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John L Markley - One of the best experts on this subject based on the ideXlab platform.
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architectural features of human mitochondrial Cysteine Desulfurase complexes from crosslinking mass spectrometry and small angle x ray scattering
Structure, 2018Co-Authors: Kai Cai, Ronnie O Frederick, Hesam Dashti, John L MarkleyAbstract:Summary Cysteine Desulfurase plays a central role in mitochondrial iron-sulfur cluster biogenesis by generating sulfur through the conversion of L-Cysteine to L-alanine and by serving as the platform for assembling other components of the biosynthetic machinery, including ISCU, frataxin, and ferredoxin. The human mitochondrial Cysteine Desulfurase complex consists of two copies each of NFS1, ISD11, and acyl carrier protein. We describe results from chemical crosslinking coupled with tandem mass spectrometry and small-angle X-ray scattering studies that are consistent with a closed NFS1 dimer rather than an open one for both the Cysteine Desulfurase-ISCU and Cysteine Desulfurase-ISCU-frataxin complexes. We present a structural model for the Cysteine Desulfurase-ISCU-frataxin complex derived from chemical crosslinking restraints in conjunction with the recent crystal structure of the Cysteine Desulfurase-ISCU-zinc complex and distance constraints from nuclear magnetic resonance.
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interactions of iron bound frataxin with iscu and ferredoxin on the Cysteine Desulfurase complex leading to fe s cluster assembly
Journal of Inorganic Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe2+ but not Fe3+. While FXN (with or without bound Fe2+) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the Cysteine Desulfurase complex ([NFS1]2:[ISD11]2:[Acp]2), abbreviated as (NIA)2, where "N" represents the Cysteine Desulfurase (NFS1), "I" represents the accessory protein (ISD11), and "A" represents acyl carrier protein (Acp). FXN binds (NIA)2 weakly in the absence of ISCU but more strongly in its presence. Fe2+-FXN binds to the (NIA)2-ISCU2 complex without release of iron. However, upon the addition of both l-Cysteine and a reductant (either reduced FDX2 or DTT), Fe2+ is released from FXN as consistent with Fe2+-FXN being the proximal source of iron for Fe-S cluster assembly.
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iscu m108i and iscu d39v differ from wild type iscu in their failure to form Cysteine Desulfurase complexes containing both frataxin and ferredoxin
Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Whereas iron–sulfur (Fe–S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human Cysteine Desulfurase complex (NIA)2, exhibits a requirement for frataxin (FXN), in yeast, ISCU variant M108I has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that variants ISCU(M108I) and ISCU(D39V) of human ISCU populate only the structured state. We have compared the properties of ISCU, ISCU(M108I), and ISCU(D39V), with and without FXN, in both the Cysteine Desulfurase step of Fe–S cluster assembly and the overall Fe–S cluster assembly reaction catalyzed by (NIA)2. In the Cysteine Desulfurase step with dithiothreitol (DTT) as the reductant, FXN was found to stimulate Cysteine Desulfurase activity with both the wild-type and structured variants, although the effect was less prominent with ISCU(D39V) than with the wild-type or ISCU(M108I). In overall Fe–S cluster assembly, f...
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mitochondrial Cysteine Desulfurase and isd11 coexpressed in escherichia coli yield complex containing acyl carrier protein
ACS Chemical Biology, 2017Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Mitochondrial Cysteine Desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human Cysteine Desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the Cysteine Desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.
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human mitochondrial ferredoxin 1 fdx1 and ferredoxin 2 fdx2 both bind Cysteine Desulfurase and donate electrons for iron sulfur cluster biosynthesis
Biochemistry, 2017Co-Authors: Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract: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 ...
Ronnie O Frederick - One of the best experts on this subject based on the ideXlab platform.
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architectural features of human mitochondrial Cysteine Desulfurase complexes from crosslinking mass spectrometry and small angle x ray scattering
Structure, 2018Co-Authors: Kai Cai, Ronnie O Frederick, Hesam Dashti, John L MarkleyAbstract:Summary Cysteine Desulfurase plays a central role in mitochondrial iron-sulfur cluster biogenesis by generating sulfur through the conversion of L-Cysteine to L-alanine and by serving as the platform for assembling other components of the biosynthetic machinery, including ISCU, frataxin, and ferredoxin. The human mitochondrial Cysteine Desulfurase complex consists of two copies each of NFS1, ISD11, and acyl carrier protein. We describe results from chemical crosslinking coupled with tandem mass spectrometry and small-angle X-ray scattering studies that are consistent with a closed NFS1 dimer rather than an open one for both the Cysteine Desulfurase-ISCU and Cysteine Desulfurase-ISCU-frataxin complexes. We present a structural model for the Cysteine Desulfurase-ISCU-frataxin complex derived from chemical crosslinking restraints in conjunction with the recent crystal structure of the Cysteine Desulfurase-ISCU-zinc complex and distance constraints from nuclear magnetic resonance.
