Iron-Sulfur Cluster

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John L Markley - One of the best experts on this subject based on the ideXlab platform.

  • human mitochondrial ferredoxin 1 fdx1 and ferredoxin 2 fdx2 both bind cysteine desulfurase and donate electrons for iron sulfur Cluster biosynthesis
    Biochemistry, 2017
    Co-Authors: Marco Tonelli, Ronnie O Frederick, John L Markley
    Abstract:

    Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) Cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur Cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur Cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for Cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S Cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to ...

  • Human Mitochondrial Ferredoxin 1 (FDX1) and Ferredoxin 2 (FDX2) Both Bind Cysteine Desulfurase and Donate Electrons for Iron–Sulfur Cluster Biosynthesis
    2016
    Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L Markley
    Abstract:

    Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) Cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur Cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur Cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for Cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S Cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does. The reduced form of each ferredoxin became oxidized in the presence of the cysteine desulfurase complex when l-cysteine was added, leading to its conversion to l-alanine and the generation of sulfide. In an in vitro reaction, the reduced form of each ferredoxin was found to support Fe–S Cluster assembly on ISCU; the rate of Cluster assembly was faster with FDX2 than with FDX1. Taken together, these results show that both FDX1 and FDX2 can function in Fe–S Cluster assembly in vitro

  • tangled web of interactions among proteins involved in iron sulfur Cluster assembly as unraveled by nmr saxs chemical crosslinking and functional studies
    Biochimica et Biophysica Acta, 2015
    Co-Authors: Jin Hae Kim, Jameson R Bothe, Reid T Alderson, John L Markley
    Abstract:

    Abstract Proteins containing iron–sulfur (Fe–S) Clusters arose early in evolution and are essential to life. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe–S Clusters efficiently so as to minimize cellular exposure to their toxic constituents: iron and sulfide ions. To date, the best studied system is the Iron-Sulfur Cluster (isc) operon of Escherichia coli, and the eight ISC proteins it encodes. Our investigations over the past five years have identified two functional conformational states for the scaffold protein (IscU) and have shown that the other ISC proteins that interact with IscU prefer to bind one conformational state or the other. From analyses of the NMR spectroscopy-derived network of interactions of ISC proteins, small-angle X-ray scattering (SAXS) data, chemical crosslinking experiments, and functional assays, we have constructed working models for Fe–S Cluster assembly and delivery. Future work is needed to validate and refine what has been learned about the E. coli system and to extend these findings to the homologous Fe–S Cluster biosynthetic machinery of yeast and human mitochondria. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

  • 2fe 2s ferredoxin binds directly to cysteine desulfurase and supplies an electron for iron sulfur Cluster assembly but is displaced by the scaffold protein or bacterial frataxin
    Journal of the American Chemical Society, 2013
    Co-Authors: Ronnie O Frederick, Nichole M Reinen, Andrew T Troupis, John L Markley
    Abstract:

    Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur Cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S Clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S Cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin...

  • 2fe 2s ferredoxin binds directly to cysteine desulfurase and supplies an electron for iron sulfur Cluster assembly but is displaced by the scaffold protein or bacterial frataxin
    Journal of the American Chemical Society, 2013
    Co-Authors: Jin Hae Kim, Ronnie O Frederick, Nichole M Reinen, Andrew T Troupis, John L Markley
    Abstract:

    Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the 'house-keeping' mechanism of Iron-Sulfur Cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S(0)) produced by the cysteine desulfurase (IscS) to sulfide (S(2-)) as required for the assembly of Fe-S Clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe-S Cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe-S Cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S(0) is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the isc operon.

Tracey A Rouault - One of the best experts on this subject based on the ideXlab platform.

  • both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron sulfur Cluster biogenesis
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Manik C Ghosh, Gennadiy Kovtunovych, Daniel R Crooks, Tracey A Rouault
    Abstract:

    Ferredoxins are iron–sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron–sulfur Cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2 — formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron–sulfur Cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron–sulfur Clusters, but not steroidogenesis. To further assess the role of the FDX–FDXR system in mammalian iron–sulfur Cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron–sulfur Cluster biogenesis. Loss of FDX1 activity disrupted activity of iron–sulfur Cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron–sulfur Cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron–sulfur Cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.

