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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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Cysteine desulfurase activity for Fe–Sassembly complexescontaining different ISCU2 variants. (A) Cysteine desulfurase activityfor SDU complexes with different ISCU2 variants compared to the nativeSDUF complex. The double mutant is ISCU2 variant C35A/C61A. (B) Cysteinedesulfurase activity for the SDUF complexes with saturating amountsof FXN and the ISCU2 variant in the presence and absence of 5 μMFe(NH4)2(SO4)2. Errorbars in panels A and B are for three independent measurements. Allassays were performed with 100 μM l-cysteine.
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fig5: Cysteine desulfurase activity for Fe–Sassembly complexescontaining different ISCU2 variants. (A) Cysteine desulfurase activityfor SDU complexes with different ISCU2 variants compared to the nativeSDUF complex. The double mutant is ISCU2 variant C35A/C61A. (B) Cysteinedesulfurase activity for the SDUF complexes with saturating amountsof FXN and the ISCU2 variant in the presence and absence of 5 μMFe(NH4)2(SO4)2. Errorbars in panels A and B are for three independent measurements. Allassays were performed with 100 μM l-cysteine.

Mentions: Next, we determined that assemblycomplexes that included C104 variants behaved differently than theother cysteine variants with respect to sulfur transfer chemistry.In the absence of FXN, all of the SDU variant complexes exhibitedtypical unstimulated cysteine desulfurase activities that were similarto the native SDU complex (Figure 5A). In thepresence of FXN, the SDUC96SF, SDUC35AF, SDUC35SF, and SDUC61AF complexes exhibited kcat values similar (9.4–11.1 min–1) to the value of 10.7 min–1 forthe native SDUF complex (Table 1). The kcat values for the SDUC61SF (5.9min–1) and SDUC35A/C61AF (6.4 min–1) complexes were slightly compromised compared tothat of the native SDUF complex, whereas complexes for the C104A (0.9min–1) and C104S (2.7 min–1) variantswere more similar to samples that lacked FXN (0.8 min–1). Previously, iron was found to further increase the FXN-based stimulationof the cysteine desulfurase activity and was observed for reactionswith the activated SDUF but not unactivated SDU Fe–S assemblycomplex.15 Here, all SDUF variant complexes,except those that include substitutions at C104, exhibited this characteristiciron-based stimulation of the cysteine desulfurase activity (Figure 5B and Table 1). The lossof Fe-based stimulation under standard conditions was not simply dueto the increased concentrations of the ISCU2 variant or FXN, whichmay compete for iron binding, but was specifically associated withthe C104 variants. For example, the SDUC61AF complex exhibitedthe most dramatic Fe-based stimulation (Figure 5B and Table 1) and includes ∼27-foldmore ISCU2 and ∼7-fold more FXN than the SDUC104AF complex. CD spectroscopy established that the C104 substitutionsdid not impart a global change in the ISCU2 structure (Figure S5 ofthe Supporting Information). These datarevealed that mutations at position C104, but not the other cysteineresidues, were compromised in their FXN-associated stimulation ofthe cysteine desulfurase reaction and suggested a link between ISCU2residue C104 and FXN activation.


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)

Cysteine desulfurase activity for Fe–Sassembly complexescontaining different ISCU2 variants. (A) Cysteine desulfurase activityfor SDU complexes with different ISCU2 variants compared to the nativeSDUF complex. The double mutant is ISCU2 variant C35A/C61A. (B) Cysteinedesulfurase activity for the SDUF complexes with saturating amountsof FXN and the ISCU2 variant in the presence and absence of 5 μMFe(NH4)2(SO4)2. Errorbars in panels A and B are for three independent measurements. Allassays were performed with 100 μM l-cysteine.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Cysteine desulfurase activity for Fe–Sassembly complexescontaining different ISCU2 variants. (A) Cysteine desulfurase activityfor SDU complexes with different ISCU2 variants compared to the nativeSDUF complex. The double mutant is ISCU2 variant C35A/C61A. (B) Cysteinedesulfurase activity for the SDUF complexes with saturating amountsof FXN and the ISCU2 variant in the presence and absence of 5 μMFe(NH4)2(SO4)2. Errorbars in panels A and B are for three independent measurements. Allassays were performed with 100 μM l-cysteine.
Mentions: Next, we determined that assemblycomplexes that included C104 variants behaved differently than theother cysteine variants with respect to sulfur transfer chemistry.In the absence of FXN, all of the SDU variant complexes exhibitedtypical unstimulated cysteine desulfurase activities that were similarto the native SDU complex (Figure 5A). In thepresence of FXN, the SDUC96SF, SDUC35AF, SDUC35SF, and SDUC61AF complexes exhibited kcat values similar (9.4–11.1 min–1) to the value of 10.7 min–1 forthe native SDUF complex (Table 1). The kcat values for the SDUC61SF (5.9min–1) and SDUC35A/C61AF (6.4 min–1) complexes were slightly compromised compared tothat of the native SDUF complex, whereas complexes for the C104A (0.9min–1) and C104S (2.7 min–1) variantswere more similar to samples that lacked FXN (0.8 min–1). Previously, iron was found to further increase the FXN-based stimulationof the cysteine desulfurase activity and was observed for reactionswith the activated SDUF but not unactivated SDU Fe–S assemblycomplex.15 Here, all SDUF variant complexes,except those that include substitutions at C104, exhibited this characteristiciron-based stimulation of the cysteine desulfurase activity (Figure 5B and Table 1). The lossof Fe-based stimulation under standard conditions was not simply dueto the increased concentrations of the ISCU2 variant or FXN, whichmay compete for iron binding, but was specifically associated withthe C104 variants. For example, the SDUC61AF complex exhibitedthe most dramatic Fe-based stimulation (Figure 5B and Table 1) and includes ∼27-foldmore ISCU2 and ∼7-fold more FXN than the SDUC104AF complex. CD spectroscopy established that the C104 substitutionsdid not impart a global change in the ISCU2 structure (Figure S5 ofthe Supporting Information). These datarevealed that mutations at position C104, but not the other cysteineresidues, were compromised in their FXN-associated stimulation ofthe cysteine desulfurase reaction and suggested a link between ISCU2residue C104 and FXN activation.

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