional adaptations in kinetoplastid monothiol Grxs. NMR structural studies of 1-C-Grx1 revealed an intrinsically disordered N-terminal extension unique to trypanosomatids (and some plants) that modulates access to the GSH-binding pocket and may influence cluster exchange dynamics (Balatti et al., 2021; Sturlese et al., 2018). Moreover, the ability of T(SH)₂ to coordinate Fe-S clusters independently of protein raises the possibility that small-molecule Fe-S complexes contribute to intracellular cluster trafficking, although in vivo evidence remains limited.

In the apicomplexan parasites Neospora caninum and Plasmodium falciparum, Fe-S cluster synthesis is critical for survival, as these cofactors support essential metabolic pathways in both the mitochondrion and the apicoplast. The mitochondrial ISC pathway supports respiratory metabolism, while the plastid-derived apicoplast houses a SUF pathway that is essential for biosynthetic processes such as isoprenoid synthesis (Gisselberg et al., 2013). CGFS Grx homologs are implicated in Fe-S cluster metabolism within both organelles, although direct evidence for Fe-S binding by apicomplexan CGFS Grxs is lacking (Loveridge & Sigala, 2026; Song et al., 2023). In Neospora caninum, mitochondrial NcGrx5 was shown to interact with ISC components, while deletion of this protein compromises mitochondrial metabolism, slows growth, and abrogates virulence in mouse models (Song et al., 2023). In contrast, the functional importance of apicoplast-localized Grxs appears to be more variable and may depend on species or life stage (Song et al., 2021). The role of CGFS Grxs in Fe-S cluster biogenesis in P. falciparum has not been directly addressed, but these proteins (known as GLP1–3) have been classified at essential or fitness-conferring in a transposon insertional mutagenesis screen, suggesting that they perform critical cellular functions (Loveridge & Sigala, 2026; Zhang et al., 2018).

CGFS-type or Grx-like proteins have been identified in a range of other protozoan parasites, where they retain Fe-S cluster-binding capacity but exhibit significant functional diversification. In Giardia intestinalis, a monothiol Grx localizes to the mitosome, a highly reduced mitochondrion-related organelle, and retains the ability to coordinate a [2Fe-2S] cluster despite the absence of canonical mitochondrial metabolism (Rada et al., 2009). In Entamoeba histolytica, a divergent Grx-like small protein (GLSP) lacking the canonical CGFS motif nonetheless binds a GSH-ligated Fe-S cluster and can partially complement yeast Grx5 deficiency, indicating functional conservation despite sequence divergence (Birocco et al., 2023). Collectively, these studies demonstrate that monothiol Grxs are conserved components of Fe-S cluster biogenesis pathways in protists, functioning primarily in cluster coordination, transfer, and stabilization. However, compared to fungal and bacterial systems, their roles appear more tightly coupled to core metabolic processes, with organism-specific adaptations reflecting differences in redox chemistry and organellar complexity. While their capacity to coordinate [2Fe-2S] clusters is retained, their precise roles remain incompletely defined and likely reflect adaptation to diverse cellular and environmental constraints.

4. Monothiol Grxs as Iron Sensors and Regulatory Factors

Previous studies investigating the integration of CGFS Grxs within ISC/SUF/NIF/CIA Fe-S cluster assembly pathways have established monothiol CGFS Grxs as central intermediates in Fe-S cluster trafficking networks across diverse organisms. However, an emerging theme across bacterial and fungal systems is that the function of these proteins extends beyond simple cluster delivery. The Fe-S clusters bound by CGFS Grxs are not static cofactors but highly dynamic and environmentally responsive entities, whose occupancy reflects cellular iron availability, redox state, and metabolic demand. Consistent with this, perturbation of CGFS Grx function frequently leads to profound defects in iron homeostasis, including iron accumulation and misregulation of iron-responsive gene expression. These observations have revealed that monothiol Grxs are components of a broader Fe-S-dependent signaling system that communicates cellular iron status to regulatory networks, rather than acting solely as carriers within Fe-S assembly pathways. In this context, CGFS Grxs and their partner proteins, particularly BolA family members, function at the interface between Fe-S cluster metabolism and transcriptional control. The following section examines how Fe-S clusters themselves act as signaling ligands and how monothiol Grxs contribute to iron sensing and regulatory adaptation across microbial systems.