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interactions of iron bound frataxin with iscu and ferredoxin on the Cysteine Desulfurase complex leading to fe s cluster assembly
Journal of Inorganic Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe2+ but not Fe3+. While FXN (with or without bound Fe2+) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the Cysteine Desulfurase complex ([NFS1]2:[ISD11]2:[Acp]2), abbreviated as (NIA)2, where "N" represents the Cysteine Desulfurase (NFS1), "I" represents the accessory protein (ISD11), and "A" represents acyl carrier protein (Acp). FXN binds (NIA)2 weakly in the absence of ISCU but more strongly in its presence. Fe2+-FXN binds to the (NIA)2-ISCU2 complex without release of iron. However, upon the addition of both l-Cysteine and a reductant (either reduced FDX2 or DTT), Fe2+ is released from FXN as consistent with Fe2+-FXN being the proximal source of iron for Fe-S cluster assembly.
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iscu m108i and iscu d39v differ from wild type iscu in their failure to form Cysteine Desulfurase complexes containing both frataxin and ferredoxin
Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Whereas iron–sulfur (Fe–S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human Cysteine Desulfurase complex (NIA)2, exhibits a requirement for frataxin (FXN), in yeast, ISCU variant M108I has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that variants ISCU(M108I) and ISCU(D39V) of human ISCU populate only the structured state. We have compared the properties of ISCU, ISCU(M108I), and ISCU(D39V), with and without FXN, in both the Cysteine Desulfurase step of Fe–S cluster assembly and the overall Fe–S cluster assembly reaction catalyzed by (NIA)2. In the Cysteine Desulfurase step with dithiothreitol (DTT) as the reductant, FXN was found to stimulate Cysteine Desulfurase activity with both the wild-type and structured variants, although the effect was less prominent with ISCU(D39V) than with the wild-type or ISCU(M108I). In overall Fe–S cluster assembly, f...
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mitochondrial Cysteine Desulfurase and isd11 coexpressed in escherichia coli yield complex containing acyl carrier protein
ACS Chemical Biology, 2017Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Mitochondrial Cysteine Desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human Cysteine Desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the Cysteine Desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.
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human mitochondrial ferredoxin 1 fdx1 and ferredoxin 2 fdx2 both bind Cysteine Desulfurase and donate electrons for iron sulfur cluster biosynthesis
Biochemistry, 2017Co-Authors: Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract: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 ...
Marco Tonelli - One of the best experts on this subject based on the ideXlab platform.
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interactions of iron bound frataxin with iscu and ferredoxin on the Cysteine Desulfurase complex leading to fe s cluster assembly
Journal of Inorganic Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe2+ but not Fe3+. While FXN (with or without bound Fe2+) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the Cysteine Desulfurase complex ([NFS1]2:[ISD11]2:[Acp]2), abbreviated as (NIA)2, where "N" represents the Cysteine Desulfurase (NFS1), "I" represents the accessory protein (ISD11), and "A" represents acyl carrier protein (Acp). FXN binds (NIA)2 weakly in the absence of ISCU but more strongly in its presence. Fe2+-FXN binds to the (NIA)2-ISCU2 complex without release of iron. However, upon the addition of both l-Cysteine and a reductant (either reduced FDX2 or DTT), Fe2+ is released from FXN as consistent with Fe2+-FXN being the proximal source of iron for Fe-S cluster assembly.
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iscu m108i and iscu d39v differ from wild type iscu in their failure to form Cysteine Desulfurase complexes containing both frataxin and ferredoxin
Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Whereas iron–sulfur (Fe–S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human Cysteine Desulfurase complex (NIA)2, exhibits a requirement for frataxin (FXN), in yeast, ISCU variant M108I has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that variants ISCU(M108I) and ISCU(D39V) of human ISCU populate only the structured state. We have compared the properties of ISCU, ISCU(M108I), and ISCU(D39V), with and without FXN, in both the Cysteine Desulfurase step of Fe–S cluster assembly and the overall Fe–S cluster assembly reaction catalyzed by (NIA)2. In the Cysteine Desulfurase step with dithiothreitol (DTT) as the reductant, FXN was found to stimulate Cysteine Desulfurase activity with both the wild-type and structured variants, although the effect was less prominent with ISCU(D39V) than with the wild-type or ISCU(M108I). In overall Fe–S cluster assembly, f...
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mitochondrial Cysteine Desulfurase and isd11 coexpressed in escherichia coli yield complex containing acyl carrier protein
ACS Chemical Biology, 2017Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Mitochondrial Cysteine Desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human Cysteine Desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the Cysteine Desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.
