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Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

Bridwell-Rabb J, Fox NG, Tsai CL, Winn AM, Barondeau DP - Biochemistry (2014)

Bottom Line: Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters.These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN.Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77842, United States.

ABSTRACT
Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.

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Human NFS1–ISCU2complex modeled from the crystal structureof the analogous IscS–IscU complex (Protein Data Bank entry 3LVL). NFS1 subunitsare colored green and cyan, whereas ISCU2 is colored magenta.
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fig1: Human NFS1–ISCU2complex modeled from the crystal structureof the analogous IscS–IscU complex (Protein Data Bank entry 3LVL). NFS1 subunitsare colored green and cyan, whereas ISCU2 is colored magenta.

Mentions: Theprimary models for the role of FXN in Fe–S cluster biosynthesisare as an iron donor and as an allosteric activator that participatesin sulfur transfer chemistry. We provide experiments to test thissecond model. The ability of FXN to enhance the cysteine desulfuraseand Fe–S assembly activities implies that it is involved insulfur mobilization and/or transfer chemistry from the cysteine desulfuraseNFS1 to the scaffold protein ISCU2. A 35S radiolabelingexperiment using Saccharomyces cerevisiae mitochondriarevealed a NFS1 persulfide species under iron-depleted conditionsand radiolabeled ferredoxin under iron-replete conditions.23 However, few details about the eukaryotic sulfurhand-off mechanism from NFS1 to ISCU2 (see Figure 1 for a model of the human NFS1–ISCU2 complex) and therole of FXN in this process are known. One advantage of this experimentalsystem compared to the bacterial ISC systems is the significant rateenhancement of the cysteine desulfurase reaction upon FXN binding.Here we build upon this observation and present experiments that supporta model in which FXN stabilizes a conformation that both acceleratesthe formation of persulfide on NFS1 and also interprotein sulfur transferfrom NFS1 to residue C104 on ISCU2 (equivalent to E. coli IscU C106) as an early step in Fe–S cluster biosynthesis.Furthermore, we establish that the persulfide formed on ISCU2 in thesulfur transfer reaction is viable in a subsequent Fe–S clustersynthesis reaction, consistent with a sulfur-first mechanism.


Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

Bridwell-Rabb J, Fox NG, Tsai CL, Winn AM, Barondeau DP - Biochemistry (2014)

Human NFS1–ISCU2complex modeled from the crystal structureof the analogous IscS–IscU complex (Protein Data Bank entry 3LVL). NFS1 subunitsare colored green and cyan, whereas ISCU2 is colored magenta.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215901&req=5

fig1: Human NFS1–ISCU2complex modeled from the crystal structureof the analogous IscS–IscU complex (Protein Data Bank entry 3LVL). NFS1 subunitsare colored green and cyan, whereas ISCU2 is colored magenta.
Mentions: Theprimary models for the role of FXN in Fe–S cluster biosynthesisare as an iron donor and as an allosteric activator that participatesin sulfur transfer chemistry. We provide experiments to test thissecond model. The ability of FXN to enhance the cysteine desulfuraseand Fe–S assembly activities implies that it is involved insulfur mobilization and/or transfer chemistry from the cysteine desulfuraseNFS1 to the scaffold protein ISCU2. A 35S radiolabelingexperiment using Saccharomyces cerevisiae mitochondriarevealed a NFS1 persulfide species under iron-depleted conditionsand radiolabeled ferredoxin under iron-replete conditions.23 However, few details about the eukaryotic sulfurhand-off mechanism from NFS1 to ISCU2 (see Figure 1 for a model of the human NFS1–ISCU2 complex) and therole of FXN in this process are known. One advantage of this experimentalsystem compared to the bacterial ISC systems is the significant rateenhancement of the cysteine desulfurase reaction upon FXN binding.Here we build upon this observation and present experiments that supporta model in which FXN stabilizes a conformation that both acceleratesthe formation of persulfide on NFS1 and also interprotein sulfur transferfrom NFS1 to residue C104 on ISCU2 (equivalent to E. coli IscU C106) as an early step in Fe–S cluster biosynthesis.Furthermore, we establish that the persulfide formed on ISCU2 in thesulfur transfer reaction is viable in a subsequent Fe–S clustersynthesis reaction, consistent with a sulfur-first mechanism.

Bottom Line: Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters.These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN.Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77842, United States.

ABSTRACT
Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.

Show MeSH
Related in: MedlinePlus