4.1. The Fe-S Cluster as a Signaling Ligand

A central conceptual advance in the study of iron homeostasis is the recognition that microbial cells may not directly sense free intracellular iron pools but instead monitor the status of iron incorporation into functional cofactors, particularly Fe-S clusters. Because Fe-S cluster assembly depends on iron availability, sulfur mobilization, cellular redox state, and (in eukaryotes) mitochondrial function, the presence, absence, or integrity of these cofactors provides a sensitive and integrated readout of the cellular metabolic state. Fe-S clusters are uniquely suited for this role, since they report not only on iron abundance, but on the capacity of the cell to generate functional iron cofactors. Unlike many metal cofactors that are tightly buried within protein structures, Fe-S clusters in regulatory proteins are often solvent-exposed, coordinatively flexible, and chemically labile. These features enable them to undergo reversible assembly, disassembly, and ligand exchange in response to environmental or metabolic perturbations. As a result, Fe-S clusters can function not only as catalytic cofactors but also as dynamic signaling ligands that modulate protein activity, protein-protein and protein-nucleotide interactions, and subcellular localization. Several paradigmatic regulatory systems illustrate this principle. In bacteria, the transcription factor IscR binds a [2Fe-2S] cluster that directly controls its DNA-binding specificity and regulatory output, allowing cells to rapidly respond to changes in iron availability, Fe-S cluster metabolism, oxygen levels, and oxidative/nitrosative stress (Mettert & Kiley, 2024). In yeast and other fungi, the primary transcription factors that control iron homeostasis are all Fe-S proteins in which Fe-S cluster binding regulates their protein-protein or protein-DNA interactions, which in turn alter their transcriptional activity (Gupta & Outten, 2020). In higher eukaryotes, aconitase/IRP1 switches between an Fe-S-bound enzymatic form and an apo-RNA-binding form that regulates intracellular iron metabolism (Rouault, 2019). These systems exemplify how Fe-S cluster occupancy can act as a molecular switch that toggles between distinct functional states.

Within this broader framework, monothiol CGFS Grxs have emerged as particularly versatile components of Fe-S-based signaling networks. Unlike classical Fe-S proteins that bind clusters as stable prosthetic groups, CGFS Grxs coordinate labile [2Fe-2S] clusters at protein-protein interfaces, often in conjunction with GSH and partner proteins such as BolA. This mode of coordination enables rapid and reversible cluster exchange, allowing CGFS Grxs to participate in dynamic networks of Fe-S cluster transfer rather than serving as static endpoints. Importantly, the Fe-S clusters associated with CGFS Grxs are highly responsive to cellular conditions. Their occupancy decreases under iron limitation or oxidative stress and increases under iron-replete conditions, reflecting the balance between cluster supply and demand. This dynamic behavior positions CGFS Grxs as sensitive reporters of intracellular Fe-S cluster availability. Another key mechanistic feature that distinguishes CGFS Grxs from other Fe-S proteins is their ability to form both homodimeric and heterodimeric complexes that differ in cluster stability and exchangeability. Homodimeric Grx complexes typically bind relatively labile clusters that are readily exchanged, consistent with roles in buffering or trafficking. In contrast, heterodimeric complexes formed with BolA proteins often exhibit altered cluster coordination environments and increased stability, suggesting specialization for regulatory functions. These distinct biochemical states provide a mechanism by which cells can partition Fe-S clusters into pools dedicated to trafficking versus signaling. Collectively, these observations support a unifying model in which Fe-S clusters act as signaling currencies, and CGFS Grxs function as dynamic exchangers that couple cluster availability to regulatory outputs. In this model, changes in Fe-S cluster occupancy within Grx or Grx-BolA complexes are transduced into alterations in transcription factor activity, protein localization, or protein-protein interactions, thereby linking iron metabolism to broader cellular responses.

4.2. Bacteria: Regulatory Integration

Although the regulatory roles of monothiol Grxs are most extensively characterized in fungi, increasing evidence indicates that bacterial CGFS Grxs also participate in the integration of Fe-S cluster metabolism with iron homeostasis and stress responses. In bacteria, these functions are mediated by CGFS Grxs alone or via interactions with BolA-family proteins.

