Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly

Abstract

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

Keywords: Iron-Sulfur Protein; Metal Homeostasis; Metals; Mitochondria; Mitochondrial Metabolism; Pyridoxal Phosphate; Sulfur; Yeast.

FIGURE 1.

Analysis of proteins expressed in bacteria. A, various proteins were expressed in bacteria in soluble forms. These include Nfs1-His₆ together with Isd11 (Nfs1·Isd11, lane 1), Nfs1-His₆ alone (lane 2), Nfs1 (K263A)-His₆ alone (Nfs1_mut, lane 3), Isd11-His₆ alone (lane 4), Yfh1-His₆ (lane 5), Yfh1 (W98G)-His₆ (Yfh1_mut, lane 6), Isu1-His₆ (lane 7), and Isu1 (M107I)-His₆ (Isu1_Sup, lane 8). These soluble proteins were purified by Ni-NTA-agarose chromatography. Proteins were analyzed on 15% gel by SDS-PAGE under reducing conditions, followed by silver staining of the gel. Std (lane 9) indicates protein standards with their molecular mass in kDa. B, reaction mixture contained the Nfs1·Isd11 complex (100 ng, lane 1), Nfs1 alone (100 ng, lane 2), or Nfs1 (100 ng) plus various proteins (100 ng each) as indicated (lanes 3–8) in HS buffer (20 mm Hepes/KOH, pH 7.5, 0.6 m sorbitol) containing 0.15 m NaCl. Samples were supplemented with 0.1 μm [³⁵S]cysteine (5 μCi) and 0.15 mm PLP in a final volume of 50 μl. After incubation at 30 °C for 15 min, proteins were precipitated with 10% TCA and analyzed on 12% gel by nonreducing SDS-PAGE and autoradiography for covalent [³⁵S]persulfide formation on Nfs1.

FIGURE 2.

Yfh1 or Isu1_Sup stimulates persulfide formation by the purified Nfs1·Isd11 complex. A, as indicated, the Nfs1·Isd11 complex (100 ng) was mixed with Yfh1 (1× = 50 ng) and/or Isu1 (100 ng) in HS buffer containing 0.15 m NaCl. Samples were supplemented with 0.1 μm [³⁵S]cysteine (5 μCi) and 0.15 mm PLP in a final volume of 50 μl. After incubation at 30 °C for 15 min, proteins were precipitated with 10% TCA and analyzed by nonreducing SDS-PAGE and autoradiography. B, Nfs1·Isd11 complex (100 ng) was mixed with Isu1_Sup (1× = 50 ng) and/or Isu1 (100 ng) as indicated, and assays were performed as in A. C, Nfs1·Isd11 complex (100 ng) in HS buffer containing 0.15 m NaCl was incubated at 30 °C for 10 min, with or without added Yfh1_mut (1× = 50 ng). As indicated, reaction mixtures were then supplemented with Yfh1 or Isu1_Sup (1× = 50 ng). Following addition of 0.1 μm [³⁵S]cysteine (5 μCi) and 0.15 mm PLP, samples were incubated at 30 °C for 15 min and analyzed as in A.

FIGURE 3.

Yfh1 or Isu1_Sup stimulates cysteine binding to purified Nfs1. A, as indicated, Nfs1 (100 ng) was mixed with Yfh1 or Isu1_Sup (1× = 50 ng) in HS buffer containing 0.15 m NaCl. Samples were then supplemented with [³⁵S]cysteine (0.1 μm; 5 μCi) and 0.15 mm PLP in a final volume of 50 μl. After incubation at 30 °C for 15 min, proteins were precipitated with ice-cold ammonium sulfate (final 67%) and centrifuged to remove excess and unbound [³⁵S]cysteine. Protein-bound radioactivity was determined by scintillation counting. Identical samples in which wild-type Nfs1 was replaced with the Nfs1 (K263A) mutant served as respective background controls. Background counts were subtracted, and data are from three separate experiments. B, Nfs1 (100 ng) was incubated at 30 °C for 15 min with increasing amounts of Yfh1 or Isu1_Sup (1× = 50 ng) in HS buffer containing NaCl (0.15 m), [³⁵S]cysteine (0.1 μm; 5 μCi) and PLP (0.15 mm) (first step). Nfs1 (K263A) served as negative control. Proteins were precipitated with ammonium sulfate and centrifuged. The protein pellets were solubilized with HS buffer containing 0.15 m NaCl and 0.15 mm PLP and incubated with Isd11 (100 ng) at 30 °C for 15 min (second step). Samples were analyzed by nonreducing SDS-PAGE and autoradiography.

FIGURE 4.

