Characterization of [2Fe-2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods.
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ABSTRACT:
SUBMITTER: Jia M
PROVIDER: S-EPMC7170024 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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Project description:Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability. Herein, we provide biophysical and biochemical evidence that the two tandem Grx-like domains in human Glrx3 form similar [2Fe-2S]-bridged complexes with human BolA2. UV-visible absorption and circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic analyses of recombinant [2Fe-2S] Glrx3 homodimers and [2Fe-2S] Glrx3-BolA2 complexes indicate that the Fe-S coordination environments in these complexes are virtually identical to those of the analogous complexes in yeast. Furthermore, we demonstrate that apo BolA2 binds to each Grx domain in the [2Fe-2S] Glrx3 homodimer forming a [2Fe-2S] BolA2-Glrx3 heterotrimer. Taken together, these results suggest that the unusual [2Fe-2S]-bridging Grx-BolA interaction is conserved in higher eukaryotes and may play a role in signaling cellular iron status in humans.
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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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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.
| S-EPMC8007109 | biostudies-literature
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
Project description: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.
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Project description:Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimer as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.
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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
Project description:In eukaryotes, iron-sulfur clusters are essential cofactors for numerous physiological processes, but these clusters are primarily biosynthesized in mitochondria. Previous studies suggest mitochondrial ABCB7-type exporters are involved in maturation of cytosolic iron-sulfur proteins. However, the molecular mechanism for how the ABCB7-type exporters participate in this process remains elusive. Here, we report a series of cryo-electron microscopy structures of a eukaryotic homolog of human ABCB7, CtAtm1, determined at average resolutions ranging from 2.8 to 3.2 Å, complemented by functional characterization and molecular docking in silico. We propose that CtAtm1 accepts delivery from glutathione-complexed iron-sulfur clusters. A partially occluded state links cargo-binding to residues at the mitochondrial matrix interface that line a positively charged cavity, while the binding region becomes internalized and is partially divided in an early occluded state. Collectively, our findings substantially increase the understanding of the transport mechanism of eukaryotic ABCB7-type proteins.
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Project description:IscR is an Fe-S cluster-containing transcription factor involved in a homeostatic mechanism that controls Fe-S cluster biogenesis in Escherichia coli. Although IscR has been proposed to act as a sensor of the cellular demands for Fe-S cluster biogenesis, the mechanism by which IscR performs this function is not known. In this study, we investigated the biochemical properties of the Fe-S cluster of IscR to gain insight into the proposed sensing activity. Mössbauer studies revealed that IscR contains predominantly a reduced [2Fe-2S](+) cluster in vivo. However, upon anaerobic isolation of IscR, some clusters became oxidized to the [2Fe-2S](2+) form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that the cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mössbauer, and nuclear magnetic resonance spectroscopies leads to the proposal that the [2Fe-2S] cluster does not have full cysteinyl ligation. Mutagenesis studies indicate that, in addition to the three previously identified cysteine residues (Cys92, Cys98, and Cys104), the highly conserved His107 residue is essential for cluster ligation. Thus, these data suggest that IscR binds the cluster with an atypical ligation scheme of three cysteines and one histidine, a feature that may be relevant to the proposed function of IscR as a sensor of cellular Fe-S cluster status.
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