Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin

Abstract

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.

Fig. 1

Characterization of iron-loaded ISCU. a Zinc content of WT ISCU as purified (gray) and after zinc repletion (blue), and of the C35S, D37A, C61A, C96S, H103A, and C104S mutants, indicated as molar ratio of zinc ion per ISCU protein. b CD spectra of Zn-ISCU with (pink) and without FXN (black) and of apo-ISCU (blue), incubated with one molar equivalent of Fe²⁺ ions. c Titration of apo-ISCU with Fe²⁺ ions monitored by CD. Amounts of iron are indicated as molar ratio of iron to ISCU. d Plot of the values of the CD signal at 270 nm from the spectra in c at each concentration of iron. e Mössbauer spectrum of apo-ISCU incubated with one equivalent of ⁵⁷Fe enriched Fe²⁺ ions. The red solid line represents the best fits of the data achieved using the components displayed as black solid lines. See Supplementary Table 1 for parameters. f CD spectra of apoISCU proteins WT (blue), C35S (orange), C61A (brown), D37A (purple), H103A (pink), and C104S (green) incubated with one molar equivalent of Fe²⁺ ions. Errors bars represent standard deviation from n = 3 independent experiments and source data for panels a, d are provided as a Source Data file

Fig. 2

Fe-S cluster assembly by Fe-ISCU and Zn-ISCU. a UV and CD spectra of Fe-S cluster reconstitution reactions by Fe-ISCU (blue) and Zn-ISCU (black) performed under catalytic conditions with one equivalent of L-cysteine. One equivalent of iron was added in the case of Zn-ISCU. b Kinetics of Fe-S cluster reconstitution by Fe-ISCU (blue) and Zn-ISCU (black) as described in a, monitored at 456 nm. c Mössbauer spectrum of reconstitution performed with apo-ISCU in the presence of two equivalents or iron and L-cysteine. The red solid line represents the best fit of the data achieved using the two components displayed as black solid lines (See Supplementary Table 1 for parameters). d Native MS spectra of reconstitution performed with apo-ISCU in the presence of two equivalents or iron and L-cysteine. e, f Titrations of [2Fe2S] clusters in ISCU for various amounts of L-cysteine (e) and iron (f) (Source data are provided in Supplementary Fig. 2b, c and as a Source Data file). g Fe-S cluster reconstitution assays by Fe-ISCU in reactions containing all components as described in a (blue) and missing FDX2 (green) or FXN (red)

Fig. 3

Sequence of Fe-S cluster assembly and functional role of FDX2. a Persulfidation of ISCU monitored by the alkylation assay in the NIAUF complex containing either Fe-ISCU or Zn-ISCU without L-cysteine, with L-cysteine and after addition of FDX2. b Persulfidation of the C104S mutant of Fe-ISCU and of apo-ISCU in the NIAUF complex upon addition of L-cysteine. c–e ESI-Q/TOF deconvoluted mass spectra of Fe-ISCU upon incubation of the NIAUF complex without (c) or with (d) L-cysteine and after Fe-S cluster reconstitution (e), analyzed under denaturing conditions (see Supplementary Fig. 4 for raw spectra and Supplementary Table 3 for expected masses). f Fe-S cluster formation monitored at 456 nm upon addition of FDX2 to the NIAUF complex containing persulfidated Fe-ISCU. g Effect of a metal chelator (DTPA) on persulfide reduction by FDX2. h Effect of FDX2 on the persulfide of NFS1 and Fe-ISCU for various amounts of L-cysteine indicated as molar ratios. i Quantifications of the gels in h expressed as the relative percentages of persulfidated (gray bar) and non-persulfidated (white bar) proteins. Error bars represent standard deviation from n = 3 independent experiments, uncropped gels underlying panels a, b, g, h and source data of panel i are provided as Source Data file

Fig. 4

Functional role of FXN. a, b Kinetics of persulfide formation on Fe-ISCU (a) and Zn-ISCU (b) in the NIAU complex upon addition of L-cysteine, in the absence (red) and presence (blue) of FXN. c Kinetics of Fe-S cluster formation upon addition of FDX2 in the absence (red) and presence (blue) of FXN to the NIAU complex pre-incubated with L-cysteine. d Plot of percentage of persulfidated Fe-ISCU determined by quantification of the gels in a. The black lines represent the fits of data using a first order equation. e Kinetics of Fe-S cluster reconstitution under stoichiometric conditions with and without FXN. The black lines represent the fits of data using a first order equation. f Rate constants of persulfide transfer (gray bars), persulfide reduction combined with formation of the [2Fe2S] cluster (green bars) and global Fe-S cluster assembly upon addition of L-cysteine (orange bars), in the absence and presence of FXN, determined from data in d, c, e using stoichiometric amounts of the NFS1-ISD11-ACP complex and Fe-ISCU (see Methods for rate constant determination). Error bars represent standard deviation from n = 3 independent experiments, uncropped gels underlying panels a, b and source data of panel d, f are provided as Source Data file

Fig. 5

Fe-S cluster reconstitution with Zn-ISCU with excess L-cysteine. a Kinetics of Fe-S cluster assembly by Zn-ISCU (100 μM) monitored by CD at 430 nm, performed under catalytic conditions with the ISC components (NFS1-ISD11-ACP, FXN, FDX2, and FDXR), iron (200 μM) and various amounts of L-cysteine as indicated. b CD spectra of Fe-S cluster reconstitution assays by Zn-ISCU as performed in a with 1 mM of L-cysteine. The spectra were normalized to the spectrum obtained with Fe-ISCU. c Kinetics of Fe-S cluster assembly by Zn-ISCU with 10 equivalents of L-cysteine in standard conditions (blue) and in reactions missing FXN (red) or FDX2 (green);

Fig. 6

Proposed model of Fe-S cluster biosynthesis. The upper part describes physiologically relevant Fe-S cluster assembly by Fe-ISCU. Upon reaction with L-cysteine, a persulfide is generated on the catalytic cysteine of NFS1 that is transferred to the cysteine C104 of Fe-ISCU and FXN accelerates this reaction. The persulfide of Fe-ISCU is then reduced into sulfide by FDX2, which leads to formation of a [2Fe2S] cluster. The [2Fe2S] cluster is putatively formed by dimerization of ISCU that is assisted by the dimeric structure of the NIA complex. The bridged [2Fe2S] cluster subsequently segregates on one subunit and is transferred to recipient apo-proteins by the HSPA9/HSC20 chaperone system. Reloading of ISCU with iron ions, by a still ill-defined chaperone, allows subsequent turnovers. The lower part describes the reaction with Zn-ISCU that is not physiologically relevant. Upon reaction with L-cysteine, a persulfide is generated on NFS1 and is transferred to the cysteine C104 of Zn-ISCU. FXN enhances the rate of this reaction, but the persulfide of Zn-ISCU is not reduced by FDX2. Upon reaction with a second molecule of L-cysteine, the persulfide of NFS1 is regenerated. This persulfide is reduced by thiols such as L-cysteine which leads to formation of free sulfide that combines with free iron to form a Fe-S cluster in Zn-ISCU in a slow and poorly efficient process akin to chemical reconstitution (orange background). FXN accelerates the reduction of the persulfide of NFS1 by thiols