Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria

► The enzyme cysteine desulfurase provides sulfur for Fe-S cluster synthesis. ► We identified a persulfide intermediate on the enzyme in isolated mitochondria. ► The formation of enzyme-bound persulfide does not require nucleotides or iron. ► The persulfide could be utilized for Fe-S cluster synthesis by mitochondria. ► The use of persulfide for Fe-S cluster assembly requires both nucleotides and iron.