4.2.1. Interplay of Bacterial Monothiol Grxs with Iron Regulatory Networks

Bacterial iron homeostasis is controlled by interconnected regulatory systems, including the ferric uptake regulator (Fur) and Fe-S-dependent transcription factors such as IscR. Although direct mechanistic links between GrxD and these regulators remain incompletely defined, multiple lines of evidence support functional integration. GrxD-deficient E. coli strains exhibit phenotypes consistent with altered Fe-S homeostasis, including aggravating genetic interactions with isc components, impaired activity of Fe-S enzymes, increases in the intracellular labile iron pool, and growth defects under iron-limiting conditions (Burschel et al., 2019; Fisher et al., 2024; Yeung et al., 2011). These phenotypes overlap with those observed in mutants affecting ISC and SUF pathways, suggesting that GrxD influences the effective availability of Fe-S clusters sensed by regulatory proteins such as IscR. Because IscR activity depends on its Fe-S cluster occupancy, changes in intracellular cluster distribution mediated by GrxD are likely to indirectly influence transcriptional programs controlling Fe-S assembly and iron utilization. In fact, holo-IscR repressor activity appears to be slightly increased upon grxD deletion (Fisher et al., 2024), reflecting a change in Fe-S cluster availability to this sensor. In parallel, Fur-regulated genes were also impacted by grxD deletion in E. coli, presumably due to expansion of the labile iron pool. Fur is a global transcriptional repressor conserved across bacteria that controls expression of iron acquisition and storage proteins, antioxidant enzymes, and pathogenesis factors. Fur is historically thought to sense intracellular iron pools via Fe²⁺ binding to trigger its repressor activity; however, recent studies have demonstrated that Fur binds a [2Fe-2S]²⁺ cluster that promotes its dimerization and DNA binding activity in vitro, similar to other divalent metals (Ding, 2025; Steingard & Helmann, 2023). It remains to be seen whether [2Fe-2S] binding is relevant to Fur activity in vivo and if GrxD directly impacts its function.

As noted in E. coli, the regulatory importance of GrxD becomes particularly evident under environmental stress conditions. Similarly, in pathogenic Pseudomonas species, deletion of grxD leads to increased sensitivity to oxidative stress and antibiotics, impaired siderophore production, and dysregulation of iron-dependent metabolic pathways. Interestingly, in the beneficial rhizobacteria Pseudomonas fluorescens, GrxD additionally influences production of the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG), which acts as a broad-spectrum antibiotic to suppress soil-borne fungal and bacterial diseases in plants (Dong et al., 2022). Consequently, P. fluorescens ΔgrxD mutant strains (or ones expressing GrxD with a mutated CGFS active site) are deficient in production of the antibiotic and exhibit impaired biocontrol activity against phytopathogens. A similar phenotype was noted for P. fluorescens strains deleted in the BolA protein IbaG (Dong et al., 2023). As in E. coli, P. fluorescens expresses two BolA proteins, IbaG and BolA, that were both shown to interact with GrxD via two-hybrid assays. However, deletion of bolA in P. fluorescens did not exhibit similar effects, demonstrating differential functions for the two BolA proteins. The effects of grxD or ibaG deletion on 2,4-DAPG biosynthesis were linked to a signaling pathway (Gac/Rsm) and transcriptional factor (PhiF) that repress expression of the biosynthetic gene cluster (Dong et al., 2022). Deletion of these negative regulatory factors in a ΔgrxD background restored 2,4-DAPG production, ruling out a direct role for GrxD in 2,4-DAPG biosynthesis. Intriguingly, since the interaction between GrxD and IbaG is dependent on conserved cysteines in each protein, and the thiol reductant dithiothreitol rescued 2,4-DAPG production in grxD deficient strains, the authors suggest that GrxD’s impact on IbaG function is redox-dependent (Dong et al., 2023). Together, these findings highlight that bacterial GrxD-BolA/IbaG complexes can affect global regulatory networks via Fe-S-dependent and redox-dependent mechanisms, with the relative contribution of each remaining context-dependent.

4.2.2. Emerging Model: Indirect but Significant Regulatory Roles

Collectively, these findings support a model in which bacterial CGFS Grxs function not as direct transcriptional regulators but as modulators of intracellular iron- and redox-dependent processes that, in turn, shape regulatory network activity. By influencing the distribution and availability of labile Fe-S clusters, GrxD can indirectly tune the activity of Fe-S-dependent sensors such as IscR and potentially Fur, as well as broader iron-responsive pathways. At the same time, observations from systems such as P. fluorescens highlight that GrxD-BolA/IbaG complexes may also exert regulatory effects through thiol-redox mechanisms that are independent of, or only partially coupled to, Fe-S cluster dynamics. The emerging interplay between GrxD and BolA-family proteins therefore suggests that distinct complexes may integrate both Fe-S cluster status and cellular redox state to modulate bacterial physiology. In contrast to the well-defined, direct Fe-S transfer mechanisms that control transcription factors in fungi, bacterial systems appear to rely on a more distributed mode of regulation in which metabolic and redox cues propagate through interconnected networks. This flexibility positions monothiol Grxs as central integrators of iron homeostasis, redox balance, and environmental stress, while underscoring that the relative contributions of Fe-S-dependent versus thiol-based mechanisms remain an important area for future investigation.