  • characterization of the human hsc20 an unusual dnaj type iii protein involved in iron sulfur Cluster biogenesis
    Human Molecular Genetics, 2010
    Co-Authors: Manik C Ghosh, Gennadiy Kovtunovych, Helge Uhrigshardt, Anamika Singh, Tracey A Rouault
    Abstract:

    The importance of mitochondrial iron–sulfur Cluster (ISC) biogenesis for human health has been well established, but the roles of some components of this critical pathway still remain uncharacterized in mammals. Among them is human heat shock cognate protein 20 (hHSC20), the putative human homolog of the specialized DnaJ type co-chaperones, which are crucial for bacterial and fungal ISC assembly. Here, we show that the human HSC20 protein can complement for its counterpart in yeast, Jac1p, and interacts with its proposed human partners, hISCU and hHSPA9. hHSC20 is expressed in various human tissues and localizes mainly to the mitochondria in HeLa cells. However, small amounts were also detected extra-mitochondrially. RNA interference-mediated depletion of hHSC20 specifically reduced the activities of both mitochondrial and cytosolic ISC-containing enzymes. The recovery of inactivated ISC enzymes was markedly delayed after an oxidative insult of hHSC20-deficient cells. Conversely, overexpression of hHSC20 substantially protected cells from oxidative insults. These results imply that hHSC20 is an integral component of the human ISC biosynthetic machinery that is particularly important in the assembly of ISCs under conditions of oxidative stress. A cysteine-rich N-terminal domain, which clearly distinguishes hHSC20 from the specialized DnaJ type III proteins of fungi and most bacteria, was found to be important for the integrity and function of the human co-chaperone.

  • splice mutation in the iron sulfur Cluster scaffold protein iscu causes myopathy with exercise intolerance
    American Journal of Human Genetics, 2008
    Co-Authors: Fanny Mochel, Tracey A Rouault, Melanie A Knight, Wing Hang Tong, Dena G Hernandez, Karen Ayyad, Tanja Taivassalo, Peter M Andersen, Andrew B Singleton, Kenneth H Fischbeck
    Abstract:

    A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the Iron-Sulfur Cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in Iron-Sulfur Cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in Iron-Sulfur Cluster biogenesis.

  • roles of the mammalian cytosolic cysteine desulfurase iscs and scaffold protein iscu in iron sulfur Cluster assembly
    Journal of Biological Chemistry, 2006
    Co-Authors: Wing Hang Tong, Robert M Hughes, Tracey A Rouault
    Abstract:

    Iron-Sulfur Clusters are prosthetic groups composed of sulfur and iron that are found in respiratory chain complexes and numerous enzymes. Iron-Sulfur Clusters are synthesized in a multistep process that utilizes cysteine desulfurases, scaffold proteins, chaperones, and iron donors. Assembly of Iron-Sulfur Clusters occurs in the mitochondrial matrix of mammalian cells, but cytosolic isoforms of three major mammalian Iron-Sulfur Cluster (ISC) assembly components have been found, raising the possibility that de novo Iron-Sulfur Cluster biogenesis also occurs in cytosol. The human cysteine desulfurase, ISCS, has two isoforms, one of which targets to the mitochondria, whereas the other less abundant form is cytosolic and nuclear. The open-reading frame of cytosolic mammalian ISCS begins at the second AUG of the transcript and lacks mitochondrial targeting information. Yeast complementation experiments have suggested that the human cytosolic ISCS isoform (c-ISCS) cannot be functional. To evaluate function of c-ISCS, we overexpressed the human cytosolic ISCS in yeast Pichia pastoris and showed that the cytosolic form of ISCS is an active cysteine desulfurase that covalently binds 35S acquired from desulfuration of radiolabeled cysteine. Human cytosolic ISCS dimerized as efficiently as bacterial ISCS and formed a complex in vitro with overexpressed cytosolic human ISCU. When incubated with iron regulatory protein 1, cysteine, and iron, the cytosolic forms of ISCS and ISCU facilitated efficient formation of a [4Fe-4S] Cluster on IRP1. Thus, the cytosolic form of ISCS is a functional cysteine desulfurase that can collaborate with cytosolic ISCU to promote de novo Iron-Sulfur Cluster formation.

