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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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35S radiolabel tracking from a cysteine substrate toa Fe–S cluster on FDX. The SDUF complex was reacted (see Experimental Procedures) with l-[35S]cysteine and (A) fractionated with a HisTrap column. (B) Fractionswere analyzed for protein (top) and radioactivity (bottom) via nonreducing14% SDS–PAGE, and (C) fractions 11–13 correspondingto [35S]SDUF were combined and analyzed for protein (top)and radioactivity (bottom) via nonreducing 6.5% Native PAGE. [35S]SDUF was then reacted with iron (see ExperimentalProcedures) and (D) fractionated on a second HisTrap column.(E) Fractions 2 and 3 from panel D were combined and analyzed vianative PAGE in the absence (labeled 1) and presence (labeled 2) ofDTT. Standards SD, ISCU2, FXN, FDX, and SDU were included for thenative gels; proteins were stained using Coomassie blue, and radioactivitywas detected using a Phosphorimager. The absorbance (blue) at 280(A) or 405 nm (D) was overlaid with a 5 to 500 mM imidazole gradient(pink).
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fig4: 35S radiolabel tracking from a cysteine substrate toa Fe–S cluster on FDX. The SDUF complex was reacted (see Experimental Procedures) with l-[35S]cysteine and (A) fractionated with a HisTrap column. (B) Fractionswere analyzed for protein (top) and radioactivity (bottom) via nonreducing14% SDS–PAGE, and (C) fractions 11–13 correspondingto [35S]SDUF were combined and analyzed for protein (top)and radioactivity (bottom) via nonreducing 6.5% Native PAGE. [35S]SDUF was then reacted with iron (see ExperimentalProcedures) and (D) fractionated on a second HisTrap column.(E) Fractions 2 and 3 from panel D were combined and analyzed vianative PAGE in the absence (labeled 1) and presence (labeled 2) ofDTT. Standards SD, ISCU2, FXN, FDX, and SDU were included for thenative gels; proteins were stained using Coomassie blue, and radioactivitywas detected using a Phosphorimager. The absorbance (blue) at 280(A) or 405 nm (D) was overlaid with a 5 to 500 mM imidazole gradient(pink).

Mentions: To establish thatthe 35S-labeled ISCU2 can functionas a Fe–S cluster assembly intermediate, we tracked the progressionof the 35S label in a two-step reaction equivalent. Inthe first step, we generated a 35S-labeled form of ISCU2by adding l-[35S]cysteine, 3 equiv of ISCU2, and3 equiv of FXN to SD that contained NFS1 labeled with a six-His tag(see Experimental Procedures). The reactionmixture was applied to a HisTrap column and washed with buffer, andproteins associated with six-His NFS1 were eluted with imidazole (Figure 4A). The 35S label was primarily in boundfractions (fractions 11–13) and corresponded to both radiolabeledNFS1 and radiolabeled ISCU2 (Figure 4B). Verylittle radioactivity was associated with the noncomplexed flow-throughfractions (Figure 4B, bottom). A nonreducingnative gel showed that the radioactivity was linked to a slower migratingband (Figure 4C) that had been previously shown15 to be associated with the SDUF complex (FigureS3 of the Supporting Information).


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)

35S radiolabel tracking from a cysteine substrate toa Fe–S cluster on FDX. The SDUF complex was reacted (see Experimental Procedures) with l-[35S]cysteine and (A) fractionated with a HisTrap column. (B) Fractionswere analyzed for protein (top) and radioactivity (bottom) via nonreducing14% SDS–PAGE, and (C) fractions 11–13 correspondingto [35S]SDUF were combined and analyzed for protein (top)and radioactivity (bottom) via nonreducing 6.5% Native PAGE. [35S]SDUF was then reacted with iron (see ExperimentalProcedures) and (D) fractionated on a second HisTrap column.(E) Fractions 2 and 3 from panel D were combined and analyzed vianative PAGE in the absence (labeled 1) and presence (labeled 2) ofDTT. Standards SD, ISCU2, FXN, FDX, and SDU were included for thenative gels; proteins were stained using Coomassie blue, and radioactivitywas detected using a Phosphorimager. The absorbance (blue) at 280(A) or 405 nm (D) was overlaid with a 5 to 500 mM imidazole gradient(pink).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: 35S radiolabel tracking from a cysteine substrate toa Fe–S cluster on FDX. The SDUF complex was reacted (see Experimental Procedures) with l-[35S]cysteine and (A) fractionated with a HisTrap column. (B) Fractionswere analyzed for protein (top) and radioactivity (bottom) via nonreducing14% SDS–PAGE, and (C) fractions 11–13 correspondingto [35S]SDUF were combined and analyzed for protein (top)and radioactivity (bottom) via nonreducing 6.5% Native PAGE. [35S]SDUF was then reacted with iron (see ExperimentalProcedures) and (D) fractionated on a second HisTrap column.(E) Fractions 2 and 3 from panel D were combined and analyzed vianative PAGE in the absence (labeled 1) and presence (labeled 2) ofDTT. Standards SD, ISCU2, FXN, FDX, and SDU were included for thenative gels; proteins were stained using Coomassie blue, and radioactivitywas detected using a Phosphorimager. The absorbance (blue) at 280(A) or 405 nm (D) was overlaid with a 5 to 500 mM imidazole gradient(pink).
Mentions: To establish thatthe 35S-labeled ISCU2 can functionas a Fe–S cluster assembly intermediate, we tracked the progressionof the 35S label in a two-step reaction equivalent. Inthe first step, we generated a 35S-labeled form of ISCU2by adding l-[35S]cysteine, 3 equiv of ISCU2, and3 equiv of FXN to SD that contained NFS1 labeled with a six-His tag(see Experimental Procedures). The reactionmixture was applied to a HisTrap column and washed with buffer, andproteins associated with six-His NFS1 were eluted with imidazole (Figure 4A). The 35S label was primarily in boundfractions (fractions 11–13) and corresponded to both radiolabeledNFS1 and radiolabeled ISCU2 (Figure 4B). Verylittle radioactivity was associated with the noncomplexed flow-throughfractions (Figure 4B, bottom). A nonreducingnative gel showed that the radioactivity was linked to a slower migratingband (Figure 4C) that had been previously shown15 to be associated with the SDUF complex (FigureS3 of the Supporting Information).

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