4.3. Fungi: Iron Regulon Control

Among microbial systems, fungi provide the most mechanistically developed framework for understanding how monothiol Grxs function as Fe-S-dependent signaling modules that couple iron availability to transcriptional regulation. In these organisms, cytosolic CGFS Grxs act at the interface between Fe-S cluster metabolism and gene expression, integrating mitochondrial Fe-S assembly with nuclear transcriptional responses.

4.3.1. Saccharomyces cerevisiae: Grx3/4-Bol2 Control of Aft1/2

In the budding yeast Saccharomyces cerevisiae, the role of cytosolic glutaredoxins Grx3 and Grx4 in regulating iron homeostasis is well established and represents a paradigm for Fe-S-dependent signaling (Gupta & Outten, 2020; Martínez-Pastor et al., 2017). Grx3 and Grx4, which are largely redundant, form complexes with the cytosolic BolA protein Bol2 (formerly Fra2), generating [2Fe-2S]-bridged Grx3/4-Bol2 heterodimers that regulate the activity of the paralogous iron-responsive transcription factors Aft1 and Aft2 (Figure 5A) (Kumanovics et al., 2008; Li et al., 2011; Li et al., 2009; Ojeda et al., 2006; Poor et al., 2014; Pujol-Carrion et al., 2006). Aft1/2 are nucleocytoplasmic DNA binding proteins that respond to iron availability by modulating the expression of genes involved in iron uptake and storage. Under iron-limiting conditions, Aft1/2 exist as monomers that translocate to the nucleus, bind DNA, and activate the iron regulon. Upon restoration of iron sufficiency, Aft1/2 dissociate from DNA and are exported to the cytosol (Ueta et al., 2007, 2012; Yamaguchi-Iwai et al., 1996; Yamaguchi-Iwai et al., 2002). Mechanistically, this transition is driven by Fe-S cluster-dependent signaling that is dependent on mitochondrial Fe-S cluster biogenesis, GSH, and Atm1, but independent of the CIA pathway (Chen et al., 2004; Kumar et al., 2011; Rutherford et al., 2005; Sipos et al., 2002). DNA binding assays demonstrated that dissociation of Aft2 from target promoters requires [2Fe-2S] cluster binding by Aft2, which induces a monomer-to-dimer transition in Aft2 that reduces DNA-binding affinity and promotes transcriptional inactivation (Figure 5A) (Li & Outten, 2019; Poor et al., 2014). A similar mechanism is proposed for Aft1, although direct experimental confirmation remains incomplete. Importantly, this regulatory process is dependent on [2Fe-2S] cluster transfer from the heterodimeric Grx3/4-Bol2 complex to Aft2. Although Grx3/4 homodimers also coordinate [2Fe-2S] clusters, they are unable to transfer the cluster to Aft proteins under comparable conditions. This functional asymmetry highlights a key principle: Fe-S cluster binding alone is insufficient for signaling competence. Instead, the specific quaternary structure of the Grx-BolA heterodimer defines the signaling-active state.

Figure 5.

Models for Grx3/4-dependent iron regulation in S. cerevisiae (A) and S. pombe (B). Under iron limitation in S. cerevisiae (A, top), Aft1 and Aft2 activate expression of iron uptake and transport genes as well as the mRNA binding proteins Cth1 and Cth2 that degrade transcripts encoding proteins in non-essential, iron-consuming metabolic pathways. During conditions of iron sufficiency (A, bottom), Grx3 and Grx4 form Fe-S-bridged heterodimers with Bol2 and deliver [2Fe-2S] clusters to Aft1/2, which promotes their DNA dissociation, dimerization, and export to the cytosol. This change, in turn, leads to deactivation of Aft1/2-regulated genes. During iron limitation in S. pombe (B, top), Php4 economizes iron usage by binding the CCAAT binding complex (CBC) and repressing transcription of the iron-responsive repressor Fep1 as well as genes encoding proteins in non-essential, iron-utilizing pathways. Grx4 maintains an interaction with Php4 but does not interfere in its association with the CBC. Fep1 dissociates from its target genes when iron is low, leading to the expression of iron uptake and transport genes and PHP4. The dissociation of Fep1 from DNA is triggered by transfer of [2Fe-2S] clusters from Fep1 to the Grx4-Fra2 heterodimer. During iron sufficiency (B, bottom), Grx4 and Php4 form a [2Fe-2S]-binding complex that promotes Php4’s dissociation from the CBC and its nuclear export, leading to derepression of the Php4 regulon. Meanwhile, [2Fe-2S] cluster binding to Fep1 (delivered by unknown factors) promotes its DNA binding activity and repression of its target genes. Created in BioRender. Outten, C. (2026) https://BioRender.com/yd3wb81