  • overexpression of iron responsive element binding protein and its analytical characterization as the rna binding form devoid of an iron sulfur Cluster
    Archives of Biochemistry and Biophysics, 1994
    Co-Authors: James P Basilion, M C Kennedy, Helmut Beinert, C M Massinople, R D Klausner, Tracey A Rouault
    Abstract:

    Abstract The iron-responsive element-binding protein (IRE-BP) has been defined and identified as an RNA-binding protein found in iron-deprived eukaryotic cells. IRE-BP binds to stem-loop structures, iron-responsive elements (IREs), which are located in the untranslated regions of the mRNAs for several genes including ferritin, and the transferrin receptor. When bound, IRE-BP prevents ferritin translation and stabilizes the transferrin receptor transcript. When cells are iron replete, an Iron-Sulfur Cluster is ligated to the IRE-BP, the protein loses RNA binding properties, and it acquires aconitase activity. Cytosolic aconitase from liver can be converted into the IRE-BP by oxidative removal of its Fe-S Cluster. We describe here overexpression of IRE-BP in baculovirus-infected insect cells which yields IRE-BP devoid of an Iron-Sulfur Cluster. We describe a one-step purification of the IRE-BP and a quantitative analysis of Fe, S2−, S0, protein, and enzyme activity on IRE-BP, as obtained in cell lysates, after purification, and after reconstitution to active aconitase. On the average not more than 3% of the over-expressed purified protein contained an intact Fe-S Cluster, and it was demonstrated that that Cluster was not lost during purification. Scatchard analysis of RNA-binding data was compatible with a single high-affinity RNA-binding form of the IRE-BP. Active aconitase could be reconstituted from the purified IRE-BP obtained from the expression system by addition of iron, thiol, and sulfide, and the characteristic epr spectrum of the 3Fe form of cytosolic aconitase was obtained after ferricyanide oxidation of the reconstituted material.

Marco Tonelli - One of the best experts on this subject based on the ideXlab platform.

  • human mitochondrial ferredoxin 1 fdx1 and ferredoxin 2 fdx2 both bind cysteine desulfurase and donate electrons for iron sulfur Cluster biosynthesis
    Biochemistry, 2017
    Co-Authors: Marco Tonelli, Ronnie O Frederick, John L Markley
    Abstract:

    Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) Cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur Cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur Cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for Cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S Cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to ...

  • Human Mitochondrial Ferredoxin 1 (FDX1) and Ferredoxin 2 (FDX2) Both Bind Cysteine Desulfurase and Donate Electrons for Iron–Sulfur Cluster Biosynthesis
    2016
    Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L Markley
    Abstract:

    Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) Cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur Cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur Cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for Cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S Cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does. The reduced form of each ferredoxin became oxidized in the presence of the cysteine desulfurase complex when l-cysteine was added, leading to its conversion to l-alanine and the generation of sulfide. In an in vitro reaction, the reduced form of each ferredoxin was found to support Fe–S Cluster assembly on ISCU; the rate of Cluster assembly was faster with FDX2 than with FDX1. Taken together, these results show that both FDX1 and FDX2 can function in Fe–S Cluster assembly in vitro

  • metamorphic protein iscu alternates conformations in the course of its role as the scaffold protein for iron sulfur Cluster biosynthesis and delivery
    FEBS Letters, 2013
    Co-Authors: John L Markley, Ronnie O Frederick, Jin Hae Kim, Jameson R Bothe, Kai Cai, Ziqi Dai, Marco Tonelli
    Abstract:

    IscU from Escherichia coli, the scaffold protein for Iron-Sulfur Cluster biosynthesis and delivery, populates a complex energy landscape. IscU exists as two slowly interconverting species: one (S) is largely structured with all four peptidyl–prolyl bonds trans; the other (D) is partly disordered but contains an ordered domain that stabilizes two cis peptidyl–prolyl peptide bonds. At pH 8.0, the S-state is maximally populated at 25 °C, but its population decreases at higher or lower temperatures or at lower pH. The D-state binds preferentially to the cysteine desulfurase (IscS), which generates and transfers sulfur to IscU cysteine residues to form persulfides. The S-state is stabilized by Fe–S Cluster binding and interacts preferentially with the DnaJ-type co-chaperone (HscB), which targets the holo-IscU:HscB complex to the DnaK-type chaperone (HscA) in its ATP-bound from. HscA is involved in delivery of Fe–S Clusters to acceptor proteins by a mechanism dependent on ATP hydrolysis. Upon conversion of ATP to ADP, HscA binds the D-state of IscU ensuring release of the Cluster and HscB. These findings have led to a more complete model for Cluster biosynthesis and delivery.

