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Defining the mechanism of the mitochondrial Atm1p [2Fe-2S] cluster exporter.

ABSTRACT: Iron-sulfur cluster proteins play key roles in a multitude of physiological processes; including gene expression, nitrogen and oxygen sensing, electron transfer, and DNA repair. Biosynthesis of iron-sulfur clusters occurs in mitochondria on iron-sulfur cluster scaffold proteins in the form of [2Fe-2S] cores that are then transferred to apo targets within metabolic or respiratory pathways. The mechanism by which cytosolic Fe-S cluster proteins mature to their holo forms remains controversial. The mitochondrial inner membrane protein Atm1p can transport glutathione-coordinated iron-sulfur clusters, which may connect the mitochondrial and cytosolic iron-sulfur cluster assembly systems. Herein we describe experiments on the yeast Atm1p/ABCB7 exporter that provide additional support for a glutathione-complexed cluster as the natural physiological substrate and a reflection of the endosymbiotic model of mitochondrial evolution. These studies provide insight on the mechanism of cluster transport and the molecular basis of human disease conditions related to ABCB7. Recruitment of MgATP following cluster binding promotes a structural transition from closed to open conformations that is mediated by coupling helices, with MgATP hydrolysis facilitating the return to the closed state.

SUBMITTER: Pearson SA

PROVIDER: S-EPMC7369126 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Evolution of the human mitochondrial ABCB7 [2Fe-2S](GS)<sub>4</sub> cluster exporter and the molecular mechanism of an E433K disease-causing mutation.

Project description:Iron-sulfur cluster proteins play key roles in a multitude of cellular processes. Iron-sulfur cofactors are assembled primarily in mitochondria and are then exported to the cytosol by use of an ABCB7 transporter. It has been shown that the yeast mitochondrial transporter Atm1 can export glutathione-coordinated iron-sulfur clusters, [2Fe-2S](SG)4, providing a source of cluster units for cytosolic iron-sulfur cluster assembly systems. This pathway is consistent with the endosymbiotic model of mitochondrial evolution where homologous bacterial heavy metal transporters, utilizing metal glutathione adducts, were adapted for use in eukaryotic mitochondria. Herein, the basis for endosymbiotic evolution of the human cluster export protein (ABCB7) is developed through a BLAST analysis of transporters from ancient proteobacteria. In addition, a functional comparison of native human protein, versus a disease-causing mutant, demonstrates a key role for residue E433 in promoting cluster transport. Dysfunction in mitochondrial export of Fe-S clusters is a likely cause of the disease condition X-linked sideroblastic anemia.

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Glutathione-coordinated [2Fe-2S] cluster: a viable physiological substrate for mitochondrial ABCB7 transport.

Project description:The glutathione-coordinated [2Fe-2S] cluster is demonstrated to be a viable and likely substrate for physiological iron-sulfur cluster transport by Atm1p, a mitochondrial ABC export protein. Flow cytometry and colorimetric assays demonstrate a quantitative methodology for study of metal translocation proteins and their proteoliposome products.

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[2Fe-2S] cluster transfer in iron-sulfur protein biogenesis.

Project description:Monothiol glutaredoxins play a crucial role in iron-sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in solution in the presence of physiological concentrations of glutathione and forming a transient, cluster-mediated protein-protein intermediate with two physiological protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.

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Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2.

Project description:Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381NFS1 to Cys138ISCU2 and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138ISCU2 rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin's molecular role in this fundamental process.

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The Arabidopsis Mitochondrial Glutaredoxin GRXS15 Provides [2Fe-2S] Clusters for ISCA-Mediated [4Fe-4S] Cluster Maturation.

Project description:Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]2+ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]2+ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]2+ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]2+ clusters, via two-electron reductive coupling of two [2Fe-2S]2+ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]2+ to [4Fe-4S]2+ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.

| S-EPMC7730481 | biostudies-literature

GLRX3 Acts as a [2Fe-2S] Cluster Chaperone in the Cytosolic Iron-Sulfur Assembly Machinery Transferring [2Fe-2S] Clusters to NUBP1.

