Mammalian iron–sulfur cluster biogenesis: Recent insights into the roles of frataxin,acyl carrier protein and ATPase-mediated transfer to recipient proteins

The recently solved crystal structures of the human cysteine desulfurase NFS1, in complex with the LYR protein ISD11, the acyl carrier protein ACP, and the main scaffold ISCU, have shed light on the molecular interactions that govern initial cluster assembly on ISCU. Here, we aim to highlight recent insights into iron–sulfur (Fe–S) cluster (ISC) biogenesis in mammalian cells that have arisen from the crystal structures of the core ISC assembly complex. We will also discuss how ISCs are delivered to recipient proteins and the challenges that remain in dissecting the pathways that deliver clusters to numerous Fe–S recipient proteins in both the mitochondrial matrix and cytosolic compartments of mammalian cells.     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.     

Di-iron proteins of the Ric family are involved in iron–sulfur cluster repair

A key element in eukaryotic immune defenses against invading microbes is the production of reactive oxygen and nitrogen species. One of the main targets of these species are the iron–sulfur clusters, which are essential prosthetic groups that confer to proteins the ability to perform crucial roles in biological processes. Microbes have developed sophisticated systems to eliminate nitrosative and oxidative species and promote the repair of the damages inflicted. The Ric (Repair of Iron Centers) proteins constitute a novel family of microbial di-iron proteins with a widespread distribution among microbes, including Gram-positive and Gram-negative bacteria, protozoa and fungi. The Ric proteins are encoded by genes that are up-regulated by nitric oxide and hydrogen peroxide. Recent studies have shown that the active di-iron center is involved in the restoration of Fe–S clusters damaged by exposure to nitric oxide and hydrogen peroxide.     

Reactive oxygen species regulates expression of iron–sulfur cluster assembly protein IscS of Leishmania donovani

The cysteine desulfurase, IscS, is a highly conserved and essential component of the mitochondrial iron–sulfur cluster (ISC) system that serves as a sulfur donor for Fe–S clusters biogenesis. Fe–S clusters are versatile and labile cofactors of proteins that orchestrate a wide array of essential metabolic processes, such as energy generation and ribosome biogenesis. However, no information regarding the role of IscS or its regulation is available in Leishmania, an evolving pathogen model with rapidly developing drug resistance. In this study, we characterized LdIscS to investigate the ISC system in AmpB-sensitive vs resistant isolates of L. donovani and to understand its regulation. We observed an upregulated Fe–S protein activity in AmpB-resistant isolates but, in contrast to our expectations, LdIscS expression was upregulated in the sensitive strain. However, further investigations showed that LdIscS expression is positively correlated with ROS level and negatively correlated with Fe–S protein activity, independent of strain sensitivity. Thus, our results suggested that LdIscS expression is regulated by ROS level with Fe–S clusters/proteins acting as ROS sensors. Moreover, the direct evidence of a mechanism, in support of our results, is provided by dose-dependent induction of LdIscS-GFP as well as endogenous LdIscS in L. donovani promastigotes by three different ROS inducers: H₂O₂, menadione, and Amphotericin B. We postulate that LdIscS is upregulated for de novo synthesis or repair of ROS damaged Fe–S clusters. Our results reveal a novel mechanism for regulation of IscS expression that may help parasite survival under oxidative stress conditions encountered during infection of macrophages and suggest a cross talk between two seemingly unrelated metabolic pathways, the ISC system and redox metabolism in L. donovani.     

Iron–sulfur protein folds,iron–sulfur chemistry,and evolution

An inventory of unique local protein folds around Fe–S clusters has been derived from the analysis of protein structure databases. Nearly 50 such folds have been identified, and over 90% of them harbor low-potential [2Fe–2S]^2+,+ or [4Fe–4S]^2+,+ clusters. In contrast, high-potential Fe–S clusters, notwithstanding their structural diversity, occur in only three different protein folds. These observations suggest that the extant population of Fe–S protein folds has to a large extent been shaped in the reducing iron- and sulfur-rich environment that is believed to have predominated on this planet until approximately two billion years ago. High-potential active sites are then surmised to be rarer because they emerged later, in a more oxidizing biosphere, in conditions where iron and sulfide had become poorly available, Fe–S clusters were less stable, and in addition faced competition from heme iron and copper active sites. Among the low-potential Fe–S active sites, protein folds hosting [4Fe–4S]^2+,+ clusters outnumber those with [2Fe–2S]^2+,+ ones by a factor of 3 at least. This is in keeping with the higher chemical stability and versatility of the tetranuclear clusters, compared with the binuclear ones. It is therefore suggested that, at least while novel Fe–S sites are evolving within proteins, the intrinsic chemical stability of the inorganic moiety may be more important than the stabilizing effect of the polypeptide chain. The discovery rate of novel Fe–S-containing protein folds underwent a sharp increase around 1995, and has remained stable to this day. The current trend suggests that the mapping of the Fe–S fold space is not near completion, in agreement with predictions made for protein folds in general. Altogether, the data collected and analyzed here suggest that the extant structural landscape of Fe–S proteins has been shaped to a large extent by primeval geochemical conditions on one hand, and iron–sulfur chemistry on the other.     

Interplay between formation of photosynthetic complexes and expression of genes for iron–sulfur cluster assembly in Rhodobacter sphaeroides?

Photosynthesis Research - Formation of photosynthetic complexes leads to a higher demand for Fe–S clusters. We hypothesized that in the facultative phototrophic alpha-proteobacterium...     

Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe–S cluster biogenesis regulation

Iron–sulfur (Fe–S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe–S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe–S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe–S cluster biogenesis does not meet cellular demands.     