  • disordered form of the scaffold protein iscu is the substrate for iron sulfur Cluster assembly on cysteine desulfurase
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Jin Hae Kim, Marco Tonelli, John L Markley
    Abstract:

    The scaffold protein for Iron-Sulfur Cluster assembly, apo-IscU, populates two interconverting conformational states, one disordered (D) and one structured (S) as revealed by extensive NMR assignments. At pH 8 and 25 °C, approximately 70% of the protein is S, and the lifetimes of the states are 1.3 s (S) and 0.50 s (D). Zn(II) and Fe(II) each bind and stabilize structured (S-like) states. Single amino acid substitutions at conserved residues were found that shift the equilibrium toward either the S or the D state. Cluster assembly takes place in the complex between IscU and the cysteine desulfurase, IscS, and our NMR studies demonstrate that IscS binds preferentially the D form of apo-IscU. The addition of 10% IscS to IscU was found to greatly increase H/D exchange at protected amides of IscU, to increase the rate of the S → D reaction, and to decrease the rate of the D → S reaction. In the saturated IscU:IscS complex, IscU is largely disordered. In vitro Cluster assembly reactions provided evidence for the functional importance of the S⇆D equilibrium. IscU variants that favor the S state were found to undergo a lag phase, not observed with the wild type, that delayed Cluster assembly; variants that favor the D state were found to assemble less stable Clusters at an intermediate rate without the lag. It appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and to convert to a structured state that stabilizes the Cluster once it is assembled.

  • structure and dynamics of the iron sulfur Cluster assembly scaffold protein iscu and its interaction with the cochaperone hscb
    Biochemistry, 2009
    Co-Authors: Anna K Fuzery, Marco Tonelli, Larry E Vickery, Dennis T Ta, William M Westler, John L Markley
    Abstract:

    IscU is a scaffold protein that functions in iron−sulfur Cluster assembly and transfer. Its critical importance has been recently underscored by the finding that a single intronic mutation in the human iscu gene is associated with a myopathy resulting from deficient succinate dehydrogenase and aconitase [Mochel, F., Knight, M. A., Tong, W. H., Hernandez, D., Ayyad, K., Taivassalo, T., Andersen, P. M., Singleton, A., Rouault, T. A., Fischbeck, K. H., and Haller, R. G. (2008) Am. J. Hum. Genet. 82, 652−660]. IscU functions through interactions with a chaperone protein HscA and a cochaperone protein HscB. To probe the molecular basis for these interactions, we have used NMR spectroscopy to investigate the solution structure of IscU from Escherichia coli and its interaction with HscB from the same organism. We found that wild-type apo-IscU in solution exists as two distinct conformations: one largely disordered and one largely ordered except for the metal binding residues. The two states interconvert on the m...

J A Cowan - One of the best experts on this subject based on the ideXlab platform.

  • role of the hspa9 hsc20 chaperone pair in promoting directional human iron sulfur Cluster exchange involving monothiol glutaredoxin 5
    Journal of Inorganic Biochemistry, 2018
    Co-Authors: Joshua A Olive, J A Cowan
    Abstract:

    Abstract Iron‑sulfur Clusters are essential cofactors found across all domains of life. Their assembly and transfer are accomplished by highly conserved protein complexes and partners. In eukaryotes a [2Fe-2S] Cluster is first assembled in the mitochondria on the iron‑sulfur Cluster scaffold protein ISCU in tandem with iron, sulfide, and electron donors. Current models suggest that a chaperone pair interacts with a Cluster-bound ISCU to facilitate Cluster transfer to a monothiol glutaredoxin. In humans this protein is glutaredoxin 5 (GLRX5) and the Cluster can then be exchanged with a variety of target apo proteins. By use of circular dichroism spectroscopy, the kinetics of Cluster exchange reactivity has been evaluated for human GLRX5 with a variety of Cluster donor and acceptor partners, and the role of chaperones determined for several of these. In contrast to the prokaryotic model, where heat-shock type chaperone proteins HscA and HscB are required for successful and efficient transfer of a [2Fe-2S] Cluster from the ISCU scaffold to a monothiol glutaredoxin. However, in the human system the chaperone homologs, HSPA9 and HSC20, are not necessary for human ISCU to promote Cluster transfer to GLRX5, and appear to promote the reverse transfer. Cluster exchange with the human iron‑sulfur Cluster carrier protein NFU1 and ferredoxins (FDX's), and the role of chaperones, has also been evaluated, demonstrating in certain cases control over the directionality of Cluster transfer. In contrast to other prokaryotic and eukaryotic organisms, NFU1 is identified as a more likely physiological donor of [2Fe-2S] Cluster to human GLRX5 than ISCU.

  • mapping cellular fe s Cluster uptake and exchange reactions divergent pathways for iron sulfur Cluster delivery to human ferredoxins
    Metallomics, 2016
    Co-Authors: Insiya Fidai, Christine Wachnowsky, J A Cowan
    Abstract:

    Ferredoxins are protein mediators of biological electron-transfer reactions and typically contain either [2Fe-2S] or [4Fe-4S] Clusters. Two ferredoxin homologues have been identified in the human genome, Fdx1 and Fdx2, that share 43% identity and 69% similarity in protein sequence and both bind [2Fe-2S] Clusters. Despite the high similarity, the two ferredoxins play very specific roles in distinct physiological pathways and cannot replace each other in function. Both eukaryotic and prokaryotic ferredoxins and homologues have been reported to receive their Fe-S Cluster from scaffold/delivery proteins such as IscU, Isa, glutaredoxins, and Nfu. However, the preferred and physiologically relevant pathway for receiving the [2Fe-2S] Cluster by ferredoxins is subject to speculation and is not clearly identified. In this work, we report on in vitro UV-visible (UV-vis) circular dichroism studies of [2Fe-2S] Cluster transfer to the ferredoxins from a variety of partners. The results reveal rapid and quantitative transfer to both ferredoxins from several donor proteins (IscU, Isa1, Grx2, and Grx3). Transfer from Isa1 to Fdx2 was also observed to be faster than that of IscU to Fdx2, suggesting that Fdx2 could receive its Cluster from Isa1 instead of IscU. Several other transfer combinations were also investigated and the results suggest a complex, but kinetically detailed map for cellular Cluster trafficking. This is the first step toward building a network map for all of the possible Iron-Sulfur Cluster transfer pathways in the mitochondria and cytosol, providing insights on the most likely cellular pathways and possible redundancies in these pathways.

  • iron sulfur Cluster exchange reactions mediated by the human nfu protein
    Journal of Biological Inorganic Chemistry, 2016
    Co-Authors: Christine Wachnowsky, Insiya Fidai, J A Cowan
    Abstract:

    Human Nfu is an iron–sulfur Cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron–sulfur Cluster binding and transfer roles. Herein, we characterize the Cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe–2S] Cluster. The mechanism of Cluster uptake from a physiologically relevant [2Fe–2S](GS)4 Cluster complex, and extraction of the Nfu-bound iron–sulfur Cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to Cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe–2S] Cluster. This Cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe–2S] Cluster trafficking and delivery.

  • iron sulfur Cluster biosynthesis thermatoga maritima iscu is a structured iron sulfur Cluster assembly protein
    Journal of Biological Chemistry, 2002
    Co-Authors: Sheref S Mansy, Kristene K Surerus, J A Cowan
    Abstract:

    Abstract Genetic evidence has indicated that Isc proteins play an important role in Iron-Sulfur Cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent Iron-Sulfur Cluster that is subsequently delivered to target Iron-Sulfur apoproteins. We report the characterization of an IscU fromThermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. Mossbauer (δ = 0.29 ± 0.03 mm/s, ΔEQ = 0.58 ± 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]2+ Cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable Cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% α-helix, 13.1% antiparallel β-sheet, 11.3% parallel β-sheet, 20.2% β-turn, and 19.1% other at 20 °C, with negligible spectral change observed at 70 °C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in1H-15N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritimaIscU-mediated reconstitution of human apoferredoxin.

