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Cysteine desulfurases generate a covalent persulfide intermediate from cysteine, and this activated form of sulfur is essential for the synthesis of iron–sulfur (Fe–S) clusters. In yeast mitochondria, there is a complete machinery for Fe–S cluster synthesis, including a cysteine desulfurase, Nfs1p. Here we show that following supplementation of isolated mitochondria with [ 35 S]cysteine, a...