4.3.2. Schizosaccharomyces pombe: Divergent Regulatory Architecture

In the evolutionarily distant fission yeast Schizosaccharomyces pombe, iron homeostasis is governed by a distinct but conceptually related regulatory circuit involving the cytosolic CGFS glutaredoxin Grx4, the BolA protein Fra2, and two iron-responsive transcriptional repressors, Fep1 and Php4 (Figure 5B). Unlike the Aft1/2 system, Fep1 and Php4 function as reciprocal regulators of iron acquisition and iron utilization, respectively, and also regulate each other’s expression in an iron-dependent manner (reviewed in (Brault et al., 2015; Gupta & Outten, 2020)). Fep1 is a GATA-type transcription factor that represses iron uptake genes under iron-replete conditions. Whereas Php4 is recruited under iron limitation to the CCAAT binding complex (Php2/3/5), where it represses genes involved in iron-consuming processes, including mitochondrial respiration, TCA cycle activity, and Fe-S cluster biogenesis. Grx4 serves as a central regulator of both pathways. Deletion of grx4 results in hypersensitivity to iron deprivation and constitutive dysregulation of both Fep1- and Php4-dependent gene expression, underscoring its essential role in coordinating iron-responsive transcription (Encinar del Dedo et al., 2015; Jbel et al., 2011; Kim et al., 2011; Mercier & Labbe, 2009).

The functional interactions between Grx4 and both Php4 and Fep1 are Fe-S cluster dependent. Biochemical and spectroscopic studies have demonstrated that Php4 does not bind Fe-S clusters alone but forms a [2Fe-2S]-bridged complex with Grx4 (Dlouhy et al., 2017). Formation of this heterocomplex under iron sufficiency is proposed to promote dissociation of Php4 from the Php2/3/5 transcriptional complex and facilitate its nuclear export via the exportin Crm1 (Figure 5B) (Encinar del Dedo et al., 2015; Mercier & Labbe, 2009; Vachon et al., 2012). These studies collectively support a model in which Fe-S cluster binding to the Grx4-Php4 complex regulates Php4 localization and activity. Interestingly, unlike the situation in S. cerevisiae where Grx3/4 works in partnership with a BolA protein to regulate Aft1/2 activity, the S. pombe cytosolic BolA protein, Fra2, is not involved in iron signaling to Php4 (Jacques et al., 2014). However, regulation of the Fep1 repressor does involve both Grx4 and Fra2. Fra2 co-immunoprecipitates with both Grx4 and Fep1 and functions cooperatively with Grx4 in iron-dependent repression of Fep1 activity (Encinar del Dedo et al., 2015; Hati et al., 2023; Jacques et al., 2014). Under iron-replete conditions, Fep1 binds DNA and represses iron uptake genes, a process that is enhanced by Fe-S cluster coordination. Under iron-depleted conditions, destabilization of the [2Fe-2S] cluster is proposed to promote transfer of the cluster from Fep1 to the Grx4-Fra2 complex, resulting in transcriptional inactivation of Fep1 (Figure 5B) (Encinar del Dedo et al., 2015; Jacques et al., 2014). Recent work has experimentally validated this model, demonstrating that Grx4 and Fra2 function as Fe-S cluster acceptors that mediate Fep1 inactivation during iron starvation. Mutational analyses further revealed that the conserved Fe-S binding Fra2 residues Cys29 and His66 are required for stable heterodimer formation with Grx4 and for proper regulation of Fep1 activity in vivo (Hati et al., 2023). These findings reinforce a broader conceptual framework in which Grx-BolA complexes function as regulated Fe-S cluster exchange modules, capable of either donating or accepting clusters depending on the regulatory context.

4.3.3. Pathogenic Fungi: Iron Sensing, Virulence, and Network Integration

Recent studies in pathogenic fungi reinforce principles established in the model yeasts S. cerevisiae and S. pombe, demonstrating that cytosolic CGFS Grxs function as Fe-S-dependent signal transducers that couple iron sensing to transcriptional and metabolic adaptation (reviewed in (Gupta & Outten, 2020; Martínez-Pastor & Puig, 2020; Misslinger et al., 2021)). These regulatory processes are critical for survival under host-imposed iron limitation or iron excess, and accordingly, disruption of cytosolic CGFS Grxs in fungal pathogens typically leads to inviability or severe attenuation of virulence. This paradigm is well illustrated in the opportunistic human pathogen Aspergillus fumigatus, where the monothiol glutaredoxin GrxD is required for iron-dependent regulation of the transcription factors HapX and SreA, orthologs of the Php4 and Fep1 regulators in S. pombe. These factors coordinately control iron acquisition and utilization programs in response to changing iron availability. Affinity purification studies demonstrate that GrxD associates with both HapX and SreA in vivo, supporting a direct role in modulating their activity through post-translational mechanisms. HapX binds [2Fe-2S] clusters with distinct affinities across four cysteine-rich motifs in vitro, and in vivo experiments suggest that sequential cluster occupancy may drive transitions between functional states that enable adaptation to iron limitation, sufficiency, and excess (Misslinger et al., 2019; Oberegger et al., 2025; Rossetto et al., 2024). However, the mechanistic basis of this regulation remains incompletely defined, particularly with respect to how cluster occupancy influences DNA binding and transcriptional activity, and whether GrxD functions as a cluster donor, acceptor, or stabilizing factor at individual sites.

