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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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Conserved cysteines arecritical for Fe–S cluster formationon ISCU2. (A) Fe–S cluster formation was monitored at 456 nmby UV–vis spectroscopy as a function of time. (B) Fe–Scluster formation was monitored by CD spectroscopy, and the 60 mintime point is displayed. Samples include SDU (yellow), SDUF (red),SDUC35AF (blue), SDUC61AF (black) SDUC96SF (purple), and SDUC104AF (green).
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fig6: Conserved cysteines arecritical for Fe–S cluster formationon ISCU2. (A) Fe–S cluster formation was monitored at 456 nmby UV–vis spectroscopy as a function of time. (B) Fe–Scluster formation was monitored by CD spectroscopy, and the 60 mintime point is displayed. Samples include SDU (yellow), SDUF (red),SDUC35AF (blue), SDUC61AF (black) SDUC96SF (purple), and SDUC104AF (green).

Mentions: We then measured Fe–S cluster formationrates for ISCU2 variants using two different spectroscopic assays.First, the increase in absorbance at 456 nm was used as a measureof the Fe–S cluster assembly activity. The ISCU2 C96S variant,which eliminates the nonconserved cysteine, had a Fe–S clusterassembly activity similar to those of the native SDU and SDUF complexes(Figure 6A), indicating that this residue isessential neither for cluster ligation nor for the mechanism of clusterformation. In contrast, SDUF complexes containing the C35A, C61A,and C104A ISCU2 variants had a dramatic loss of activity relativeto that of native SDUF (Figure 6A). Interestingly,there is a low-level increase in absorbance for samples containingthe C35A, C61A, and C104A variants or native ISCU2 in the absenceof FXN, which would be consistent with sulfide- and iron-dependentsolution chemistry. Consistent with this idea, the samples with thelowest cysteine desulfurase activity, SDU and SDUC104AF(Table 1), exhibited the slowest increase inabsorbance (Figure 6A). Second, we monitoredthe 300–600 nm region under DTT-free conditions using circulardichroism (CD) spectroscopy, which is sensitive to the PLP cofactorand to protein-bound Fe–S cluster species. A CD signal withmaxima at 330 and 430 nm developed for samples of SDUF containingthe native ISCU2 or the C96S variant (Figure 6B) that appeared to be similar to the [2Fe-2S] cluster bound to bacterialIscU.14 The development of this Fe–Ssignal was absent for assembly complex samples that contained theC35A, C61A, and C104A variants. These samples had a CD signal witha maximum at 420 nm, but this signal is due to the PLP cofactor ofNFS1 rather than an Fe–S cluster. Together, these data indicatethat C96 is not required for the Fe–S assembly reaction andthat all three conserved cysteines (C35, C61, and C104) are essentialfor enzymatic Fe–S cluster formation.


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)

Conserved cysteines arecritical for Fe–S cluster formationon ISCU2. (A) Fe–S cluster formation was monitored at 456 nmby UV–vis spectroscopy as a function of time. (B) Fe–Scluster formation was monitored by CD spectroscopy, and the 60 mintime point is displayed. Samples include SDU (yellow), SDUF (red),SDUC35AF (blue), SDUC61AF (black) SDUC96SF (purple), and SDUC104AF (green).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Conserved cysteines arecritical for Fe–S cluster formationon ISCU2. (A) Fe–S cluster formation was monitored at 456 nmby UV–vis spectroscopy as a function of time. (B) Fe–Scluster formation was monitored by CD spectroscopy, and the 60 mintime point is displayed. Samples include SDU (yellow), SDUF (red),SDUC35AF (blue), SDUC61AF (black) SDUC96SF (purple), and SDUC104AF (green).
Mentions: We then measured Fe–S cluster formationrates for ISCU2 variants using two different spectroscopic assays.First, the increase in absorbance at 456 nm was used as a measureof the Fe–S cluster assembly activity. The ISCU2 C96S variant,which eliminates the nonconserved cysteine, had a Fe–S clusterassembly activity similar to those of the native SDU and SDUF complexes(Figure 6A), indicating that this residue isessential neither for cluster ligation nor for the mechanism of clusterformation. In contrast, SDUF complexes containing the C35A, C61A,and C104A ISCU2 variants had a dramatic loss of activity relativeto that of native SDUF (Figure 6A). Interestingly,there is a low-level increase in absorbance for samples containingthe C35A, C61A, and C104A variants or native ISCU2 in the absenceof FXN, which would be consistent with sulfide- and iron-dependentsolution chemistry. Consistent with this idea, the samples with thelowest cysteine desulfurase activity, SDU and SDUC104AF(Table 1), exhibited the slowest increase inabsorbance (Figure 6A). Second, we monitoredthe 300–600 nm region under DTT-free conditions using circulardichroism (CD) spectroscopy, which is sensitive to the PLP cofactorand to protein-bound Fe–S cluster species. A CD signal withmaxima at 330 and 430 nm developed for samples of SDUF containingthe native ISCU2 or the C96S variant (Figure 6B) that appeared to be similar to the [2Fe-2S] cluster bound to bacterialIscU.14 The development of this Fe–Ssignal was absent for assembly complex samples that contained theC35A, C61A, and C104A variants. These samples had a CD signal witha maximum at 420 nm, but this signal is due to the PLP cofactor ofNFS1 rather than an Fe–S cluster. Together, these data indicatethat C96 is not required for the Fe–S assembly reaction andthat all three conserved cysteines (C35, C61, and C104) are essentialfor enzymatic Fe–S cluster formation.

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