Iron-sulfur Cluster Assembly Machinery Research Articles

Reactivating PTEN to impair glioma stem cells by inhibiting cytosolic iron-sulfur assembly.

Glioblastoma, the most lethal primary brain tumor, harbors glioma stem cells (GSCs) that not only initiate and maintain malignant phenotypes but also enhance therapeutic resistance. Although frequently mutated in glioblastomas, the function and regulation of PTEN in PTEN-intact GSCs are unknown. Here, we found that PTEN directly interacted with MMS19 and competitively disrupted MMS19-based cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) machinery in differentiated glioma cells. PTEN was specifically succinated at cysteine (C) 211 in GSCs compared with matched differentiated glioma cells. Isotope tracing coupled with mass spectrometry analysis confirmed that fumarate, generated by adenylosuccinate lyase (ADSL) in the de novo purine synthesis pathway that is highly activated in GSCs, promoted PTEN C211 succination. This modification abrogated the interaction between PTEN and MMS19, reactivating the CIA machinery pathway in GSCs. Functionally, inhibiting PTEN C211 succination by reexpressing a PTEN C211S mutant, depleting ADSL by shRNAs, or consuming fumarate by the US Food and Drug Administration-approved prescription drug N-acetylcysteine (NAC) impaired GSC maintenance. Reexpressing PTEN C211S or treating with NAC sensitized GSC-derived brain tumors to temozolomide and irradiation, the standard-of-care treatments for patients with glioblastoma, by slowing CIA machinery-mediated DNA damage repair. These findings reveal an immediately practicable strategy to target GSCs to treat glioblastoma by combination therapy with repurposed NAC.

Components of iron–Sulfur cluster assembly machineries are robust phylogenetic markers to trace the origin of mitochondria and plastids

Establishing the origin of mitochondria and plastids is key to understand 2 founding events in the origin and early evolution of eukaryotes. Recent advances in the exploration of microbial diversity and in phylogenomics approaches have indicated a deep origin of mitochondria and plastids during the diversification of Alphaproteobacteria and Cyanobacteria, respectively. Here, we strongly support these placements by analyzing the machineries for assembly of iron–sulfur ([Fe–S]) clusters, an essential function in eukaryotic cells that is carried out in mitochondria by the ISC machinery and in plastids by the SUF machinery. We assessed the taxonomic distribution of ISC and SUF in representatives of major eukaryotic supergroups and analyzed the phylogenetic relationships with their prokaryotic homologues. Concatenation datasets of core ISC proteins show an early branching of mitochondria within Alphaproteobacteria, right after the emergence of Magnetococcales. Similar analyses with the SUF machinery place primary plastids as sister to Gloeomargarita within Cyanobacteria. Our results add to the growing evidence of an early emergence of primary organelles and show that the analysis of essential machineries of endosymbiotic origin provide a robust signal to resolve ancient and fundamental steps in eukaryotic evolution.

Essential mitochondrial role in iron-sulfur cluster assembly of the cytoplasmic isopropylmalate isomerase Leu1 in Saccharomyces cerevisiae

Iron-sulfur (Fe-S) cluster assembly in mitochondria and cytoplasm is essential for cell viability. In the yeast S. cerevisiae, Leu1 [4Fe-4S] is the cytoplasmic isopropylmalate isomerase involved in leucine biosynthesis. Using permeabilized Δleu1 cells and recombinant apo-Leu1R, here we show that the [4Fe-4S] cluster assembly on Leu1R can be reconstituted in a physiologic manner requiring both mitochondria and cytoplasm, as judged by conversion of the inactive enzyme to an active form. The mitochondrial contribution to this reconstitution assay is abrogated by inactivating mutations in the mitochondrial ISC (iron-sulfur cluster assembly) machinery components (such as Nfs1 cysteine desulfurase and Ssq1 chaperone) or the mitochondrial exporter Atm1. Likewise, depletion of a CIA (cytoplasmic iron-sulfur protein assembly) component Dre2 leads to impaired Leu1R reconstitution. Mitochondria likely make and export an intermediate, called X-S or (Fe-S)int, that is needed for cytoplasmic Fe-S cluster biosynthesis. Here we show that once exported, the same intermediate can be used for both [2Fe-2S] and [4Fe-4S] cluster biogenesis in the cytoplasm, with no further requirement of mitochondria. Our data also suggest that the exported intermediate can activate defective/latent CIA components in cytoplasm isolated from nfs1 or Δatm1 mutant cells. These findings may provide a way to isolate X-S or (Fe-S)int.

Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins

The importance of mitochondria in mammalian cells is widely known. Several biochemical reactions and pathways take place within mitochondria: among them, there are those involving the biogenesis of the iron–sulfur (Fe-S) clusters. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a variety of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation. The synthesis and distribution of Fe-S clusters are strictly controlled cellular processes that involve several mitochondrial proteins that specifically interact each other to form a complex machinery (Iron Sulfur Cluster assembly machinery, ISC machinery hereafter). This machinery ensures the correct assembly of both [2Fe-2S] and [4Fe-4S] clusters and their insertion in the mitochondrial target proteins. The present review provides a structural and molecular overview of the rare diseases associated with the genes encoding for the accessory proteins of the ISC machinery (i.e., GLRX5, ISCA1, ISCA2, IBA57, FDX2, BOLA3, IND1 and NFU1) involved in the assembly and insertion of [4Fe-4S] clusters in mitochondrial proteins. The disease-related missense mutations were mapped on the 3D structures of these accessory proteins or of their protein complexes, and the possible impact that these mutations have on their specific activity/function in the frame of the mitochondrial [4Fe-4S] protein biogenesis is described.

Protein-Interaction Affinity Gradient Drives [4Fe-4S] Cluster Insertion in Human Lipoyl Synthase.

Human lipoyl synthase (LIAS) is an enzyme containing two [4Fe–4S] clusters (named FeSRS and FeSaux) involved in the biosynthesis of the lipoyl cofactor. The mechanism by which a [4Fe–4S] cluster is inserted into LIAS has thus far remained elusive. Here we show that NFU1 and ISCA1 of the mitochondrial iron–sulfur cluster assembly machinery, via forming a heterodimeric complex, are the key factors for the insertion of a [4Fe–4S] cluster into the FeSRS site of LIAS. In this process, the crucial actor is the C-domain of NFU1, which, by exploiting a protein-interaction affinity gradient increasing from ISCA1 to LIAS, drives the cluster to its final destination.

Sulfur Administration in Fe-S Cluster Homeostasis.

Mitochondria are the key organelles of Fe–S cluster synthesis. They contain the enzyme cysteine desulfurase, a scaffold protein, iron and electron donors, and specific chaperons all required for the formation of Fe–S clusters. The newly formed cluster can be utilized by mitochondrial Fe–S protein synthesis or undergo further transformation. Mitochondrial Fe–S cluster biogenesis components are required in the cytosolic iron–sulfur cluster assembly machinery for cytosolic and nuclear cluster supplies. Clusters that are the key components of Fe–S proteins are vulnerable and prone to degradation whenever exposed to oxidative stress. However, once degraded, the Fe–S cluster can be resynthesized or repaired. It has been proposed that sulfurtransferases, rhodanese, and 3-mercaptopyruvate sulfurtransferase, responsible for sulfur transfer from donor to nucleophilic acceptor, are involved in the Fe–S cluster formation, maturation, or reconstitution. In the present paper, we attempt to sum up our knowledge on the involvement of sulfurtransferases not only in sulfur administration but also in the Fe–S cluster formation in mammals and yeasts, and on reconstitution-damaged cluster or restoration of enzyme’s attenuated activity.

The iron-sulfur scaffold protein HCF101 unveils the complexity of organellar evolution in SAR, Haptista and Cryptista

