Fe-S cluster biogenesis in isolated mammalian mitochondria: coordinated use of persulfide sulfur and iron and requirements for GTP, NADH, and ATP.
Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [(35)S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-(35)S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the (35)S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia.
From the Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101 and.
Alok
A
Pandey
From the Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101 and.
Jayashree
J
Pain
From the Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101 and.
Arnab K
AK
Ghosh
the Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Andrew
A
Dancis
From the Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, New Jersey 07101 and painde@njms.rutgers.edu.
Debkumar
D
Pain
D000154:Aconitate Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013440:Sulfides / Q000737:chemistry* / Q000378:metabolism; D013462:Sulfur Radioisotopes
D005288:Ferredoxins; D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D013440:Sulfides; D013462:Sulfur Radioisotopes; C051552:persulfides; D009243:NAD; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D007501:Iron; D000154:Aconitate Hydratase; D013437:Carbon-Sulfur Lyases; C513493:Nfs1 protein, mouse; D003545:Cysteine
Friedreich Ataxia; Iron; Iron Metabolism; Iron-Sulfur Protein; Metal Homeostasis; Mitochondria; Mitochondrial Aconitase; Mitochondrial Metabolism; Sulfur; Yeast