High potential iron–sulfur proteins and their role as soluble electron carriers in bacterial photosynthesis: tale of a discovery

This review is an attempt to retrace the chronicle of the discovery of the role of high-potential iron–sulfur proteins (HiPIPs) as electron carriers in the photosynthetic chain of bacteria. Data and facts are presented through the magnifying lenses of the authors, using their best judgment to filter and elaborate on the many facets of the research carried out on this class of proteins over the years. The tale is divided into four main periods: the seeds, the blooming, the ripening, and the harvest, representing the times from the discovery of these proteins to the most recent advancements in the understanding of the relationship between their structure and their function.     

Metabolic regulation of oxygen and redox homeostasis by p53: Lessons from evolutionary biology?

The genetic links between p53 and metabolic processes such as oxidative phosphorylation are being studied with increasing interest given that cellular metabolism seems to play an important role in tumorigenesis. This review focuses on how p53 regulation of various metabolic genes may influence redox homeostasis, as the genome is constantly susceptible to oxidative damage, a consequence of living in an aerobic environment. Because p53-like genetic sequences are also found in life forms that may not necessarily benefit from tumor suppression, an evolutionary introduction is given in an attempt to understand why p53 might regulate a basic cellular activity such as metabolism. The presented epidemiologic and experimental data suggest that one reason may be for the homeostatic regulation of oxygen, the essential substrate for reactive oxygen species generation.     

Loss of Ssq1 leads to mitochondrial dysfunction,activation of autophagy and cell cycle arrest due to iron overload triggered by mitochondrial iron–sulfur cluster assembly defects in Candida albicans

Iron–sulfur clusters perform essential functions in enzymatic catalysis and homeostatic regulation. Here we for the first time identified Ssq1 as an essential component for iron–sulfur cluster assembly in Candida albicans. Ssq1 played an important role in cell growth. Shutting off SSQ1 led to accumulation of intracellular iron, especially in mitochondria, and disorder of intracellular iron regulation. In tetO-SSQ1, iron overloading triggered the oxidative damage of mitochondrial function. Surprisingly, disruption of SSQ1 activated autophagic pathway. The mitochondrial dysfunction was further aggravated when CCZ1 (which is essential for autophagy) and SSQ1 was simultaneously deleted, suggesting that autophagy played a critical role in maintenance of mitochondrial function in tetO-SSQ1. In addition, double deletion of SSQ1 and CCZ1 further elevated cellular iron levels in comparison with tetO-SSQ1, indicating that autophagy participated in maintenance of iron homeostasis. Furthermore, we found that loss of SSQ1 led to increasing protein expression of Rnr1 and redistribution of Rnr2 from the nucleus to cytoplasm, and further resulted in cell cycle arrest. The results implied that cell cycle arrest was caused by activating the checkpoint pathway because of impairing the iron–sulfur cluster assembly in tetO-SSQ1. Shutting off SSQ1 led to a significant defect in filamentous development. Interestingly, the tetO-SSQ1ccz1Δ/Δ growth was inhibited on hyphae-inducing solid media. Both tetO-SSQ1 and tetO-SSQ1ccz1Δ/Δ exhibited extremely attenuated virulence, indicating that Ssq1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of iron–sulfur cluster assembly-related gene in C. albicans.     

Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron–sulfur cluster

Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (³⁹³CX₂CX₃CX₁₄C) that was found in only six other proteobacteria (CX₂CX₃CX_11–14C). The cysteine motif is likely to coordinate an unprecedented [Fe–S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe–4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe–4S] cluster centered at g = 2.02. The [3Fe–4S] signal was observed independently of the [4Fe–4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe–4S] and [3Fe–4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe–4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.     

Iron–sulfur cluster exchange reactions mediated by the human Nfu protein

Human Nfu is an iron–sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron–sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe–2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe–2S](GS)₄ cluster complex, and extraction of the Nfu-bound iron–sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe–2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe–2S] cluster trafficking and delivery.     

Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase

Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX_31–39CCX_35–36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) ⁵⁷Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four ⁵⁷Fe hyperfine couplings to the cluster in all three proteins. ¹³C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. ⁵⁷Fe resonances in all three systems revealed unusually large ⁵⁷Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster’s unique magnetic properties arise from the CCG binding motif.     

Expanding horizons in iron chelation and the treatment of cancer: Role of iron in the regulation of ER stress and the epithelial–mesenchymal transition

Cancer is a major public health issue and, despite recent advances, effective clinical management remains elusive due to intra-tumoural heterogeneity and therapeutic resistance. Iron is a trace element integral to a multitude of metabolic processes, including DNA synthesis and energy transduction. Due to their generally heightened proliferative potential, cancer cells have a greater metabolic demand for iron than normal cells. As such, iron metabolism represents an important “Achilles' heel” for cancer that can be targeted by ligands that bind and sequester intracellular iron. Indeed, novel thiosemicarbazone chelators that act by a “double punch” mechanism to both bind intracellular iron and promote redox cycling reactions demonstrate marked potency and selectivity in vitro and in vivo against a range of tumours. The general mechanisms by which iron chelators selectively target tumour cells through the sequestration of intracellular iron fall into the following categories: (1) inhibition of cellular iron uptake/promotion of iron mobilisation; (2) inhibition of ribonucleotide reductase, the rate-limiting, iron-containing enzyme for DNA synthesis; (3) induction of cell cycle arrest; (4) promotion of localised and cytotoxic reactive oxygen species production by copper and iron complexes of thiosemicarbazones (e.g., Triapine^® and Dp44mT); and (5) induction of metastasis and tumour suppressors (e.g., NDRG1 and p53, respectively). Emerging evidence indicates that chelators can further undermine the cancer phenotype via inhibiting the epithelial–mesenchymal transition that is critical for metastasis and by modulating ER stress. This review explores the “expanding horizons” for iron chelators in selectively targeting cancer cells.