BackgroundNbp35-like proteins (Nbp35, Cfd1, HCF101, Ind1, and AbpC) are P-loop NTPases that serve as components of iron-sulfur cluster (FeS) assembly machineries. In eukaryotes, Ind1 is present in mitochondria, and its function is associated with the assembly of FeS clusters in subunits of respiratory Complex I, Nbp35 and Cfd1 are the components of the cytosolic FeS assembly (CIA) pathway, and HCF101 is involved in FeS assembly of photosystem I in plastids of plants (chHCF101). The AbpC protein operates in Bacteria and Archaea. To date, the cellular distribution of these proteins is considered to be highly conserved with only a few exceptions.ResultsWe searched for the genes of all members of the Nbp35-like protein family and analyzed their targeting sequences. Nbp35 and Cfd1 were predicted to reside in the cytoplasm with some exceptions of Nbp35 localization to the mitochondria; Ind1was found in the mitochondria, and HCF101 was predicted to reside in plastids (chHCF101) of all photosynthetically active eukaryotes. Surprisingly, we found a second HCF101 paralog in all members of Cryptista, Haptista, and SAR that was predicted to predominantly target mitochondria (mHCF101), whereas Ind1 appeared to be absent in these organisms. We also identified a few exceptions, as apicomplexans possess mHCF101 predicted to localize in the cytosol and Nbp35 in the mitochondria. Our predictions were experimentally confirmed in selected representatives of Apicomplexa (Toxoplasma gondii), Stramenopila (Phaeodactylum tricornutum, Thalassiosira pseudonana), and Ciliophora (Tetrahymena thermophila) by tagging proteins with a transgenic reporter. Phylogenetic analysis suggested that chHCF101 and mHCF101 evolved from a common ancestral HCF101 independently of the Nbp35/Cfd1 and Ind1 proteins. Interestingly, phylogenetic analysis supports rather a lateral gene transfer of ancestral HCF101 from bacteria than its acquisition being associated with either α-proteobacterial or cyanobacterial endosymbionts.ConclusionOur searches for Nbp35-like proteins across eukaryotic lineages revealed that SAR, Haptista, and Cryptista possess mitochondrial HCF101. Because plastid localization of HCF101 was only known thus far, the discovery of its mitochondrial paralog explains confusion regarding the presence of HCF101 in organisms that possibly lost secondary plastids (e.g., ciliates, Cryptosporidium) or possess reduced nonphotosynthetic plastids (apicomplexans).

A novel role of the mitochondrial iron-sulfur cluster assembly protein ISCU-1/ISCU in longevity and stress response.

As an ancient cellular co-factor ubiquitously present in all domains of life, nearly all iron-sulfur ([Fe-S]) clusters are assembled in the mitochondrion. Although multiple mitochondrion-derived signalings are known to be key players in longevity regulation, whether the mitochondrial [Fe-S] cluster assembly machinery modulates lifespan is previously unknown. Here, we find that ISCU-1, the C. elegans ortholog of the evolutionarily conserved iron-sulfur cluster (ISC) assembly machinery central protein ISCU, regulates longevity and stress response. Specifically, ISCU-1 accelerates aging in the intestine. Moreover, we identify the Nrf2 transcription factor SKN-1 and a nuclear hormone receptor NHR-49 as the downstream factors of ISCU-1. Lastly, a mitochondrial outer membrane protein phosphatase PGAM-5 appears to link ISCU-1 to SKN-1 and NHR-49 in lifespan regulation. Together, we have identified a novel function of mitochondrial ISC assembly machinery in longevity modulation and stress response.

A Highly Conserved Iron-Sulfur Cluster Assembly Machinery between Humans and Amoeba Dictyostelium discoideum: The Characterization of Frataxin.

Several biological activities depend on iron–sulfur clusters ([Fe-S]). Even though they are well-known in several organisms their function and metabolic pathway were poorly understood in the majority of the organisms. We propose to use the amoeba Dictyostelium discoideum, as a biological model to study the biosynthesis of [Fe-S] at the molecular, cellular and organism levels. First, we have explored the D. discoideum genome looking for genes corresponding to the subunits that constitute the molecular machinery for Fe-S cluster assembly and, based on the structure of the mammalian supercomplex and amino acid conservation profiles, we inferred the full functionality of the amoeba machinery. After that, we expressed the recombinant mature form of D. discoideum frataxin protein (DdFXN), the kinetic activator of this pathway. We characterized the protein and its conformational stability. DdFXN is monomeric and compact. The analysis of the secondary structure content, calculated using the far-UV CD spectra, was compatible with the data expected for the FXN fold, and near-UV CD spectra were compatible with the data corresponding to a folded protein. In addition, Tryptophan fluorescence indicated that the emission occurs from an apolar environment. However, the conformation of DdFXN is significantly less stable than that of the human FXN, (4.0 vs. 9.0 kcal mol−1, respectively). Based on a sequence analysis and structural models of DdFXN, we investigated key residues involved in the interaction of DdFXN with the supercomplex and the effect of point mutations on the energetics of the DdFXN tertiary structure. More than 10 residues involved in Friedreich’s Ataxia are conserved between the human and DdFXN forms, and a good correlation between mutational effect on the energetics of both proteins were found, suggesting the existence of similar sequence/function/stability relationships. Finally, we integrated this information in an evolutionary context which highlights particular variation patterns between amoeba and humans that may reflect a functional importance of specific protein positions. Moreover, the complete pathway obtained forms a piece of evidence in favor of the hypothesis of a shared and highly conserved [Fe-S] assembly machinery between Human and D. discoideum.