Ronnie O Frederick - One of the best experts on this subject based on the ideXlab platform.

  • human mitochondrial ferredoxin 1 fdx1 and ferredoxin 2 fdx2 both bind cysteine desulfurase and donate electrons for iron sulfur Cluster biosynthesis
    Biochemistry, 2017
    Co-Authors: Marco Tonelli, Ronnie O Frederick, John L Markley
    Abstract:

    Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) Cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur Cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur Cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for Cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S Cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to ...

  • Human Mitochondrial Ferredoxin 1 (FDX1) and Ferredoxin 2 (FDX2) Both Bind Cysteine Desulfurase and Donate Electrons for Iron–Sulfur Cluster Biosynthesis
    2016
    Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L Markley
    Abstract:

    Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) Cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur Cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur Cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for Cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S Cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does. The reduced form of each ferredoxin became oxidized in the presence of the cysteine desulfurase complex when l-cysteine was added, leading to its conversion to l-alanine and the generation of sulfide. In an in vitro reaction, the reduced form of each ferredoxin was found to support Fe–S Cluster assembly on ISCU; the rate of Cluster assembly was faster with FDX2 than with FDX1. Taken together, these results show that both FDX1 and FDX2 can function in Fe–S Cluster assembly in vitro

  • 2fe 2s ferredoxin binds directly to cysteine desulfurase and supplies an electron for iron sulfur Cluster assembly but is displaced by the scaffold protein or bacterial frataxin
    Journal of the American Chemical Society, 2013
    Co-Authors: Ronnie O Frederick, Nichole M Reinen, Andrew T Troupis, John L Markley
    Abstract:

    Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur Cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S Clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S Cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin...

  • 2fe 2s ferredoxin binds directly to cysteine desulfurase and supplies an electron for iron sulfur Cluster assembly but is displaced by the scaffold protein or bacterial frataxin
    Journal of the American Chemical Society, 2013
    Co-Authors: Jin Hae Kim, Ronnie O Frederick, Nichole M Reinen, Andrew T Troupis, John L Markley
    Abstract:

    Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the 'house-keeping' mechanism of Iron-Sulfur Cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S(0)) produced by the cysteine desulfurase (IscS) to sulfide (S(2-)) as required for the assembly of Fe-S Clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe-S Cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe-S Cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S(0) is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the isc operon.

  • metamorphic protein iscu alternates conformations in the course of its role as the scaffold protein for iron sulfur Cluster biosynthesis and delivery
    FEBS Letters, 2013
    Co-Authors: John L Markley, Ronnie O Frederick, Jin Hae Kim, Jameson R Bothe, Kai Cai, Ziqi Dai, Marco Tonelli
    Abstract:

    IscU from Escherichia coli, the scaffold protein for Iron-Sulfur Cluster biosynthesis and delivery, populates a complex energy landscape. IscU exists as two slowly interconverting species: one (S) is largely structured with all four peptidyl–prolyl bonds trans; the other (D) is partly disordered but contains an ordered domain that stabilizes two cis peptidyl–prolyl peptide bonds. At pH 8.0, the S-state is maximally populated at 25 °C, but its population decreases at higher or lower temperatures or at lower pH. The D-state binds preferentially to the cysteine desulfurase (IscS), which generates and transfers sulfur to IscU cysteine residues to form persulfides. The S-state is stabilized by Fe–S Cluster binding and interacts preferentially with the DnaJ-type co-chaperone (HscB), which targets the holo-IscU:HscB complex to the DnaK-type chaperone (HscA) in its ATP-bound from. HscA is involved in delivery of Fe–S Clusters to acceptor proteins by a mechanism dependent on ATP hydrolysis. Upon conversion of ATP to ADP, HscA binds the D-state of IscU ensuring release of the Cluster and HscB. These findings have led to a more complete model for Cluster biosynthesis and delivery.