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human mitochondrial ferredoxin 1 fdx1 and ferredoxin 2 fdx2 both bind Cysteine Desulfurase and donate electrons for iron sulfur cluster biosynthesis
Biochemistry, 2017Co-Authors: Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract: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 ...
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Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein
2017Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Mitochondrial Cysteine Desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human Cysteine Desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the Cysteine Desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue
Kai Cai - One of the best experts on this subject based on the ideXlab platform.
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architectural features of human mitochondrial Cysteine Desulfurase complexes from crosslinking mass spectrometry and small angle x ray scattering
Structure, 2018Co-Authors: Kai Cai, Ronnie O Frederick, Hesam Dashti, John L MarkleyAbstract:Summary Cysteine Desulfurase plays a central role in mitochondrial iron-sulfur cluster biogenesis by generating sulfur through the conversion of L-Cysteine to L-alanine and by serving as the platform for assembling other components of the biosynthetic machinery, including ISCU, frataxin, and ferredoxin. The human mitochondrial Cysteine Desulfurase complex consists of two copies each of NFS1, ISD11, and acyl carrier protein. We describe results from chemical crosslinking coupled with tandem mass spectrometry and small-angle X-ray scattering studies that are consistent with a closed NFS1 dimer rather than an open one for both the Cysteine Desulfurase-ISCU and Cysteine Desulfurase-ISCU-frataxin complexes. We present a structural model for the Cysteine Desulfurase-ISCU-frataxin complex derived from chemical crosslinking restraints in conjunction with the recent crystal structure of the Cysteine Desulfurase-ISCU-zinc complex and distance constraints from nuclear magnetic resonance.
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interactions of iron bound frataxin with iscu and ferredoxin on the Cysteine Desulfurase complex leading to fe s cluster assembly
Journal of Inorganic Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe2+ but not Fe3+. While FXN (with or without bound Fe2+) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the Cysteine Desulfurase complex ([NFS1]2:[ISD11]2:[Acp]2), abbreviated as (NIA)2, where "N" represents the Cysteine Desulfurase (NFS1), "I" represents the accessory protein (ISD11), and "A" represents acyl carrier protein (Acp). FXN binds (NIA)2 weakly in the absence of ISCU but more strongly in its presence. Fe2+-FXN binds to the (NIA)2-ISCU2 complex without release of iron. However, upon the addition of both l-Cysteine and a reductant (either reduced FDX2 or DTT), Fe2+ is released from FXN as consistent with Fe2+-FXN being the proximal source of iron for Fe-S cluster assembly.
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iscu m108i and iscu d39v differ from wild type iscu in their failure to form Cysteine Desulfurase complexes containing both frataxin and ferredoxin
Biochemistry, 2018Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Whereas iron–sulfur (Fe–S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human Cysteine Desulfurase complex (NIA)2, exhibits a requirement for frataxin (FXN), in yeast, ISCU variant M108I has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured and one dynamically disordered. We show here that variants ISCU(M108I) and ISCU(D39V) of human ISCU populate only the structured state. We have compared the properties of ISCU, ISCU(M108I), and ISCU(D39V), with and without FXN, in both the Cysteine Desulfurase step of Fe–S cluster assembly and the overall Fe–S cluster assembly reaction catalyzed by (NIA)2. In the Cysteine Desulfurase step with dithiothreitol (DTT) as the reductant, FXN was found to stimulate Cysteine Desulfurase activity with both the wild-type and structured variants, although the effect was less prominent with ISCU(D39V) than with the wild-type or ISCU(M108I). In overall Fe–S cluster assembly, f...
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mitochondrial Cysteine Desulfurase and isd11 coexpressed in escherichia coli yield complex containing acyl carrier protein
ACS Chemical Biology, 2017Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Mitochondrial Cysteine Desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human Cysteine Desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the Cysteine Desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.
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Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein
2017Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, John L MarkleyAbstract:Mitochondrial Cysteine Desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human Cysteine Desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the Cysteine Desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue
Jin Hae Kim - One of the best experts on this subject based on the ideXlab platform.