CIAO3 protein forms a stable ternary complex with two key players of the human cytosolic iron-sulfur cluster assembly machinery.

The CIAO3 protein operates at a crossroad of the cytosolic iron-sulfur protein assembly (CIA) machinery. Although the functional role of CIAO3 has been recently characterized, a description of its interaction network is still not complete. Literature data suggested that CIAO3 interacts individually with CIA2A and CIAO1 protein, with the latter two interacting each other. However, no experimental data are available yet showing the formation of a possible ternary complex composed by CIAO3, CIAO1, and CIA2A. This work shows, for the first time, via size exclusion chromatography coupled with multiangle light scattering, UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopies, the formation of a stable, [4Fe-4S]-bound, complex, composed by CIAO3 and the hetero-CIA2A-CIAO1 complex. Moreover, site-directed mutagenesis data suggested a structural role for the C-terminal [4Fe-4S] cluster of the CIAO3 protein. These findings can provide solid bases for further investigation of the molecular mechanisms involving these CIA machinery proteins.

Mechanisms of Mitochondrial Iron-Sulfur Protein Biogenesis.

Mitochondria are essential in most eukaryotes and are involved in numerous biological functions including ATP production, cofactor biosyntheses, apoptosis, lipid synthesis, and steroid metabolism. Work over the past two decades has uncovered the biogenesis of cellular iron-sulfur (Fe/S) proteins as the essential and minimal function of mitochondria. This process is catalyzed by the bacteria-derived iron-sulfur cluster assembly (ISC) machinery and has been dissected into three major steps: de novo synthesis of a [2Fe-2S] cluster on a scaffold protein; Hsp70 chaperone-mediated trafficking of the cluster and insertion into [2Fe-2S] target apoproteins; and catalytic conversion of the [2Fe-2S] into a [4Fe-4S] cluster and subsequent insertion into recipient apoproteins. ISC components of the first two steps are also required for biogenesis of numerous essential cytosolic and nuclear Fe/S proteins, explaining the essentiality of mitochondria. This review summarizes the molecular mechanisms underlying the ISC protein-mediated maturation of mitochondrial Fe/S proteins and the importance for human disease.

A pathway for assembling [4Fe-4S]2+ clusters in mitochondrial iron-sulfur protein biogenesis.

During its late steps, the mitochondrial iron-sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe-4S] clusters. Invivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe-4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late-acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe-4S]2+ cluster on NFU1. We showed that the [2Fe-2S]2+ GLRX5-BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe-4S]2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe-2S]2+ GLRX5, and thus, the [4Fe-4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an 'assembler' of [4Fe-4S] clusters, that is, a protein able of converting two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. Finally, we found that the [4Fe-4S]2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur-containing ligands, as is typically observed in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster-exchange reaction.

Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly machinery

In mitochondria, a complex protein machinery is devoted to the maturation of iron-sulfur cluster proteins. Structural information on the last steps of the machinery, which involve ISCA1, ISCA2 and IBA57 proteins, needs to be acquired in order to define how these proteins cooperate each other. We report here the use of an integrative approach, utilizing information from small-angle X-ray scattering (SAXS) and bioinformatics-driven docking prediction, to determine a low-resolution structural model of the human mitochondrial [2Fe-2S]2+ ISCA2-IBA57 complex. In the applied experimental conditions, all the data converge to a structural organization of dimer of dimers for the [2Fe-2S]2+ ISCA2-IBA57 complex with ISCA2 providing the homodimerization core interface. The [2Fe-2S] cluster is out of the ISCA2 core while being shared with IBA57 in the dimer. The specific interaction pattern identified from the dimeric [2Fe-2S]2+ ISCA2-IBA57 structural model allowed us to define the molecular grounds of the pathogenic Arg146Trp mutation of IBA57. This finding suggests that the dimeric [2Fe-2S] ISCA2-IBA57 hetero-complex is a physiologically relevant species playing a role in mitochondrial [4Fe-4S] protein biogenesis.

