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

• Published in: Mitochondrion 2012-09, Vol.12 (5), p.539-549
• Main Authors: Pandey, Alok, Yoon, Heeyong, Lyver, Elise R, Dancis, Andrew, Pain, Debkumar
• Format: Article
• Language: English
• Subjects: Aconitate Hydratase - metabolism; Cysteine - metabolism; Isotope Labeling; Mitochondria - enzymology; Mitochondria - metabolism; Mitochondrial Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Sulfides - metabolism; Sulfur Radioisotopes - metabolism; Sulfurtransferases - metabolism
• ISSN: 1567-7249; 1872-8278
• DOI: 10.1016/j.mito.2012.07.103

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 radiolabeled persulfide could be detected on Nfs1p. The persulfide persisted under conditions that did not permit Fe-S cluster formation, such as nucleotide and/or iron depletion of mitochondria. By contrast, under permissive conditions, the radiolabeled Nfs1p persulfide was greatly reduced and radiolabeled aconitase was formed, indicating transfer of persulfide to downstream Fe-S cluster recipients. Nfs1p in mitochondria was found to be relatively more resistant to inactivation by N-ethylmaleimide (NEM) as compared with a prokaryotic cysteine desulfurase. Mitochondria treated with NEM (1 mM) formed the persulfide on Nfs1p but failed to generate Fe-S clusters on aconitase, likely due to inactivation of downstream recipient(s) of the Nfs1p persulfide. Thus the Nfs1p-bound persulfide as described here represents a precursor en route to Fe-S cluster synthesis in mitochondria.