Protein purification
Homo sapiens ISCU2 (Q9H1K1-1), (NIA) 2 (Q9Y697, Q9HD34 and O14561), FXN (Q16595), FDXR (P22570) and wild-type and mutant FDX2 (Q6P4F2) proteins were prepared as previously described18. BL21 Star (DE3) cells were transformed with the corresponding plasmids: pCDFDuet-His 6 -Thr-NFS1 (site 1), ISD11 (site 2) with pET21(+)-ACP for (NIA) 2 , pET28a(+)-His 6- Thr-ISCU2 for ISCU2, pET28a(+)-His 6- Thr-FDX2 for wild-type and mutant FDX2 constructs, pET28a(+)-His 6 -Thr-FXN for FXN, and pCDFDuet-His 6 -FDXR with pET28a(+)-GroEL for FDXR, where His 6 corresponds to a 6× histidine tag and Thr corresponds to a thrombin cleavage site with sequence SQDLVPRGS. Cells were cultured in LB with appropriate antibiotics at 37 °C at 180 rpm. Cell culture transformed with wild-type and mutant FDX2 were supplemented with FeSO 4 to a final concentration of 10 µM, and FDXR growth was supplemented with riboflavin to a final concentration of 3.75 µM. Protein overexpression was induced by IPTG to a final concentration of 1 mM. (NIA) 2 and FDXR were induced when the optical density at 600 nm (OD 600 ) reached 0.6, and incubated overnight at 25 °C (FDXR) or 18 °C ((NIA) 2 ). ISCU2, FXN and FDX2 were induced when the OD 600 reached 0.7 and incubated for 3.5 h at 30 °C. Cells were collected by centrifugation at 4,500 rpm for 20 min at 4 °C with a Beckman Coulter JLA-10.500 rotor, and resuspended in His buffer A (20 mM Tris-HCl, 250 mM NaCl and 5 mM imidazole, pH 8.0) with the addition of protease inhibitor (cOmplete Protease Inhibitor Cocktail). Cells were lysed by sonication and cell debris was removed by centrifugation at 45,000 rpm for 45 min at 4 °C with a Beckman Coulter Type 70 Ti rotor. Supernatant was loaded to a Cytiva 5-ml HisTrap HP pre-equilibrated with His buffer A and eluted over a linear gradient of 0–100% His buffer B (20 mM Tris-HCl, 250 mM NaCl and 500 mM imidazole, pH 8.0). Fractions were pooled and concentrated by ultracentrifugation using Amicon Ultra-15 Centrifugal Filter Units with a molecular-mass cut-off of 10 kDa (ISCU2, FXN, FDX2 and FDXR) or 50 kDa for (NIA) 2 . Proteins (ISCU2, FXN, FDX2 and (NIA) 2 ) were buffer-exchanged into SEC buffer (20 mM Tris-HCl and 250 mM NaCl, pH 8.0) using a NAP-5 desalting column, and FDXR was buffer-exchanged into ion-exchange buffer A (20 mM Tris-HCl and 25 mM NaCl, pH 8.0). ISCU2, FXN and FDX2 were treated with recombinant thrombin protease to remove the His 6 -tag. ISCU2 was incubated with 50 molar eq. DTT and 100 eq. DTPA for 60 min at 20 °C. (NIA) 2 was incubated with 50 eq. DTT, 2 eq. PLP and 4 eq. TCEP for 60 min at 20 °C. Proteins were subjected to size-exclusion chromatography using HiLoad 16/600 columns packed with Superdex 75 pg (ISCU2, FXN and FDX2) or 200 pg for (NIA) 2 pre-equilibrated with SEC buffer (FXN and FDX2) or SEC buffer + 10 mM DTT (ISCU2 and (NIA) 2 ). Proteins were eluted and fractions were pooled. FDXR was loaded onto a Cytiva 5-ml HiTrap Q-Sepharose column and eluted with a linear gradient of 0–100% ion-exchange buffer B (20 mM Tris-HCl, 500 mM NaCl, pH 8.0). Proteins were concentrated to 0.5 ml using the corresponding Amicon Ultra-15 Centrifugal Filter Unit and buffer-exchanged under anaerobic conditions (< 2 ppm O 2 ) using a NAP-5 column into either degassed Tris buffer (20 mM Tris-HCl and 100 mM NaCl, pH 8.0) or sodium phosphate buffer (50 mM Na 2 HPO 4 and 150 mM NaCl, pH 8.0) sparged with argon for 60 min. Protein concentrations were determined by UV-visible spectroscopy at 280 nm in urea buffer (100 mM Tris-HCl and 8 M urea, pH 7.5) using absorption coefficients of 52,260 M−1 cm−1, 9,970 M−1 cm−1, 26,930 M−1 cm−1 and 44,920 M−1 cm−1 for (NIA) 2 , ISCU2, FXN and FDXR, respectively. The FDX2 protein concentration was determined by UV-visible spectroscopy at 456 nm in Tris buffer using an absorption coefficient of 10,000 M−1 cm−1 based on the concentration of the [2Fe–2S] cluster as determined using the ferrozine method. Final preparations were aliquoted, flash-frozen in liquid N 2 and stored in liquid N 2 .
