Mechanism of frataxin "bypass" in human iron-sulfur cluster biosynthesis with implications for Friedreich's ataxia

Abstract

In humans, mitochondrial iron-sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron-sulfur cluster assembly scaffold protein (ISCU2). The protein frataxin (FXN) is an allosteric activator that binds the assembly complex and stimulates the cysteine desulfurase and iron-sulfur cluster assembly activities. FXN depletion causes loss of activity of iron-sulfur-dependent enzymes and the development of the neurodegenerative disease Friedreich's ataxia. Recently, a mutation that suppressed the loss of the FXN homolog in Saccharomyces cerevisiae was identified that encodes an amino acid substitution equivalent to the human variant ISCU2 M140I. Here, we developed iron-sulfur cluster synthesis and transfer functional assays and determined that the human ISCU2 M140I variant can substitute for FXN in accelerating the rate of iron-sulfur cluster formation on the monothiol glutaredoxin (GRX5) acceptor protein. Incorporation of both FXN and the M140I substitution had an additive effect, suggesting an acceleration of distinct steps in iron-sulfur cluster biogenesis. In contrast to the canonical role of FXN in stimulating the formation of [2Fe-2S]-ISCU2 intermediates, we found here that the M140I substitution in ISCU2 promotes the transfer of iron-sulfur clusters to GRX5. Together, these results reveal an unexpected mechanism that replaces FXN-based stimulation of the iron-sulfur cluster biosynthetic pathway and suggest new strategies to overcome the loss of cellular FXN that may be relevant to the development of therapeutics for Friedreich's ataxia.

Keywords: ISCU2 M140I; analytical ultracentrifugation; circular dichroism (CD); cysteine labeling; enzyme kinetics; fluorescence anisotropy; iron–sulfur assembly; iron–sulfur protein; mitochondrial disease; neurodegeneration.

Figure 1.

Sulfur transfer and [2Fe–2S] synthesis reactions.

Figure 2.

ISCU2 and ISCU2^M140I are primarily monomeric in solution. AUC experiments revealed that ISCU2 (88.3 μm red and 29.5 μm green) and ISCU2^M140I (88.3 μm blue and 29.5 μm black) were primarily monomeric species with little evidence of aggregate or higher order oligomer. Inset, ISCU2 and ISCU2^M140I samples were eluted from an analytical size exclusion chromatography as a single peak (elution volume = 17.2 ml) prior to the AUC analysis. The estimated molecular mass from the AUC experiment is 15 kDa, consistent with the expected mass of 14.35 kDa.

Figure 3.

The M140I substitution does not significantly affect the ability of ISCU2 to form the iron–sulfur assembly complex. Fluorophore-labeled FXN (0.1 μm) was combined with 100 μm l-cysteine, 200 μm Fe²⁺, and either 30 μm ISCU2 (black) or ISCU2^M140I (red). Fluorescence anisotropy (excitation, 560 nm; emission, 605) was measured upon titration with the SDA_ec complex. Each measurement is an average from three experiments with a maximum standard deviation of 5 millianisotropy units. The dashed lines are the fit to a binding curve. The K_d values for SDA_ecUF and SDA_ecU^M140IF are 1.5 ± 0.1 and 2.8 ± 0.5 μm, respectively.

Figure 4.

FXN and the ISCU2^M140I substitution accelerate different steps in iron–sulfur cluster biosynthesis. The kinetics of iron–sulfur cluster synthesis and transfer to GRX5 were monitored for different assembly complexes by the change in ellipticity at 450 nm. The average change in ellipticity (n = 3; maximum error of 1.8 in ellipticity) is plotted and fit with an exponential rise equation. The final CD spectra are shown in Fig. S3. The color scheme is as follows: red, SDA_ecU; blue, SDA_ecUF; black, SDA_ecU^M140I; and green, SDA_ecU^M140IF.

Figure 5.

Iron–sulfur assembly complexes containing native ISCU2 or ISCU2^M140I exhibit similar kinetic parameters for the cysteine desulfurase reaction. Cysteine desulfurase activities for complexes without (a, SDA_ecU in red and SDA_ecU^M140I in black) and with FXN (b, SDA_ecUF in blue and SDA_ecU^M140IF in green) at different concentrations of l-cysteine. The lines through the data are the fits to the Michaelis–Menten equation. The experiments were performed in triplicate. The native SDA_ecU and SDA_ecUF data are reported in Ref. and shown here for comparison.

Figure 6.

FXN and the ISCU2^M140I substitution accelerate different steps in iron–sulfur cluster biosynthesis. a, iron–sulfur cluster assembly reactions on ISCU2 were monitored for assembly complexes by the change in ellipticity at 330 nm. The average change in ellipticity (n = 3; maximum error of 1.8 in ellipticity) is plotted and fit with a linear equation. The complete time course of the reactions and final CD spectra are shown in Fig. S4. b, kinetics of cluster transfer from preformed holo-ISCU2 to apo-GRX5 were monitoring by the change in ellipticity at 450 nm. The reactions were initiated (time = 0) by the anaerobic injection of GRX5 (with or without FXN). The average change in ellipticity (n = 3; maximum error of 0.8 in ellipticity) is plotted and fit with a linear equation. The CD spectra before the addition of GRX5 and the complete time course of the reaction are shown in Figs. S5 and S6. The color scheme is as follows: red, SDA_ecU; blue, SDA_ecUF; black, SDA_ecU^M140I; and green, SDA_ecU^M140IF.