In-Depth Quantitative Proteomic Analysis of Trophozoites and Pseudocysts of Trichomonas vaginalis.

Trichomonas vaginalis is a sexually transmitted anaerobic parasite that infects humans causing trichomoniasis, a common and ubiquitous sexually transmitted disease. The life cycle of this parasite possesses a trophozoite form without a cystic stage. However, the presence of nonproliferative and nonmotile, yet viable and reversible spherical forms with internalized flagella, denominated pseudocysts, has been commonly observed for this parasite. To understand the mechanisms involved in the formation of pseudocysts, we performed a mass spectrometry-based high-throughput quantitative proteomics study using a label-free approach and functional assays by biochemical and flow cytometric methods. We observed that the morphological transformation of trophozoite to pseudocysts is coupled to (i) a metabolic shift toward a less glycolytic phenotype; (ii) alterations in the abundance of hydrogenosomal iron-sulfur cluster (ISC) assembly machinery; (iii) increased abundance of regulatory particles of the ubiquitin-proteasome system; (iv) significant alterations in proteins involved in adhesion and cytoskeleton reorganization; and (v) arrest in G2/M phase associated with alterations in the abundance of regulatory proteins of the cell cycle. These data demonstrate that pseudocysts experience important physiological and structural alterations for survival under unfavorable environmental conditions.

Respiratory chain supercomplexes associate with the cysteine desulfurase complex of the iron-sulfur cluster assembly machinery.

Mitochondria are the powerhouses of eukaryotic cells. The activity of the respiratory chain complexes generates a proton gradient across the inner membrane, which is used by the F1FO-ATP synthase to produce ATP for cellular metabolism. In baker's yeast, Saccharomyces cerevisiae, the cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) associate in respiratory chain supercomplexes. Iron-sulfur clusters (ISC) form reactive centers of respiratory chain complexes. The assembly of ISC occurs in the mitochondrial matrix and is essential for cell viability. The cysteine desulfurase Nfs1 provides sulfur for ISC assembly and forms with partner proteins the ISC-biogenesis desulfurase complex (ISD complex). Here, we report an unexpected interaction of the active ISD complex with the cytochrome bc1 complex and cytochrome c oxidase. The individual deletion of complex III or complex IV blocks the association of the ISD complex with respiratory chain components. We conclude that the ISD complex binds selectively to respiratory chain supercomplexes. We propose that this molecular link contributes to coordination of iron-sulfur cluster formation with respiratory activity.

The Molecular Bases of the Dual Regulation of Bacterial Iron Sulfur Cluster Biogenesis by CyaY and IscX.

IscX (or YfhJ) is a protein of unknown function which takes part in the iron-sulfur cluster assembly machinery, a highly specialized and essential metabolic pathway. IscX binds to iron with low affinity and interacts with IscS, the desulfurase central to cluster assembly. Previous studies have suggested a competition between IscX and CyaY, the bacterial ortholog of frataxin, for the same binding surface of IscS. This competition could suggest a link between the two proteins with a functional significance. Using a hybrid approach based on nuclear magnetic resonance, small angle scattering and biochemical methods, we show here that IscX is a modulator of the inhibitory properties of CyaY: by competing for the same site on IscS, the presence of IscX rescues the rates of enzymatic cluster formation which are inhibited by CyaY. The effect is stronger at low iron concentrations, whereas it becomes negligible at high iron concentrations. These results strongly suggest the mechanism of the dual regulation of iron sulfur cluster assembly under the control of iron as the effector.

Biochemical Reconstitution and Spectroscopic Analysis of Iron-Sulfur Proteins.