Project description:Human cytosolic monothiol glutaredoxin-3 (GLRX3) is a protein essential for the maturation of cytosolic [4Fe-4S] proteins. We show here that dimeric cluster-bridged GLRX3 transfers its [2Fe-2S]2+ clusters to the human P-loop NTPase NUBP1, an essential early component of the cytosolic iron-sulfur assembly (CIA) machinery. Specifically, we observed that [2Fe-2S]2+ clusters are transferred from GLRX3 to monomeric apo NUBP1 and reductively coupled to form [4Fe-4S]2+ clusters on both N-terminal CX13CX2CX5C and C-terminal CPXC motifs of NUBP1 in the presence of glutathione that acts as a reductant. In this process, cluster binding to the C-terminal motif of NUBP1 promotes protein dimerization, while cluster binding to the N-terminal motif does not affect the quaternary structure of NUBP1. The cluster transfer/assembly process is not complete on both N- and C-terminal motifs and indeed requires a reductant stronger than GSH to increase its efficiency. We also showed that the [4Fe-4S]2+ cluster formed at the N-terminal motif of NUBP1 is tightly bound, while the [4Fe-4S]2+ cluster bound at the C-terminal motif is labile. Our findings provide the first evidence for GLRX3 acting as a [2Fe-2S] cluster chaperone in the early stage of the CIA machinery.

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

Project description: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.

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A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF.

Project description:[FeFe]-hydrogenases are nature's most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron-sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly.

| S-EPMC5501864 | biostudies-literature

Cluster exchange reactivity of [2Fe-2S] cluster-bridged complexes of BOLA3 with monothiol glutaredoxins.

Project description:The [2Fe-2S] cluster-bridged complex of BOLA3 with GLRX5 has been implicated in cluster trafficking, but cluster exchange involving this heterocomplex has not been reported. Herein we describe an investigation of the cluster exchange reactivity of holo BOLA3-GLRX complexes using two different monothiol glutaredoxins, H.s. GLRX5 and S.c. Grx3, which share significant identity. We observe that a 1?:?1 mixture of apo BOLA3 and glutaredoxin protein is able to accept a cluster from donors such as ISCU and a [2Fe-2S](GS)4 complex, with preferential formation of the cluster-bridged heterodimer over the plausible holo homodimeric glutaredoxin. Holo BOLA3-GLRX5 transfers clusters to apo acceptors at rates comparable to other Fe-S cluster trafficking proteins. Isothermal titration calorimetry experiments with apo proteins demonstrated a strong binding of BOLA3 with both GLRX5 and Grx3, while binding with an alternative mitochondrial partner, NFU1, was weak. Cluster exchange and calorimetry experiments resulted in a very similar behavior for yeast Grx3 (cytosolic) and human GLRX5 (mitochondrial), indicating conservation across the monothiol glutaredoxin family for interactions with BOLA3 and supporting a functional role for the BOLA3-GLRX5 heterocomplex relative to the previously proposed BOLA3-NFU1 interaction. The results also demonstrate rapid formation of the heterocomplexed holo cluster via delivery from a glutathione-complexed cluster, again indicative of the physiological relevance of the [2Fe-2S](GS)4 complex in the cellular labile iron pool.

| S-EPMC6146392 | biostudies-literature

Model of the MitoNEET [2Fe-2S] Cluster Shows Proton Coupled Electron Transfer.

Project description:MitoNEET is an outer membrane protein whose exact function remains unclear, though a role of this protein in redox and iron sensing as well as in controlling maximum mitochondrial respiratory rates has been discussed. It was shown to contain a redox active and acid labile [2Fe-2S] cluster which is ligated by one histidine and three cysteine residues. Herein we present the first synthetic analogue with biomimetic {SN/S2} ligation which could be structurally characterized in its diferric form, 52-. In addition to being a high fidelity structural model for the biological cofactor, the complex is shown to mediate proton coupled electron transfer (PCET) at the {SN} ligated site, pointing at a potential functional role of the enzyme's unique His ligand. Full PCET thermodynamic square schemes for the mitoNEET model 52- and a related homoleptic {SN/SN} capped [2Fe-2S] cluster 42- are established, and kinetics of PCET reactivity are investigated by double-mixing stopped-flow experiments for both complexes. While the N-H bond dissociation free energy (BDFE) of 5H2- (230 ± 4 kJ mol-1) and the free energy ?G°PCET for the reaction with TEMPO (-48.4 kJ mol-1) are very similar to values for the homoleptic cluster 4H2- (232 ± 4 kJ mol-1, -46.3 kJ mol-1) the latter is found to react significantly faster than the mitoNEET model (data for 5H2-: k = 135 ± 27 M-1 s-1, ?H‡ = 17.6 ± 3.0 kJ mol-1, ?S‡ = -143 ± 11 J mol-1 K-1, and ?G‡ = 59.8 kJ mol-1 at 293 K). Comparison of the PCET efficiency of these clusters emphasizes the relevance of reorganization energy in this process.

| S-EPMC5812485 | biostudies-literature

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