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Role of IscX in Iron–Sulfur Cluster Biogenesis in Escherichia coli
2015Co-Authors: Jin Hae Kim, Jameson R. Bothe, Ronnie O. Frederick, Johneisa C. Holder, John L MarkleyAbstract:The Escherichia coli isc operon encodes key proteins involved in the biosynthesis of iron–sulfur (Fe–S) clusters. Whereas extensive studies of most ISC proteins have revealed their functional properties, the role of IscX (also dubbed YfhJ), a small acidic protein encoded by the last gene in the operon, has remained in question. Previous studies showed that IscX binds iron ions and interacts with the Cysteine Desulfurase (IscS) and the scaffold protein for cluster assembly (IscU), and it has been proposed that IscX functions either as an iron supplier or a regulator of Fe–S cluster biogenesis. We have used a combination of NMR spectroscopy, small-angle X-ray scattering (SAXS), chemical cross-linking, and enzymatic assays to enlarge our understanding of the interactions of IscX with iron ions, IscU, and IscS. We used chemical shift perturbation to identify the binding interfaces of IscX and IscU in their complex. NMR studies showed that Fe2+ from added ferrous ammonium sulfate binds IscX much more avidly than does Fe3+ from added ferric ammonium citrate and that Fe2+ strengthens the interaction between IscX and IscU. We found that the addition of IscX to the IscU–IscS binary complex led to the formation of a ternary complex with reduced Cysteine Desulfurase activity, and we determined a low-resolution model for that complex from a combination of NMR and SAXS data. We postulate that the inhibition of Cysteine Desulfurase activity by IscX serves to reduce unproductive conversion of Cysteine to alanine. By incorporating these new findings with results from prior studies, we propose a detailed mechanism for Fe–S cluster assembly in which IscX serves both as a donor of Fe2+ and as a regulator of Cysteine Desulfurase activity
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human mitochondrial chaperone mthsp70 and Cysteine Desulfurase nfs1 bind preferentially to the disordered conformation whereas co chaperone hsc20 binds to the structured conformation of the iron sulfur cluster scaffold protein iscu
Journal of Biological Chemistry, 2013Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, Jin Hae Kim, Nichole M Reinen, John L MarkleyAbstract:Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human Cysteine Desulfurase complex (NFS1 and ISD11) separately and used NMR spectroscopy to investigate their interaction with ISCU. We found that ISD11 does not interact directly with ISCU. By contrast, NFS1 binds preferentially to the D-state of ISCU as does the NFS1-ISD11 complex. An in vitro Fe-S cluster assembly assay showed that [2Fe-2S] and [4Fe-4S] clusters are assembled on ISCU when catalyzed by NFS1 alone and at a higher rate when catalyzed by the NFS1-ISD11 complex. The DnaK-type chaperone (mtHSP70) and DnaJ-type co-chaperone (HSC20) are involved in the transfer of clusters bound to ISCU to acceptor proteins in an ATP-dependent reaction. We found that the ATPase activity of mtHSP70 is accelerated by HSC20 and further accelerated by HSC20 plus ISCU. NMR studies have shown that mtHSP70 binds preferentially to the D-state of ISCU and that HSC20 binds preferentially to the S-state of ISCU.
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human mitochondrial chaperone mthsp70 and Cysteine Desulfurase nfs1 bind preferentially to the disordered conformation whereas co chaperone hsc20 binds to the structured conformation of the iron sulfur cluster scaffold protein iscu
Journal of Biological Chemistry, 2013Co-Authors: Kai Cai, Marco Tonelli, Ronnie O Frederick, Jin Hae Kim, Nichole M Reinen, John L MarkleyAbstract:Background: Iron-sulfur cluster biosynthesis involves a scaffold protein (ISCU), Cysteine Desulfurase (NFS1), chaperone (mtHSP70), and co-chaperone (HSC20). Results: Human mitochondrial ISCU populates structured (S) and disordered (D) conformational states. S interacts preferentially with NFS1 and mtHSP70; D interacts preferentially with HSC20. Conclusion: Shifts in the S ⇄ D equilibrium reveal functional states. Significance: The scaffold protein metamorphic property seen in Escherichia coli is conserved in humans. Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human Cysteine Desulfurase complex (NFS1 and ISD11) separately and used NMR spectroscopy to investigate their interaction with ISCU. We found that ISD11 does not interact directly with ISCU. By contrast, NFS1 binds preferentially to the D-state of ISCU as does the NFS1-ISD11 complex. An in vitro Fe-S cluster assembly assay showed that [2Fe-2S] and [4Fe-4S] clusters are assembled on ISCU when catalyzed by NFS1 alone and at a higher rate when catalyzed by the NFS1-ISD11 complex. The DnaK-type chaperone (mtHSP70) and DnaJ-type co-chaperone (HSC20) are involved in the transfer of clusters bound to ISCU to acceptor proteins in an ATP-dependent reaction. We found that the ATPase activity of mtHSP70 is accelerated by HSC20 and further accelerated by HSC20 plus ISCU. NMR studies have shown that mtHSP70 binds preferentially to the D-state of ISCU and that HSC20 binds preferentially to the S-state of ISCU.
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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, 2013Co-Authors: Jin Hae Kim, Ronnie O Frederick, Nichole M Reinen, Andrew T Troupis, John L MarkleyAbstract: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.
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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, 2012Co-Authors: Jin Hae Kim, Marco Tonelli, John L MarkleyAbstract: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.