Persulfide formation by purified Nfs1 from already bound cysteine: no significant effect of added Yfh1 or Isu1_Sup. Nfs1 (100 ng) or the Nfs1 (K263A) mutant (100 ng) was incubated with [³⁵S]cysteine (0.1 μm; 5 μCi) and PLP (0.15 mm) in HS buffer containing 0.15 m NaCl at 30 °C for 15 min (first step). Proteins were precipitated with ammonium sulfate and centrifuged to remove excess and unbound [³⁵S]cysteine. The protein pellet containing Nfs1 with bound [³⁵S]cysteine was dissolved in HS buffer plus 0.15 m NaCl and 0.15 mm PLP. As indicated, reaction mixtures were supplemented with increasing amounts of Yfh1 or Isu1_Sup (1× = 50 ng). Following addition of Isd11 (100 ng), samples were incubated at 30 °C for 15 min (second step) and subsequently analyzed by nonreducing SDS-PAGE and autoradiography.

FIGURE 5.

Yfh1 or Isu1_Sup does not require iron for stimulating cysteine binding to purified Nfs1. Nfs1 (100 ng) was mixed with [³⁵S]cysteine (0.1 μm; 5 μCi) and 0.15 mm PLP in the absence or presence of Yfh1 (100 ng) or Isu1_Sup (100 ng) in 50 μl of HS buffer containing 0.15 m NaCl. As indicated, reaction mixtures were supplemented with ferrous ascorbate (5–50 μm) or EDTA (5–50 mm). After incubation at 30 °C for 15 min (first step), proteins were precipitated with ammonium sulfate and centrifuged to remove excess and unbound [³⁵S]cysteine, PLP, and ferrous ascorbate (or EDTA). The protein pellets were solubilized with HS buffer containing 0.15 m NaCl and 0.15 mm PLP. After addition of Isd11 (100 ng), reaction mixtures were incubated at 30 °C for 15 min (second step). Samples were analyzed by nonreducing SDS-PAGE and autoradiography.

FIGURE 6.

Cysteine interaction with purified Nfs1-bound cofactor: stimulatory effects of added Yfh1 or Isu1_Sup. A, Nfs1 (100 ng) in HS buffer containing 0.15 m NaCl was incubated at 30 °C for 10 min, with or without PLP (0.15 mm) (first step). Proteins were precipitated with ammonium sulfate and centrifuged to remove excess and unbound PLP. The protein pellets were dissolved in HS buffer plus 0.15 m NaCl. Samples were then supplemented with increasing amounts of Yfh1 as indicated (1× = 50 ng). Following addition of [³⁵S]cysteine (0.1 μm; 5 μCi) and Isd11 (100 ng), reaction mixtures were incubated at 30 °C for 15 min in the absence or presence of added PLP (0.15 mm) (second step). Samples were analyzed by nonreducing SDS-PAGE and autoradiography. B, same as in A except Yfh1 was replaced with Isu1_Sup during the second step of the assay.

FIGURE 7.

Yfh1 or Isu1_Sup exposes the substrate-binding sites of purified Nfs1 for effective interaction with cysteine. Nfs1 (100 ng) was incubated at 30 °C for 15 min with unlabeled cysteine (10 or 100 μm) in HS buffer containing NaCl (0.15 m) and PLP (0.15 mm) (first step). After ammonium sulfate precipitation, the protein pellets were solubilized with HS buffer plus 0.15 m NaCl, and Yfh1 or Isu1_Sup (1× = 50 ng) was added as indicated. Samples were incubated with [³⁵S]cysteine (0.1 μm; 5 μCi), PLP (0.15 mm), and Isd11 (100 ng) at 30 °C for 15 min (second step) and then analyzed by nonreducing SDS-PAGE and autoradiography.

FIGURE 8.

Yfh1 or Isu1_Sup does not promote exchange of Nfs1-bound substrate with free substrate. A, Nfs1 (100 ng) was supplemented with unlabeled cysteine (10 μm) and/or Yfh1 (100 ng) as indicated. All reaction mixtures contained PLP (0.15 mm). Samples were incubated at 30 °C for 15 min (first step). After ammonium sulfate precipitation, the protein pellets were solubilized with HS buffer plus 0.15 m NaCl, and Yfh1 (100 ng) was added to some samples as indicated. Following addition of [³⁵S]cysteine (1× = 0.1 μm; 5 μCi), reaction mixtures were incubated at 30 °C for 10 min prior to addition of Isd11 (100 ng). The incubation at 30 °C was continued for another 15 min (second step). Following TCA precipitation, proteins were analyzed by nonreducing SDS-PAGE and autoradiography. B, same as in A except Yfh1 was replaced with Isu1_Sup in both first and second steps of the assay.

FIGURE 9.

