FIGURE 9.
Nucleotide-, NADH-, and iron-dependent use of the NFS1 persulfide for Fe-S cluster biogenesis of aconitase in mammalian mitochondria. A, nucleotide/NADH-depleted CAD mitochondria (200 μg of proteins) were incubated with or without 2 mm o-phenanthroline (o-Phe) for 10 min on ice. Samples were diluted with HS buffer and centrifuged to remove excess chelator. The mitochondrial pellets thus obtained were resuspended in buffer A containing 0.1 mg/ml BSA and the master mix. Ferrous ascorbate (Fe2+, 10 μm) was added as indicated. After addition of [35S]cysteine (10 μCi), reaction mixtures were incubated for 30 min at 30 °C. Identical samples were analyzed by non-reducing SDS-PAGE and autoradiography for [35S]persulfide levels (top panel) and also by native PAGE and autoradiography for Fe-35S cluster loading of aconitase (bottom panel). B, nucleotide/NADH-depleted CAD mitochondria (200 μg of proteins) were incubated with or without 2 mm o-phenanthroline for 10 min on ice. Samples were diluted with HS buffer and centrifuged. The mitochondrial pellets thus obtained were resuspended in buffer A containing 0.1 mg/ml BSA. As indicated, αKG (2 mm), NADH (2 mm), and ferrous ascorbate (10 μm) were added. After addition of [35S]cysteine (10 μCi), reaction mixtures were incubated for 30 min at 30 °C. Identical samples were analyzed for [35S]persulfide levels (top panel) and also for Fe-35S cluster loading of aconitase (bottom panel) as in A.