A similar regulatory architecture is observed in the commensal human pathogen Candida albicans, although with notable differences in physiological outcome. The cytosolic monothiol glutaredoxin Grx3 is not essential for viability but is required for optimal growth under iron limitation and oxidative stress, and is critical for virulence in mammalian infection models (Attarian et al., 2018; Zhang et al., 2017). Grx3 regulates the activity of the iron-responsive transcription factors Sfu1 (a GATA-type repressor) and Hap43 (a HapX/Php4-like regulator) through protein-protein interactions that modulate Sfu1 DNA binding and control the nuclear-cytoplasmic localization of Hap43 in an iron-dependent manner. These findings highlight a conserved role for CGFS Grxs in coordinating transcriptional responses, while also illustrating organism-specific differences in the requirement for these proteins.

This regulatory logic extends to basidiomycete pathogens such as Cryptococcus neoformans, where the sole cytosolic CGFS glutaredoxin, Grx4, governs iron homeostasis through interaction with the transcription factor Cir1, a GATA-type regulator that controls genes involved in iron acquisition, virulence, and associated signaling pathways (Attarian et al., 2018; Jung et al., 2006). Grx4 is required for growth under low-iron conditions and for the expression of multiple virulence traits, including thermotolerance, melanin production, and capsule formation. Consistent with a central regulatory role, grx4 mutant strains are avirulent in mouse models of infection. Importantly, the Fe-S cluster-binding capacity of Grx4 is essential for its function, supporting a model in which Fe-S coordination modulates Cir1 activity in a manner analogous to GATA-type regulators in other fungi (Gupta & Outten, 2020).

In plant pathogenic fungi, CGFS Grxs similarly integrate iron sensing with transcriptional regulation and virulence, while also revealing additional layers of regulatory complexity. In the corn smut fungus, Ustilago maydis, Grx4 controls the expression of genes involved in iron acquisition and utilization, GSH metabolism, Fe-S cluster biogenesis, and melanin production (McCotter et al., 2023). Grx4 is essential for both growth and pathogenesis, as conditional mutants fail to infect maize seedlings and induce tumor formation. These phenotypes are consistent with Grx4 regulating the iron-responsive transcription factor Urbs1 (an ortholog of C. neoformans Cir1), although a direct physical interaction remains to be demonstrated. In contrast, in the major cereal pathogen F. graminearum, the cytosolic glutaredoxin FgGrx4 is not essential but is required for adaptation to iron-replete conditions and for full virulence during host infection (Sun et al., 2023). Notably, this fungus encounters iron excess rather than limitation during host colonization, highlighting a distinct environmental context for iron regulation. FgGrx4 interacts with both major iron-responsive transcriptional factors, FgSreA and FgHapX, coordinating their activities by modulating their occupancy at target promoters and facilitating recruitment of epigenetic regulators (Sun et al., 2023; Wang et al., 2019). These findings reveal an additional layer of regulation in which Fe-S cluster sensing is directly coupled to chromatin remodeling and transcriptional reprogramming, revealing an emerging interface between iron metabolism and epigenetic control in eukaryotic pathogens (Sun et al., 2023).

Collectively, these studies establish cytosolic CGFS Grxs as conserved regulatory hubs that translate Fe-S cluster status into coordinated transcriptional and chromatin-level responses. While the core regulatory logic linking Fe-S cluster coordination to transcription factor activity is broadly conserved, the physiological consequences vary across species, ranging from essential roles in viability to more specialized functions in stress adaptation and virulence. Through this integration of iron sensing, redox state, and gene regulation, CGFS Grxs enable fungal pathogens to dynamically reprogram their physiology in response to host-imposed stresses, thereby supporting successful infection across diverse fungal lineages (Figure 6).

Figure 6.