Addition of purified Yfh1 enhances cysteine binding to Nfs1 in mitochondrial lysate. A, mitochondria were isolated from a wild-type (WT) and a hypomorphic nfs1 mutant (MA14) yeast strains. Intact mitochondria (200 μg of proteins) in HS buffer were incubated at 30 °C for 10 min to deplete endogenous nucleotides. Mitochondria were recovered by centrifugation and resuspended in 20 mm Hepes/KOH, pH 7.5. Mitochondrial membranes were ruptured, and the lysate thus obtained was supplemented with [³⁵S]cysteine (0.1 μm; 10 μCi), NaCl (0.15 m), KOAc (40 mm), and Mg(OAc)₂ (10 mm). The final volume was 100 μl. After incubation at 30 °C for 15 min, proteins were precipitated with 10% TCA and analyzed by nonreducing SDS-PAGE and autoradiography. B, mitochondria were isolated from the Gal-Isd11 strain after a 9 h depletion of Isd11 in galactose-free medium. Intact Isd11-depleted mitochondria (Isd11▾) in HS buffer were incubated at 30 °C for 10 min and recovered, and then membranes were ruptured under hypotonic conditions. The lysate was supplemented with Yfh1 (1× = 50 ng) as indicated, and [³⁵S]cysteine (0.1 μm; 10 μCi) was added. The reaction mixture was adjusted to 100 μl with final concentrations of 20 mm Hepes/KOH, pH 7.5, 0.15 m NaCl, 40 mm KOAc, and 10 mm Mg(OAc)₂ and incubated at 30 °C for 15 min (first step). Proteins were precipitated with ammonium sulfate (final 67%) and centrifuged to remove free [³⁵S]cysteine. Protein pellets with bound [³⁵S]cysteine were resuspended in 20 mm Hepes/KOH, pH 7.5, 0.15 m NaCl, 40 mm KOAc, and 10 mm Mg(OAc)₂. Isd11 (100 ng) was added, and samples were incubated at 30 °C for 15 min, with or without added Yfh1 (second step). Proteins were precipitated with 10% TCA and analyzed by nonreducing SDS-PAGE and autoradiography.

FIGURE 10.

Addition of purified Yfh1 or Isu1_Sup exposes the substrate-binding sites of Nfs1 for effective interaction with cysteine in mitochondrial lysate. Mitochondria were isolated from the Gal-Yfh1 strain after an 18 h depletion of Yfh1 in galactose-free medium. Intact Yfh1-depleted mitochondria (Yfh1▾; 200 μg of proteins) in HS buffer were incubated at 30 °C for 10 min in the presence of unlabeled cysteine (10 μm) (first step). After dilution with HS buffer, mitochondria were recovered by centrifugation, removing excess and free cysteine. The mitochondrial pellet was resuspended in 20 mm Hepes/KOH, pH 7.5, and membranes were ruptured. The lysate thus obtained was supplemented with purified Yfh1 or Isu1_Sup (1× = 50 ng) as indicated. The reaction mixtures were adjusted to 100 μl with final concentrations of 20 mm Hepes/KOH, pH 7.5, 0.1 μm [³⁵S]cysteine (10 μCi), 0.15 m NaCl, 40 mm KOAc, and 10 mm Mg(OAc)₂, and samples were incubated at 30 °C for 15 min (second step) (lanes 1–7). As control (lane 8), a mixture of purified Nfs1·Isd11 complex (100 ng) and Yfh1 (100 ng) was incubated with 0.1 μm [³⁵S]cysteine (5 μCi) and 0.15 mm PLP in a final volume of 50 μl. Proteins were precipitated with 10% TCA and analyzed by nonreducing SDS-PAGE followed by autoradiography.

FIGURE 11.

Model for two-tier regulation of persulfide formation by Nfs1. The enzyme Nfs1 is shown by a green line. The Lys²⁶³ residue of the Nfs1 substrate-binding site forms a Schiff base with the cofactor PLP. Nfs1 by itself inefficiently binds the substrate cysteine (Cys-SH, sulfur indicated in red). Frataxin/Yfh1 interacts with Nfs1, inducing a conformational change in the enzyme (Change #1) and exposing new sites with Lys²⁶³-PLP, now able to efficiently bind the substrate cysteine. Isd11 then interacts with Nfs1 with substrate cysteine already bound, inducing a second conformational change (Change #2), and bringing the substrate cysteine (red sulfur) into proximity to the Cys³⁸⁵ (sulfur indicated in blue) in the peptide backbone of the Nfs1 active site loop. A nucleophilic attack by the thiolate anion of the active site Cys³⁸⁵ extracts the –SH group of the substrate, forming a covalent persulfide (Nfs1-S-SH). The substrate cysteine bound to PLP after Change #1 is shown as an external aldimine. However, this has not been shown experimentally, and it is possible that at this stage the substrate cysteine interacts with PLP still bound to the Lys²⁶³ residue of Nfs1 as an internal aldimine.