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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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Cartoon model of FXN activation of the Fe–S assemblycomplex.(A) SDU complexes exist as an equilibrium mixture between a stableinactive (helix) and less stable active (coil) conformation. (B) FXNbinds to the coil conformation for the C-terminal helix and shiftsthe equilibrium from the inactive to active form. (C) NFS1 reactswith l-cysteine to form a persulfide species on residue C381.(D) Sulfur is transferred from NFS1 to ISCU2 residue C104. (E) Additionof the remaining substrates results in [2Fe-2S] cluster formationon ISCU2. (F) The Fe–S cluster is transferred to an apo target,and the active SDUF assembly complex is re-formed. This last stepmay involve subunit dissociation and/or chaperone proteins.
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fig7: Cartoon model of FXN activation of the Fe–S assemblycomplex.(A) SDU complexes exist as an equilibrium mixture between a stableinactive (helix) and less stable active (coil) conformation. (B) FXNbinds to the coil conformation for the C-terminal helix and shiftsthe equilibrium from the inactive to active form. (C) NFS1 reactswith l-cysteine to form a persulfide species on residue C381.(D) Sulfur is transferred from NFS1 to ISCU2 residue C104. (E) Additionof the remaining substrates results in [2Fe-2S] cluster formationon ISCU2. (F) The Fe–S cluster is transferred to an apo target,and the active SDUF assembly complex is re-formed. This last stepmay involve subunit dissociation and/or chaperone proteins.

Mentions: Accumulating evidence suggests FXNfunctions at the sulfur transferstep in eukaryotic Fe–S cluster biosynthesis,15,26,41 and that this sulfur transferreaction initiates Fe–S cluster biosynthesis (Figure 4). Moreover, these FXN-dependent effects cannotbe explained by the iron donor hypothesis and are consistent withthe loss of Fe–S enzyme activity phenotype upon FXN depletion.We propose that a key aspect of the FXN-based activation of the cysteinedesulfurase and Fe–S biosynthesis reactions revolves aroundthe conformation of the C-terminal α-helix of ISCU2 (Figure 7). We propose an equilibrium mixture between nonfunctional(helix) and functional (coil) conformational states of this α-helixand that FXN functions as an allosteric activator by binding and stabilizingthe coil conformation. This proposal is reminiscent of the differentconformations for the C-terminal helix in the Aquifex aeolicus IscU crystal structure,42 and the mixtureof ordered and disordered states proposed for E. coli IscU by the Markley group.43,44 More specifically,we suggest this conformational change is responsible for the enhancedPLP-dependent chemistry on NFS1 and may also facilitate the transferof sulfur from NFS1 C381 to ISCU2 C104 as an initiating step in Fe–Scluster biosynthesis. Finally, we hypothesize that iron binds to theactivated (coil) conformation of the SDU complex and is incorporatedinto the active site after the transfer of sulfur from NFS1 to ISCU2residue C104, consistent with the link between C104 and the iron-basedstimulation in the cysteine desulfurase activity (Table 1). Future experiments will focus on testing and expandingupon this model with the ultimate goal of developing new strategiesfor treating FRDA.


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)

Cartoon model of FXN activation of the Fe–S assemblycomplex.(A) SDU complexes exist as an equilibrium mixture between a stableinactive (helix) and less stable active (coil) conformation. (B) FXNbinds to the coil conformation for the C-terminal helix and shiftsthe equilibrium from the inactive to active form. (C) NFS1 reactswith l-cysteine to form a persulfide species on residue C381.(D) Sulfur is transferred from NFS1 to ISCU2 residue C104. (E) Additionof the remaining substrates results in [2Fe-2S] cluster formationon ISCU2. (F) The Fe–S cluster is transferred to an apo target,and the active SDUF assembly complex is re-formed. This last stepmay involve subunit dissociation and/or chaperone proteins.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Cartoon model of FXN activation of the Fe–S assemblycomplex.(A) SDU complexes exist as an equilibrium mixture between a stableinactive (helix) and less stable active (coil) conformation. (B) FXNbinds to the coil conformation for the C-terminal helix and shiftsthe equilibrium from the inactive to active form. (C) NFS1 reactswith l-cysteine to form a persulfide species on residue C381.(D) Sulfur is transferred from NFS1 to ISCU2 residue C104. (E) Additionof the remaining substrates results in [2Fe-2S] cluster formationon ISCU2. (F) The Fe–S cluster is transferred to an apo target,and the active SDUF assembly complex is re-formed. This last stepmay involve subunit dissociation and/or chaperone proteins.
Mentions: Accumulating evidence suggests FXNfunctions at the sulfur transferstep in eukaryotic Fe–S cluster biosynthesis,15,26,41 and that this sulfur transferreaction initiates Fe–S cluster biosynthesis (Figure 4). Moreover, these FXN-dependent effects cannotbe explained by the iron donor hypothesis and are consistent withthe loss of Fe–S enzyme activity phenotype upon FXN depletion.We propose that a key aspect of the FXN-based activation of the cysteinedesulfurase and Fe–S biosynthesis reactions revolves aroundthe conformation of the C-terminal α-helix of ISCU2 (Figure 7). We propose an equilibrium mixture between nonfunctional(helix) and functional (coil) conformational states of this α-helixand that FXN functions as an allosteric activator by binding and stabilizingthe coil conformation. This proposal is reminiscent of the differentconformations for the C-terminal helix in the Aquifex aeolicus IscU crystal structure,42 and the mixtureof ordered and disordered states proposed for E. coli IscU by the Markley group.43,44 More specifically,we suggest this conformational change is responsible for the enhancedPLP-dependent chemistry on NFS1 and may also facilitate the transferof sulfur from NFS1 C381 to ISCU2 C104 as an initiating step in Fe–Scluster biosynthesis. Finally, we hypothesize that iron binds to theactivated (coil) conformation of the SDU complex and is incorporatedinto the active site after the transfer of sulfur from NFS1 to ISCU2residue C104, consistent with the link between C104 and the iron-basedstimulation in the cysteine desulfurase activity (Table 1). Future experiments will focus on testing and expandingupon this model with the ultimate goal of developing new strategiesfor treating FRDA.

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