Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry

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.

Figure 1

Human NFS1–ISCU2 complex modeled from the crystal structure of the analogous IscS–IscU complex (Protein Data Bank entry 3LVL). NFS1 subunits are colored green and cyan, whereas ISCU2 is colored magenta.

Figure 2

FXN enhances the accumulation of sulfur on NFS1 and ISCU2. Radiolabeled sulfur incorporation from l-[³⁵S]cysteine substrate on NFS1 with subsequent transfer to ISCU2 was monitored by nonreducing SDS–PAGE separation coupled to phosphor imaging. Samples of 3 μM SD and 3–40 equiv of ISCU2 (relative to SD) without FXN and with 9 μM FXN were incubated for 2 min with l-[³⁵S]cysteine and analyzed by SDS–PAGE. The first three lanes correspond to SD, ISCU2, or FXN controls that were incubated for 2 min with l-[³⁵S]cysteine.

Figure 3

FRDA variants decrease the level of accumulation of sulfur on NFS1 and ISCU2. SD (3 μM) was reacted with 9 μM ISCU2 and 9 μM FXN variants (native FXN, N146K, Q148R, I154F, W155R, and R165C) and analyzed as described in the legend of Figure 2.

Figure 4

³⁵S radiolabel tracking from a cysteine substrate to a 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) Fractions were analyzed for protein (top) and radioactivity (bottom) via nonreducing 14% SDS–PAGE, and (C) fractions 11–13 corresponding to [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 Experimental Procedures) and (D) fractionated on a second HisTrap column. (E) Fractions 2 and 3 from panel D were combined and analyzed via native PAGE in the absence (labeled 1) and presence (labeled 2) of DTT. Standards SD, ISCU2, FXN, FDX, and SDU were included for the native gels; proteins were stained using Coomassie blue, and radioactivity was 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).

Figure 5

Cysteine desulfurase activity for Fe–S assembly complexes containing different ISCU2 variants. (A) Cysteine desulfurase activity for SDU complexes with different ISCU2 variants compared to the native SDUF complex. The double mutant is ISCU2 variant C35A/C61A. (B) Cysteine desulfurase activity for the SDUF complexes with saturating amounts of FXN and the ISCU2 variant in the presence and absence of 5 μM Fe(NH₄)₂(SO₄)₂. Error bars in panels A and B are for three independent measurements. All assays were performed with 100 μM l-cysteine.

Figure 6

Conserved cysteines are critical for Fe–S cluster formation on ISCU2. (A) Fe–S cluster formation was monitored at 456 nm by UV–vis spectroscopy as a function of time. (B) Fe–S cluster formation was monitored by CD spectroscopy, and the 60 min time point is displayed. Samples include SDU (yellow), SDUF (red), SDU_C35AF (blue), SDU_C61AF (black) SDU_C96SF (purple), and SDU_C104AF (green).

Figure 7

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