Assays of [2Fe–2S] cluster assembly
Fe–S cluster assembly reactions were performed under anaerobic conditions (< 2 ppm O 2 ) in a glove box. Kinetic assays were routinely composed of 20 µM apo-ISCU2 incubated with 20 µM ferrous ammonium sulfate (Fe-(NH 4 ) 2 (SO 4 ) 2 ), 2 µM of the (NFS1–ISD11–ACP) complex, 1 µM FDXR and 40 µM NADPH. Titrations were performed with 0–50 molar equivalents of FXN (corresponding to 0–100 µM, where one equivalent is relative to the concentration of the (NFS1–ISD11–ACP) complex), and 0–10 eq. of either wild-type or mutant FDX2 (0–20 µM). The reaction mix was transferred into a 384-well plate and incubated at 25 °C in a Tecan Spark microplate reader. Biosynthesis of [2Fe–2S] was initiated by the injection of 30 µM -cysteine to a final volume of 100 µl and kinetics were measured at 456 nm at 25 °C. The data were collected using SparkControl Magellan 3.0. The kinetic rates of [2Fe–2S] were calculated on the basis of the slope at the start of the curve corresponding to the maximum rate.
ARBS assays
Assays were routinely performed in sodium phosphate buffer (50 mM Na 2 HPO 4 and 150 mM NaCl, pH 8.0) at 25 °C with a concentration of ISCU2 and equimolar ferrous ammonium sulfate and/or the (NFS1–ISD11–ACP) complex at 7.5 µM. Concentrations of FDX2 and FXN ranged from 0–10 eq. (0–75 µM) as indicated within the text. For studies following persulfide formation on NFS1, reactions were initiated with 7.5 µM l-cysteine. Assays following persulfide transfer from NFS1 to ISCU2 were initiated with 15 µM l-cysteine. Persulfide reduction assays were initiated with 7.5 µM l-cysteine followed by the addition of a preincubated mixture of FDX2, FDXR and NADPH to final concentrations of 7.5 µM, 3.75 µM and 40 µM, respectively. Reactions were sampled at respective timepoints by mixing 15 µl of the reaction mix with 5 µl of a stop mix making up a fivefold molar excess of mal-dPEG relative to the total thiol concentration (including l-cysteine and total cysteine residues), a 2.5-fold molar excess of EDTA relative to the total iron concentration and a final concentration of 1% SDS. To monitor the persulfidation state of ISCU2 in the presence of FDX2 and prevent overlap of alkylated ISCU2 and FDX2 on gel, which both contain four cysteine residues and have similar molecular weights of 14.4 kDa and 14.3 kDa, respectively, we identified an optimal concentration of SDS in which the solvent-accessible cysteine residues of ISCU2 were available for alkylation, whereas the cysteine residues of FDX2 ligating the [2Fe–2S] cofactor remained partially buried and shielded from alkylation (Supplementary Fig. 1). A final concentration of 0.08% SDS was found to be optimal for this detection. After 30 min of reaction with the stop mix, leading to full alkylation of ISCU2 and/or NFS1, 10 µl of reducing loading dye (60 mM Tris-HCl, 25% glycerol, 2% SDS, 700 mM 2-mercaptoethanol and 0.1% bromophenol blue) was added. Reaction aliquots were analysed by SDS–PAGE on 8% (NFS1) and 14% (ISCU2) acrylamide/bis-acrylamide 19:1 gels. The gels were imaged using an Odyssey Clx scanner (Li-COR) and the data were collected and analysed using Image Studio 5.2.