Schematic depicting the roles of CGFS Grxs in fungal pathogenesis and survival under variable iron bioavailability conditions. See text for details. Created in BioRender. Outten, C. (2026) https://BioRender.com/ze1ffoa

4.4. Protists: Emerging Regulatory Roles

In contrast to fungi, where CGFS Grxs function as well-defined Fe-S-dependent signaling modules, the regulatory roles of monothiol Grxs in protists remain comparatively underdeveloped. A key distinction lies in the regulatory architecture of these organisms. Many protists, including kinetoplastids and apicomplexans, lack conventional iron-responsive transcription factors and instead rely on post-transcriptional or post-translational mechanisms to control gene expression (Comini et al., 2013; Loveridge & Sigala, 2026). As a result, the canonical model observed in fungi, where monothiol Grxs or Grx-BolA complexes modulate transcription factor activity in an Fe-S cluster-dependent manner, does not readily translate to these systems. Instead, monothiol Grxs in protists appear to function primarily as metabolic integrators, maintaining Fe-S cluster homeostasis under conditions of fluctuating iron availability and oxidative stress. In kinetoplastids, the essential mitochondrial monothiol glutaredoxin 1-C-Grx1 is required for parasite infectivity and Fe-S metabolism (Comini et al., 2008; Manta et al., 2013). While this underscores its physiological importance, there is currently no evidence that it directly regulates gene expression through Fe-S-dependent signaling. Similarly, cytosolic multidomain Grxs such as 1-C-Grx3 are expressed across multiple life stages, but their roles appear to be linked to redox balance and Fe-S cluster handling rather than transcriptional control. Apicomplexan parasites present a similarly incomplete picture. In Plasmodium falciparum, CGFS-type Grx homologs (e.g., GLP proteins) are classified as essential or fitness-conferring in genetic screens, although biochemical studies have not yet confirmed their capacity to bind Fe-S clusters (Loveridge & Sigala, 2026; Tripathi et al., 2008). Direct connections to iron-responsive regulatory pathways have not been established. This gap is compounded by an incomplete understanding of iron homeostasis in these parasites, particularly during blood-stage infection, where iron is abundant yet tightly compartmentalized.

Despite the lack of direct evidence for transcriptional regulation, several observations suggest that monothiol Grxs in protists may still contribute to cellular signaling in broader ways. Their Fe-S cluster occupancy is likely sensitive to changes in iron availability, redox state, and metabolic demand, positioning them as potential sensors of intracellular conditions. In organisms where gene expression is regulated post-transcriptionally, such signals could be transduced through effects on protein stability, localization, or interaction networks rather than DNA binding. Furthermore, the ability of T(SH)₂ and other thiols to participate in Fe-S cluster coordination raises the possibility that small-molecule-mediated signaling mechanisms operate alongside protein-based pathways. Overall, protists represent an emerging frontier in the study of monothiol Grxs. While their roles in Fe-S cluster metabolism are well supported, their potential functions in regulatory networks remain largely unexplored. Determining whether CGFS-type Grxs in these organisms act solely as metabolic factors or also participate in non-canonical signaling pathways will be an important direction for future research, particularly given their essentiality and relevance to parasite biology.

5. Comparative Synthesis: Conserved Logic and Divergent Outcomes

Across bacteria, fungi, and protists, monothiol CGFS-type Grxs exhibit a striking combination of biochemical conservation and functional diversification. At their core, these proteins share a common molecular capability: the reversible coordination and exchange of labile [2Fe-2S] clusters. However, the physiological roles built upon this shared biochemical foundation vary substantially across lineages, reflecting differences in cellular organization, regulatory architecture, and environmental pressures (Table 1).

Table 1.

Conserved and divergent roles of monothiol CGFS Grxs across microbial systems

Feature	Bacteria	Fungi	Protists
Representative systems	E. coli, Pseudomonas	S. cerevisiae, S. pombe, pathogenic fungi	Trypanosoma, Plasmodium, Giardia
CGFS Grx types	Single domain (e.g., GrxD)	Mitochondrial single (Grx5) and cytosolic multidomain (Grx3/4-type)	Multiple isoforms with variable architectures
Role in Fe-S biogenesis pathways	Downstream of ISC/SUF; interacts with A-type carriers; cluster trafficking and buffering	Linking ISC and CIA pathways; cluster trafficking (Grx5) and cytosolic buffering/distribution (Grx3/4)	Integrated into ISC/CIA-like systems (including reduced organelles); cluster coordination and maintenance
Regulatory function	Indirect (modulates Fe-S availability sensed by regulators)	Direct (Fe-S-dependent control of transcription factors)	Limited or unclear; likely indirect or non-transcriptional
Key partners	A-type carriers (ErpA), BolA proteins	BolA proteins, transcription factors (e.g. Aft1/2, Fep1, Php4, HapX, SreA), CIA components, metallothionein	ISC/CIA components; few defined regulatory partners
Physiological roles	Stress adaptation, iron homeostasis, metabolic maintenance	Iron regulon control, intracellular iron distribution, virulence	Parasite viability, stress adaptation, metabolic regulation
Essentiality	Conditional (important under stress)	Frequently essential or fitness-conferring	Frequently essential or fitness-conferring

