Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.

Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5\'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the β-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the two ACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.

In oxygenic photosynthetic systems, the cytochrome b6f (Cytb6f) complex (plastoquinol:plastocyanin oxidoreductase) is a heart of the hub that provides connectivity between photosystems (PS) II and I. In this review, the structure and function of the Cytb6f complex are briefly outlined, being focused on the mechanisms of a bifurcated (two-electron) oxidation of plastoquinol (PQH2). In plant chloroplasts, under a wide range of experimental conditions (pH and temperature), a diffusion of PQH2 from PSII to the Cytb6f does not limit the intersystem electron transport. The overall rate of PQH2 turnover is determined mainly by the first step of the bifurcated oxidation of PQH2 at the catalytic site Qo, i.e., the reaction of electron transfer from PQH2 to the Fe2S2 cluster of the high-potential Rieske iron-sulfur protein (ISP). This point has been supported by the quantum chemical analysis of PQH2 oxidation within the framework of a model system including the Fe2S2 cluster of the ISP and surrounding amino acids, the low-potential heme b6L, Glu78 and 2,3,5-trimethylbenzoquinol (the tail-less analog of PQH2). Other structure-function relationships and mechanisms of electron transport regulation of oxygenic photosynthesis associated with the Cytb6f complex are briefly outlined: pH-dependent control of the intersystem electron transport and the regulatory balance between the operation of linear and cyclic electron transfer chains.

Iron-sulfur (Fe-S) clusters are ancient protein cofactors ubiquitously exist in organisms. They are involved in many important life processes. Plastids are semi-autonomous organelles with a double membrane and it is believed to originate from a cyanobacterial endosymbiont. By learning form the research in cyanobacteria, a Fe-S cluster biosynthesis and delivery pathway has been proposed and partly demonstrated in plastids, including iron uptake, sulfur mobilization, Fe-S cluster assembly and delivery. Fe-S clusters are essential for the downstream Fe-S proteins to perform their normal biological functions. Because of the importance of Fe-S proteins in plastid, researchers have made a lot of research progress on this pathway in recent years. This review summarizes the detail research progress made in recent years. In addition, the scientific problems remained in this pathway are also discussed.

To ensure proper utilization of iron and avoid its toxicity, cells are equipped with iron-sensing proteins to maintain cellular iron homeostasis. We showed previously that nuclear receptor coactivator 4 (NCOA4), a ferritin-specific autophagy adapter, intricately regulates the fate of ferritin; upon binding to Fe3+, NCOA4 forms insoluble condensates and regulates ferritin autophagy in iron-replete conditions. Here, we demonstrate an additional iron-sensing mechanism of NCOA4. Our results indicate that the insertion of an iron-sulfur (Fe-S) cluster enables preferential recognition of NCOA4 by the HERC2 (HECT and RLD domain containing E3 ubiquitin protein ligase 2) ubiquitin ligase in iron-replete conditions, resulting in degradation by the proteasome and subsequent inhibition of ferritinophagy. We also found that both condensation and ubiquitin-mediated degradation of NCOA4 can occur in the same cell, and the cellular oxygen tension determines the selection of these pathways. Fe-S cluster-mediated degradation of NCOA4 is enhanced under hypoxia, whereas NCOA4 forms condensates and degrades ferritin at higher oxygen levels. Considering the involvement of iron in oxygen handling, our findings demonstrate that the NCOA4-ferritin axis is another layer of cellular iron regulation in response to oxygen levels.

Iron regulatory protein 1 (IRP1) is a bifunctional protein with mutually exclusive RNA-binding or enzymatic activities that depend on the presence of a 4Fe-4S cluster. While IRP1 is a well-established cytosolic protein, work in a Drosophila model suggested that it may also exhibit nuclear localization. Herein, we addressed whether mammalian IRP1 can likewise translocate to the nucleus. We utilized primary cells and tissues from wild type and Irp1-/- mice, as well as human cell lines and tissue biopsy sections. IRP1 subcellular localization was analyzed by Western blotting, immunofluorescence and immunohistochemistry. We did not detect presence of nuclear IRP1 in wild type mouse embryonic fibroblasts (MEFs), primary hepatocytes or whole mouse liver. However, we observed IRP1-positive nuclei in human liver but not ovary sections. Biochemical fractionation studies revealed presence of IRP1 in the nucleus of human Huh7 and HepG2 hepatoma cells, but not HeLa cervical cancer cells. Importantly, nuclear IRP1 was only evident in iron-replete cells and disappeared following pharmacological iron chelation. These data provide the first experimental evidence for nuclear IRP1 expression in mammals, which appears to be species- and cell-specific. Furthermore, they suggest that the nuclear translocation of IRP1 is mediated by an iron-dependent mechanism.

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.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O2-stable [2Fe-2S] cluster form converts to an O2-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction-oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]2+/1+ couple (-520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.

In mitochondria, cysteine desulfurase (Nfs1) plays a central role in the biosynthesis of iron-sulfur (FeS) clusters, cofactors critical for activity of many cellular proteins. Nfs1 functions both as a sulfur donor for cluster assembly and as a binding platform for other proteins functioning in the process. These include not only the dedicated scaffold protein (Isu1) on which FeS clusters are synthesized but also accessory FeS cluster biogenesis proteins frataxin (Yfh1) and ferredoxin (Yah1). Yfh1 has been shown to activate cysteine desulfurase enzymatic activity, whereas Yah1 supplies electrons for the persulfide reduction. While Yfh1 interaction with Nfs1 is well understood, the Yah1-Nfs1 interaction is not. Here, based on the results of biochemical experiments involving purified WT and variant proteins, we report that in Saccharomyces cerevisiae, Yah1 and Yfh1 share an evolutionary conserved interaction site on Nfs1. Consistent with this notion, Yah1 and Yfh1 can each displace the other from Nfs1 but are inefficient competitors when a variant with an altered interaction site is used. Thus, the binding mode of Yah1 and Yfh1 interacting with Nfs1 in mitochondria of S. cerevisiae resembles the mutually exclusive binding of ferredoxin and frataxin with cysteine desulfurase reported for the bacterial FeS cluster assembly system. Our findings are consistent with the generally accepted scenario that the mitochondrial FeS cluster assembly system was inherited from bacterial ancestors of mitochondria.