Iron-sulfur (Fe/S) proteins are involved in numerous key biological functions such as respiration, metabolic processes, protein translation, DNA synthesis, and DNA repair. The simplest types of Fe/S clusters include [2Fe-2S], [3Fe-4S], and [4Fe-4S] forms that sometimes are present in multiple copies. De novo assembly of Fe/S cofactors and their insertion into apoproteins in living cells requires complex proteinaceous machineries that are frequently highly conserved. In eukaryotes such as yeast and mammals, the mitochondrial iron-sulfur cluster assembly machinery and the cytosolic iron-sulfur protein assembly system consist of more than 30 components that cooperate in the generation of some 50 cellular Fe/S proteins. Both the mechanistic dissection of the intracellular Fe/S protein assembly pathways and the identification and characterization of Fe/S proteins rely on tool boxes of in vitro and in vivo methods. These cell biological, biochemical, and biophysical techniques help to determine the extent, stability, and type of bound Fe/S cluster. They also serve to distinguish bona fide Fe/S proteins from other metal-binding proteins containing similar cofactor coordination motifs. Here, we present a collection of in vitro methods that have proven useful for basic biochemical and biophysical characterization of Fe/S proteins. First, we describe the chemical assembly of [2Fe-2S] or [4Fe-4S] clusters on purified apoproteins. Then, we summarize a reconstitution system reproducing the de novo synthesis of a [2Fe-2S] cluster in mitochondria. Finally, we explain the use of UV-vis, CD, electron paramagnetic resonance, and Mössbauer spectroscopy for the routine characterization of Fe/S proteins.

Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe-2S] cluster optical spectra and transfer chemistry.

Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions todisrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.

4Fe-4S] Cluster Assembly in Mitochondria and Its Impairment by Copper.

The cellular toxicity of copper is usually associated with its ability to generate reactive oxygen species. However, recent studies in bacterial organisms showed that copper toxicity is also strictly connected to iron-sulfur cluster proteins and to their assembly processes. Mitochondria of eukaryotic cells contain a labile copper(I) pool localized in the matrix where also the mitochondrial iron-sulfur (Fe/S) cluster assembly machinery resides to mature mitochondrial Fe/S cluster-containing proteins. Misregulation of copper homeostasis might therefore damage mitochondrial Fe/S protein maturation. To describe, from a molecular perspective, the effects of copper(I) toxicity on such a maturation process, we have here investigated the still unknown mechanism of [4Fe-4S] cluster formation conducted by the mitochondrial ISCA1/ISCA2 and GLRX5 proteins, and defined how copper(I) can impair this process. The molecular model here proposed indicates that the copper(I) and Fe/S protein maturation cellular pathways need to be strictly regulated to avoid copper(I) ion from blocking mitochondrial [4Fe-4S] protein maturation.

The Function of the Iron–Sulfur Cluster Assembly Machinery is involved in Electron Transport Chain Activity and Oxidative Stress in Saccharomyces cerevisiae

For their function, many proteins depend on cofactor binding to specific sequence motifs. Fe‐S clusters are conserved and versatile inorganic cofactors that participate in a variety of processes and functions. In eukaryotes, biogenesis and recycling of Fe‐S clusters play important roles in the mechanisms of iron homeostasis involved in mitochondrial function. These Fe‐S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The present study aimed to determine the effects of mutations in the ISC genes on mitochondrial functionality and iron homeostasis under oxidative stress in S. cerevisiae BY4741 and its KanMX4 interruption ISC genes mutants: ssq1Δ, grx5Δ and isa1Δ. Intracellular ROS, Fe2+ and mitochondrial membrane potential in yeast cultures were determined using oxidant‐sensitive, cell‐permeant fluorescent probes (DHE, PGFL and Rho123). Fluorescence was quantified by flow cytometry, viewed and colocalized by confocal microscopy. Mitochondria were isolated and permeabilized for spectrophotometric determination of respiratory complexes activities. Oxygen consumption rate (OCR) was measured with a Clark‐type oxygen electrode coupled to a biological oxygen monitor. Electron transport chain (ETC) supercomplexes were identified by BN‐PAGE and Raman spectroscopy was performed to detect mitochondrial Fe‐S content. The results indicate that ROS generation caused by oxidizing agents like menadione is increased by ISC system dysfunction, due to loss of iron homeostasis (i.e. augmented free Fe2+), suggesting that ROS and Fe2+ from Fe‐S cluster proteins react creating a strong oxidative status in mitochondria. This was associated to an impairment on the activity of the complexes II, III and IV from the ETC in the ISC mutants. Indeed, increased oxygen generation instead consumption was observed in SSQ1 and ISA1 mutants suggesting a massive generation of ROS and its detoxification by superoxide dismutase and catalase. In mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased along with III2IV2 respiratory supercomplex formation, but not in the iron‐deficient ATX1 (cytosolic copper metallochaperone) and MRS4 (mitochondrial iron transporter) mutants, used as controls. In conclusion these results indicate that the ISC system is important in iron‐homeostasis, ROS stress and in the assembly of the supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.