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Mitochondria harbor the bacteria-inherited iron-sulfur cluster assembly (ISC) machinery to generate [2Fe-2S; iron-sulfur (Fe-S)] and [4Fe-4S] proteins. In yeast, assembly of [4Fe-4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe-4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe-4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe-4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe-4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins.

Biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is an essential process involving the mitochondrial iron-sulfur cluster (ISC) assembly and export machineries and the cytosolic iron/sulfur protein assembly (CIA) apparatus. To define the integration of Fe/S protein biogenesis into cellular homeostasis, we compared the global transcriptional responses to defects in the three biogenesis systems in Saccharomyces cerevisiae using DNA microarrays. Depletion of a member of the CIA machinery elicited only weak (up to 2-fold) alterations in gene expression with no clear preference for any specific cellular process. In contrast, depletion of components of the mitochondrial ISC assembly and export systems induced strong and largely overlapping transcriptional responses of more than 200 genes (2-100-fold changes). These alterations were strikingly similar, yet not identical, to the transcriptional profiles developed upon iron starvation. Hence, mitochondria and their ISC systems serve as primary physiological regulators exerting a global control of numerous iron-dependent processes. First, ISC depletion activates the iron-responsive transcription factors Aft1/2p leading to increased cellular iron acquisition. Second, respiration and heme metabolism are repressed ensuring the balanced utilization of iron by the two major iron-consuming processes, iron-sulfur protein and heme biosynthesis. Third, the decreased respiratory activity is compensated by induction of genes involved in glucose acquisition. Finally, transcriptional remodeling of the citric acid cycle and the biosyntheses of ergosterol and biotin reflect the iron dependence of these pathways. Together, our data suggest a model in which mitochondria perform a global regulatory role in numerous cellular processes linked to iron homeostasis.

The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.

Two transcriptional activators, Aft1 and Aft2, regulate iron homeostasis in Saccharomyces cerevisiae. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 is abrogated in cells defective for Fe-S cluster biogenesis within the mitochondrial matrix (Chen, O. S., Crisp, R. J., Valachovic, M., Bard, M., Winge, D. R., and Kaplan, J. (2004) J. Biol. Chem. 279, 29513-29518). To determine whether iron sensing by Aft1/Aft2 requires the function of the mitochondrial Fe-S export and cytosolic Fe-S protein assembly systems, we evaluated the expression of the iron regulon in cells depleted of glutathione and in cells depleted of Atm1, Nar1, Cfd1, and Nbp35. The iron regulon is induced in cells depleted of Atm1 with Aft1 largely responsible for the induced gene expression. Aft2 is activated at a later time in Atm1-depleted cells. Likewise, the iron regulon is induced in cells depleted of glutathione. In contrast, repression of NAR1, CFD1, or NBP35 fails to induce the iron regulon despite strong inhibition of cytosolic/nuclear Fe-S protein assembly. Thus, iron sensing by Aft1/Aft2 is not linked to the maturation of cytosolic/nuclear Fe-S proteins, but the mitochondrial inner membrane transporter Atm1 is important to transport the inhibitory signal. Although Aft1 and Aft2 sense a signal emanating from the Fe-S cluster biogenesis pathway, there is no indication that the proteins are inhibited by direct binding of an Fe-S cluster.

Previous studies have indicated that the essential protein Nfs1 performs a crucial role in cellular iron-sulfur (Fe/S) protein maturation. The protein is located predominantly in mitochondria, yet low amounts are present in cytosol and nucleus. Here we examined several aspects concerning the molecular function of yeast Nfs1p as a model protein. First, we demonstrated that purified Nfs1p facilitates the in vitro assembly of Fe/S proteins by using cysteine as its specific substrate. Thus, eukaryotic Nfs1 is a functional orthologue of the bacterial cysteine desulfurase IscS. Second, we showed that only the mitochondrial version but not the extramitochondrial version of Nfs1p is functional in generating cytosolic and nuclear Fe/S proteins. Mutation of the nuclear targeting signal of Nfs1p did not affect the maturation of cytosolic and nuclear Fe/S proteins, despite a severe growth defect under this condition. Nfs1p could not assemble an Fe/S cluster on the Isu scaffold proteins when they were located in the yeast cytosol. The lack of function of these central Fe/S cluster assembly components suggests that the maturation of extramitochondrial Fe/S protein does not involve functional copies of the mitochondrial Fe/S cluster assembly machinery in the yeast cytosol. Third, the extramitochondrial version of Nfs1p was shown to play a direct role in the thiomodification of tRNAs. Finally, we identified a highly conserved N-terminal beta-sheet of Nfs1p as a functionally essential part of the protein. The implication of these findings for the structural stability of Nfs1p and for its targeting mechanism to mitochondria and cytosol/nucleus will be discussed.

