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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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FRDA variants decrease the level of accumulation of sulfuron NFS1and ISCU2. SD (3 μM) was reacted with 9 μM ISCU2 and 9μM FXN variants (native FXN, N146K, Q148R, I154F, W155R, andR165C) and analyzed as described in the legend of Figure 2.
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fig3: FRDA variants decrease the level of accumulation of sulfuron NFS1and ISCU2. SD (3 μM) was reacted with 9 μM ISCU2 and 9μM FXN variants (native FXN, N146K, Q148R, I154F, W155R, andR165C) and analyzed as described in the legend of Figure 2.

Mentions: Binding of FXN dramatically increases the cysteinedesulfurase and Fe–S cluster biosynthesis activities of theSDU complex.15 To test the hypothesis thatFXN activation promotes the formation of persulfide species on ISCU2,we monitored the transfer of radioactive sulfur from an l-[35S]cysteine substrate to ISCU2 in the presence of theSD complex and the presence or absence of FXN (see Experimental Procedures). The samples were incubated withthe radiolabeled substrate; excess label was removed anaerobicallywith a desalting column, and then the individual subunits of the SDUor SDUF complexes were analyzed via nonreducing SDS–PAGE. Labelingof both NFS1 and ISCU2 indicated the formation of a covalent adduct[likely a persulfide species (see Discussion)] and was promoted by the addition of FXN (Figure 2). SDUF samples exhibited >3-fold larger amounts of labelon NFS1 and >2-fold larger amounts of label on ISCU2 than SDU samples(Figure S1 of the Supporting Information). Next, the abilities of FRDA FXN variants were tested using thesame radiolabeling procedure. Samples of the SDU and SDUF complexeswere compared to SDUF samples containing the N146K, Q148R, I154F,W155R, and R165C FRDA FXN variants (Figure 3). FXN variants exhibited 50–70% lower levels of label incorporationon NFS1 and 45–76% lower levels of label incorporation on ISCU2relative to the SDUF sample (Figure S2 of the Supporting Information). Interestingly, the R165C FXN variantwas also labeled in this experiment, although this is probably a nonphysiologicalevent. Together, these results indicate an increased level of covalentincorporation of sulfur into both NFS1 and ISCU2 in the presence of FXN and that this levelof incorporation is reduced for FRDA FXN variants. Alone, these datado not distinguish if the FXN-dependent increase in the level of incorporationof sulfur into ISCU2 is solely due to the promotion of the NFS1 cysteinedesulfurase activity or also to the acceleration of the transfer ofsulfur between NFS1 and ISCU2.


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)

FRDA variants decrease the level of accumulation of sulfuron NFS1and ISCU2. SD (3 μM) was reacted with 9 μM ISCU2 and 9μM FXN variants (native FXN, N146K, Q148R, I154F, W155R, andR165C) and analyzed as described in the legend of Figure 2.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: FRDA variants decrease the level of accumulation of sulfuron NFS1and ISCU2. SD (3 μM) was reacted with 9 μM ISCU2 and 9μM FXN variants (native FXN, N146K, Q148R, I154F, W155R, andR165C) and analyzed as described in the legend of Figure 2.
Mentions: Binding of FXN dramatically increases the cysteinedesulfurase and Fe–S cluster biosynthesis activities of theSDU complex.15 To test the hypothesis thatFXN activation promotes the formation of persulfide species on ISCU2,we monitored the transfer of radioactive sulfur from an l-[35S]cysteine substrate to ISCU2 in the presence of theSD complex and the presence or absence of FXN (see Experimental Procedures). The samples were incubated withthe radiolabeled substrate; excess label was removed anaerobicallywith a desalting column, and then the individual subunits of the SDUor SDUF complexes were analyzed via nonreducing SDS–PAGE. Labelingof both NFS1 and ISCU2 indicated the formation of a covalent adduct[likely a persulfide species (see Discussion)] and was promoted by the addition of FXN (Figure 2). SDUF samples exhibited >3-fold larger amounts of labelon NFS1 and >2-fold larger amounts of label on ISCU2 than SDU samples(Figure S1 of the Supporting Information). Next, the abilities of FRDA FXN variants were tested using thesame radiolabeling procedure. Samples of the SDU and SDUF complexeswere compared to SDUF samples containing the N146K, Q148R, I154F,W155R, and R165C FRDA FXN variants (Figure 3). FXN variants exhibited 50–70% lower levels of label incorporationon NFS1 and 45–76% lower levels of label incorporation on ISCU2relative to the SDUF sample (Figure S2 of the Supporting Information). Interestingly, the R165C FXN variantwas also labeled in this experiment, although this is probably a nonphysiologicalevent. Together, these results indicate an increased level of covalentincorporation of sulfur into both NFS1 and ISCU2 in the presence of FXN and that this levelof incorporation is reduced for FRDA FXN variants. Alone, these datado not distinguish if the FXN-dependent increase in the level of incorporationof sulfur into ISCU2 is solely due to the promotion of the NFS1 cysteinedesulfurase activity or also to the acceleration of the transfer ofsulfur between NFS1 and ISCU2.

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