ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1.
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ABSTRACT:
SUBMITTER: Cai K
PROVIDER: S-EPMC7261492 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]2+ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]2+ clusters, two [2Fe-2S]2+ clusters are converted into a [4Fe-4S]2+ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]2+ cluster from ISCA1-ISCA2 complex. A structural view of the protein-protein recognition events occurring along the [4Fe-4S]2+ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]2+ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]2+ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]2+ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]2+ cluster transfer.
| S-EPMC10388178 | biostudies-literature
Project description:Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N- and C-terminal domains of human NFU1 by nuclear magnetic resonance spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermolecular disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-containing form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer. Holo-NFU1 consists of a complex of three of these dimers as shown by molecular weight estimates from SAXS and size-exclusion chromatography. The SAXS-derived structural model indicates that one N-terminal region from each of the three dimers forms a tripartite interface. The activity of the holo-NFU1 preparation was verified by demonstrating its ability to activate apo-aconitase.
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Project description:Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe(2+)) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen- or iron-sensitive coordination of the Fe-S cluster.
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Project description:NFU1, a late-acting iron-sulfur (Fe-S) cluster carrier protein, has a key role in the pathogenesis of the disease, multiple mitochondrial dysfunctions syndrome. In this work, using genetic and biochemical approaches, we identified the initial scaffold protein, mitochondrial ISCU (ISCU2) and the secondary carrier, ISCA1, as the direct donors of Fe-S clusters to mitochondrial NFU1, which appears to dimerize and reductively mediate the formation of a bridging [4Fe-4S] cluster, aided by ferredoxin 2. By monitoring the abundance of target proteins that acquire their Fe-S clusters from NFU1, we characterized the effects of several novel pathogenic NFU1 mutations. We observed that NFU1 directly interacts with each of the Fe-S cluster scaffold proteins known to ligate [2Fe-2S] clusters, ISCU2 and ISCA1, and we mapped the site of interaction to a conserved hydrophobic patch of residues situated at the end of the C-terminal alpha-helix of NFU1. Furthermore, we showed that NFU1 lost its ability to acquire its Fe-S cluster when mutagenized at the identified site of interaction with ISCU2 and ISCA1, which thereby adversely affected biochemical functions of proteins that are thought to acquire their Fe-S clusters directly from NFU1, such as lipoic acid synthase, which supports the Fe-S-dependent process of lipoylation of components of multiple key enzyme complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and the glycine cleavage complex.
| S-EPMC7689295 | biostudies-literature
Project description:The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtb's physiology are not fully understood. Since NO damages iron-sulfur (Fe-S) clusters of essential enzymes, the mechanism(s) involved in regulating Fe-S cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA-sequencing of MtbΔsufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of MtbΔsufR. Lastly, MtbΔsufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics.
| S-EPMC8371249 | biostudies-literature
Project description:Proteins incorporating iron-sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.
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Project description:Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster.
| S-EPMC6380689 | biostudies-literature
Project description:Proteins incorporating iron-sulfur (Fe-S) cofactors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries located in the cytosol, mitochondria and chloroplasts. After de novo formation on scaffold proteins, Fe-S centers are loaded on client proteins by use of Fe-S cluster transfer proteins. Among plastidial representatives of this latter category, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters found in photosystem I subunits acting upstream of HCF101. NFU2 is additionally required for the maturation of the [2Fe-2S] dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 is able to solely assemble a [4Fe-4S] cluster per homodimer. We also provide insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins by performing co-immunoprecipitation experiments and assaying the physical interaction of NFU1 with many [4Fe-4S]-containing plastidial proteins using binary yeast two-hybrid assays. Interactions with two proteins involved in isoprenoid and thiamine biosynthesis, respectively 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (ISPG) and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THIC), have been further confirmed by bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments. The additional interactions detected with SUFD and SUFA allowed building a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves for the maturation of [4Fe-4S] client proteins.
2020-07-06 | PXD015295 | Pride
Project description:The all-ferrous, carbene-capped Fe(4)S(4) cluster, synthesized by Deng and Holm (DH complex), has been studied with density functional theory (DFT). The geometry of the complex was optimized for several electronic configurations. The lowest energy was obtained for the broken-symmetry (BS) configuration derived from the ferromagnetic state by reversing the spin projection of one of the high spin (S(i) = 2) irons. The optimized geometry of the latter configuration contains one unique and three equivalent iron sites, which are both structurally and electronically clearly distinguishable. For example, a distinctive feature of the unique iron site is the diagonal Fe···S distance, which is 0.3 Å longer than for the equivalent irons. The calculated (57)Fe hyperfine parameters show the same 1:3 pattern as observed in the Mössbauer spectra and are in good agreement with experiment. BS analysis of the exchange interactions in the optimized geometry for the 1:3, M(S) = 4, BS configuration confirms the prediction of an earlier study that the unique site is coupled to the three equivalent ones by strong antiferromagnetic exchange (J > 0 in J ?(j<4)?(4)·?(j)) and that the latter are mutually coupled by ferromagnetic exchange (J' < 0 in J' ?(i<j<4)?(i)·?(j)). In combination, these exchange couplings stabilize an S = 4 ground state in which the composite spin of the three equivalent sites (S(123) = 6) is antiparallel to the spin (S(4) = 2) of the unique site. Thus, DFT analysis supports the idea that the unprecedented high value of the spin of the DH complex and, by analogy, of the all-ferrous cluster of the Fe-protein of nitrogenase, results from a remarkably strong dependence of the exchange interactions on cluster core geometry. The structure dependence of the exchange-coupling constants in the Fe(II)-(?(3)-S)(2)-Fe(II) moieties of the all-ferrous clusters is compared with the magneto-structural correlations observed in the data for dinuclear copper complexes. Finally, we discuss two all-ferric clusters in the light of the results for the all-ferrous cluster.
| S-EPMC3107574 | biostudies-literature
Project description:Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins.
| S-EPMC2805927 | biostudies-literature