[2Fe-2S]-Ferredoxin Binds Directly to Cysteine Desulfurase and Supplies an Electron for Iron–Sulfur Cluster Assembly but Is Displaced by the Scaffold Protein or Bacterial Frataxin

Abstract

Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe–S cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S0 is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the isc operon.

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin

“…A role of the ferredoxin chain in the first step is supported by the impaired in vivo Fe/S cluster assembly on Isu1 upon depletion of Yah1 27 . Consistent with this, bacterial Fdx, both in reduced and oxidized form, was recently shown to interact with IscS 34 . Mixing of reduced Fdx with IscS, IscU, Cys and iron supported the oxidation of Fdx and led to Fe/S cluster formation on IscU.…”

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

“…More likely, since FXN specifically activates sulfur transfer to ISCU, the persulfuration of ISCU might have a functional role in Fe-S cluster assembly but a dedicated reductase is lacking in the Fe-S cluster reconstitution assays. The human ferredoxin FDX2, which provides electrons for Fe-S cluster biogenesis 38,39 , is a potential candidate for the reduction of ISCU. However, under our conditions, we were not able to measure any persulfide reductase activity with FDX2.…”

“…ARTICLE L-cysteine and GSH reduce NFS1 persulfide. Using the maleimide-peptide assay, we assayed persulfide reductase activities of two electron donors postulated to enable persulfide reduction [37][38][39] , ferrous iron (Fe 2 þ ) and mammalian mitochondrial ferredoxin 2, FDX2, in its reduced state. Neither iron alone or together with FDX2 had an impact on NFS1 or ISCU persulfides ( Supplementary Fig.…”

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

“…Frataxin, a mitochondrial protein associated with the human neurodegenerative disease Friedreich ataxia (18), has been proposed previously as a putative iron chaperone for iron-sulfur cluster biogenesis (19,20). Frataxin is highly conserved from bacteria to humans and interacts with ironsulfur protein aconitase (21), mitochondrial electron transfer components (22), and the iron-sulfur cluster assembly proteins IscS (23)(24)(25)(26)(27) and IscU (28). However, frataxin has a weak ironbinding activity under physiological conditions (29 -31), and deletion of frataxin has little or no effect on iron-sulfur proteins in Saccharomyces cerevisiae (32), Salmonella enterica (33), and Escherichia coli (34).…”