Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria.
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.
Department of Pharmacology and Physiology, UMDNJ, New Jersey Medical School, Newark, NJ 07101, United States.
Alok
A
Pandey
Heeyong
H
Yoon
Elise R
ER
Lyver
Andrew
A
Dancis
Debkumar
D
Pain
D000154:Aconitate Hydratase / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D007553:Isotope Labeling; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013440:Sulfides / Q000378:metabolism*; D013462:Sulfur Radioisotopes / Q000378:metabolism; D013466:Sulfurtransferases / Q000378:metabolism*
D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D013440:Sulfides; D013462:Sulfur Radioisotopes; C051552:persulfides; D013466:Sulfurtransferases; C115912:NFS1 protein, S cerevisiae; D000154:Aconitate Hydratase; D003545:Cysteine