5.1. Conserved Core Functions

In all systems examined, CGFS Grxs function as intermediates within Fe-S cluster trafficking networks. They receive clusters from scaffold or carrier proteins and facilitate their redistribution to downstream targets. This role is conserved from bacterial ISC/SUF pathways to mitochondrial ISC and cytosolic CIA systems in eukaryotes, as well as in reduced organelles of protists. A second conserved feature is their ability to form [2Fe-2S]-bridged complexes, either as homodimers or in association with BolA-family proteins. Although some Grxs homodimers can accommodate alternate cluster forms (e.g., [4Fe-4S]²⁺ or linear [3Fe-4S]⁺), [2Fe-2S]²⁺ clusters are the predominant cluster type coordinated at the dimer interface. Grx homodimers and Grx-BolA heterodimers differ in stability and exchangeability, providing a biochemical basis for functional specialization.

5.2. Divergence in Regulatory Integration

Despite this shared core, the degree to which CGFS Grxs are integrated into regulatory networks differs markedly. In bacteria, monothiol Grxs primarily function as metabolic modulators, indirectly influencing regulatory systems such as Fur and IscR through their effects on Fe-S cluster availability and redox balance. Direct roles in transcriptional regulation remain limited and mechanistically unresolved. In fungi, CGFS Grxs have evolved into central regulatory hubs. Functioning alone or in partnership with BolA proteins, they directly mediate Fe-S-dependent control of transcription factors, linking mitochondrial Fe-S cluster biogenesis to nuclear gene expression. This paradigm represents the most mechanistically defined example of Fe-S-based signaling. In protists, current evidence supports a primary role in Fe-S cluster maintenance and parasite viability, with regulatory functions remaining speculative. The apparent absence of canonical iron-responsive transcription factors in some lineages suggests that CGFS Grxs may operate within alternative regulatory frameworks, potentially involving post-transcriptional control or metabolic signaling.

5.3. A Unifying Model: CGFS Grxs as Fe-S Rheostats

These observations support a unifying conceptual framework in which monothiol Grxs function as Fe-S cluster rheostats. Rather than acting solely as carriers, CGFS Grxs dynamically sense and redistribute labile Fe-S clusters in response to cellular conditions. Changes in cluster occupancy, driven by iron availability, oxidative stress, or metabolic demand, are translated into functional outputs through shifts in protein-protein interactions, cluster transfer reactions, or regulatory complex formation. A key mechanistic principle underlying this model is the existence of distinct functional states: Grx homodimers, which bind relatively labile clusters, are well suited for buffering and trafficking. Grx-BolA heterodimers, which generally stabilize clusters, are often specialized for regulatory signaling. The balance between these states may enable cells to partition Fe-S clusters between metabolic needs and signaling functions.

5.4. Evolutionary Implications and Future Directions

The diversification of CGFS Grx function highlights how a conserved biochemical scaffold can be repurposed to meet organism-specific demands. In simpler systems, such as bacteria, CGFS Grxs remain closely tied to metabolic homeostasis. In more complex eukaryotes, particularly fungi, they have been integrated into sophisticated regulatory circuits that coordinate iron metabolism at the transcriptional level. Protists represent an intermediate and largely unexplored space, where essential metabolic roles are established but regulatory functions remain to be defined.

Several key questions emerge from this comparative perspective:

• What determines the partitioning of CGFS Grxs between trafficking and signaling roles?

• Do alternative Fe-S cluster species contribute to regulatory specificity in vivo?

• How do CGFS Grxs interface with other metal homeostasis pathways?

• What regulatory mechanisms substitute for transcriptional control in protists?

Addressing these questions will be critical for understanding how Fe-S cluster biology is integrated into cellular physiology across diverse microorganisms.

Abbreviations:

Fe-S

iron-sulfur

GSH

glutathione

GSSG

oxidized glutathione

Trx

thioredoxin

Grx

glutaredoxin

CGFS

Cys-Gly-Phe-Ser

EPR

electron paramagnetic resonance

CD

circular dichroism

ISC

iron-sulfur cluster

SUF

sulfur mobilization factor

NIF

nitrogen fixation

CIA

cytosolic iron-sulfur assembly

T(SH)₂

trypanothione

Fur

ferric uptake regulator

2,4-DAPG

2,4-diacetylphloroglucinol