FIDA
Stock solutions of labelled FXN and FDX2, referred to as FXN ALC and FDX2 ALC , were prepared by conjugation with ALC 480 using the corresponding Fidabio Protein Labelling Kit (Fida Biosystems). For labelling reactions, FXN and FDX2 were first buffer-exchanged into sodium phosphate buffer (50 mM Na 2 HPO 4 and 150 mM NaCl, pH 8.0) using a NAP-5 column. Then, 75 µl of protein sample (FXN or FDX2) at 100 µM was incubated with 7.5 µl 1 M sodium bicarbonate. A 4 mg ml−1 reactive dye stock solution was prepared by mixing 100 µg ALC 480 with 25 µl DMSO. Then, 7.5 µl of the dye stock solution was incubated at 21 °C for 30 min with the protein sample, protected from light, to induce labelling using a fivefold molar ratio of dye to protein. Excess dye was removed and proteins were transferred to Tris buffer (20 mM Tris-HCl and 100 mM NaCl, pH 8.0) using a NAP-5 column. The concentration of FXN ALC was measured by UV-visible spectroscopy at 280 nm using an absorption coefficient of 26,930 M−1 cm−1. Because the ALC 480 dye interferes at wavelengths 400–540 nm, the concentration of FDX2 ALC was measured by UV-visible spectroscopy at 320 nm using an absorption coefficient of 20,450 M−1 cm−1 as determined by the ferrozine method. Labelled proteins were aliquoted, flash-frozen in liquid N 2 and stored in liquid N 2 . As a control to ensure that the label was not interfering with protein function, we performed [2Fe–2S] cluster assembly kinetics, and observed that the efficiencies of both FXN ALC and FDX2 ALC were comparable with those of the unlabelled proteins (Extended Data Fig. 2b).
Binding curves of FXN ALC or FDX2 ALC with the (NIAU) 2 complex were generated using a premix method in which the indicator sample (the labelled protein) is preincubated with the analyte (the binding partner (NIAU) 2 ) before analysis by FIDA. For the FXN ALC binding curve, data points are gathered by incubating a fixed concentration of 20 nM FXN ALC with a series of (NIAU) concentrations spanning 0–16 µM (corresponding to 0–8 µM of the dimerized (NIAU) 2 complex) in a final volume of 20 µl. The data points of FDX2 ALC with the (NIAU) 2 complex is prepared by incubating a fixed concentration of 20 nM FDX2 ALC with a titration of 0–128 µM of the (NIAU) complex (corresponding to 0–64 µM of the dimerized (NIAU) 2 complex) in a final volume of 20 µl. Protein samples were prepared in Tris buffer (20 mM Tris-HCl and 100 mM NaCl, pH 8.0) and samples were incubated for a minimum of 10 min to ensure binding equilibrium is reached before assay.
For competition between FXN ALC and either wild-type or mutant FDX2 constructs for the (NIAU) 2 complex, a fixed concentration of FXN ALC (20 nM) and (NIAU) (2 µM) was titrated by unlabelled FDX2 spanning 0–64 µM. For measurements with reduced FDX2, deoxygenated solutions of the analyte, indicator and buffer were prepared under anaerobic conditions in a glove box. Then, 10 molar eq. of dithionite were added to each FDX2 sample to reduce and keep it reduced throughout the experiment. The full reduction of FDX2 was checked by UV-visible spectroscopy at 456 nm. The analyte, indicator and buffer were loaded into capped vials sealed with tape and placed into a septum sealed tube before analysis. We checked by UV-visible spectroscopy that FDX2 remained reduced under the conditions and duration of FIDA analysis after removal of the tape on top of the cap while keeping tape around the seal. For the competition between FDX2 ALC and FXN for the (NIAU) 2 complex, a fixed concentration of FDX2 ALC (20 nM) and (NIAU) (16 µM) was titrated by 0–256 µM unlabelled FXN.
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