Mitochondria are responsible for the synthesis of both iron-sulfur clusters and heme, but the potential connection between the two major iron-consuming pathways is unknown. Here, we have shown that mutants in the yeast mitochondrial iron-sulfur cluster (ISC) assembly machinery displayed reduced cytochrome levels and diminished activity of the heme-containing cytochrome c oxidase, in addition to iron-sulfur protein defects. In contrast, mutants in components of the mitochondrial ISC export machinery, which are specifically required for maturation of cytosolic iron-sulfur proteins, were not decreased in heme synthesis or cytochrome levels. Heme synthesis does not involve the function of mitochondrial ISC components, because immunological depletion of various ISC proteins from mitochondrial extracts did not affect the formation and amounts of heme. The heme synthesis defects of ISC mutants were found in vivo in isolated mitochondria and in mitochondrial detergent extracts and were confined to an inhibition of ferrochelatase, the enzyme catalyzing the insertion of iron into protoporphyrin IX. In support of these findings, immunopurification of ferrochelatase from ISC mutants restored its activity to wild-type levels. We conclude that the reversible inhibition of ferrochelatase is the molecular reason for the heme deficiency in ISC assembly mutants. This inhibitory mechanism may be used for regulation of iron distribution between the two iron-consuming processes.

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.

To study the biochemical requirements for maturation of iron-sulfur (Fe/S) proteins, we have reconstituted the process in vitro using detergent extracts from Saccharomyces cerevisiae mitochondria. Efficient assembly of biotin synthase as a model Fe/S protein required anaerobic conditions, dithiothreitol, cysteine, ATP, and NADH. Cysteine is utilized by the cysteine desulfurase Nfs1p to release sulfan sulfur; ATP presumably reflects the function of the Hsp70 family chaperone Ssq1p; and NADH is used for reduction of the ferredoxin Yah1p involved in Fe/S protein biogenesis. Hence, our assay system faithfully reproduces the in vivo pathway. We have further investigated the involvement of various mitochondrial proteins suspected to participate in Fe/S protein biogenesis. In mitochondrial extracts depleted in Isa1p, Fe/S protein formation was severely decreased. A similar strong decline was observed with extracts from Delta yfh1 mitochondria, indicating that both Isa1p and the yeast frataxin homologue, Yfh1p, are crucial for biogenesis of mitochondrial Fe/S proteins. Conversely, the activities of mitochondrial extracts from Delta nfu1 cells were only moderately reduced, suggesting a dispensable role for Nfu1p. Finally, iron utilized for Fe/S protein formation was imported into the matrix of intact mitochondria in ferrous form in a membrane potential-dependent transport step. Our results represent the first in vitro reconstitution of the entire pathway of Fe/S protein maturation.

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

A strain of the cyanobacterium Synechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene. The transformants express a new PsaF subunit similar in size to PsaF from C. reinhardtii that is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700(+) reduction by C. reinhardtii cytochrome c6 or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI. The corresponding optimum second-order rate constants (k2 = 4.0 and 1.7 x 10(7) M1 s1 for cytochrome c6 and plastocyanin) are similar to those of PSI from C. reinhardtii. The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI from S. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c6, a fast first-order kinetic component (t(1)/(2) = 4 microseconds) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochrome c6. This first-order phase is characteristic for P700(+) reduction by cytochrome c6 or plastocyanin in algae and higher plants. However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild type S. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c6 or plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c6 or plastocyanin.