Friedreich ataxia is caused by reduced activity of frataxin, a conserved iron-binding protein of the mitochondrial matrix, thought to supply iron for formation of Fe-S clusters on the scaffold protein Isu. Frataxin binds Isu in an iron-dependent manner in vitro. However, the biological relevance of this interaction and whether in vivo the interaction between frataxin and Isu is mediated by adaptor proteins is a matter of debate. Here, we report that alterations of conserved, surface-exposed residues of yeast frataxin, which have deleterious effects on cell growth, impair Fe-S cluster biogenesis and interaction with Isu while altering neither iron binding nor oligomerization. Our results support the idea that the surface of the -sheet, adjacent to the acidic, iron binding ridge, is important for interaction of Yfh1 with the Fe-S cluster scaffold and point to a critical role for frataxin in Fe-S cluster biogenesis.Friedreich ataxia (FRDA), 2 a progressive neurodegenerative disease, is caused by a decrease in the level, and in some cases activity, of frataxin, a highly conserved iron-binding protein of the mitochondrial matrix (1-3). The effects of deficiency of frataxin in human cells and Yfh1 in yeast are strikingly similar: decrease in the activity of Fe-S-containing enzymes and increase in intramitochondrial iron levels (4, 5). Not surprisingly considering their 65% amino acid identity, the tertiary structures of frataxin and Yfh1 are very similar, a -sheet packed against two ␣-helices (6 -8). The larger helix and part of the adjacent -strand form an acidic ridge that binds iron. While a Yfh1 monomer can bind 2 ferrous irons (8, 9), it also undergoes iron-dependent oligomerization to form trimers and ultimately oligomers having up to 48 subunits that bind Ͼ2000 iron atoms (10, 11). Alterations in the acidic residues of the ridge can affect oligomerization and thus robust iron binding, alleviating any ability to serve an iron storage function (12, 13).Evidence indicates that Yfh1/frataxin plays an important role in the formation of Fe-S clusters (5, 14) on the highly conserved scaffold Isu prior to transfer to recipient apoproteins (15). Its proposed role as an iron donor is supported by its high affinity, iron-dependent interaction with Isu and its ability to donate iron for cluster formation on Isu in vitro (16). In addition, evidence strongly indicates that Nfs1, an essential cysteine desulfurase, which is required for Fe-S cluster formation on Isu in vivo, functions as the sulfur donor (17, 18). Another essential protein, Isd11, is also required for in vivo Fe-S cluster formation (15,19). It forms a stable complex with Nfs1 and is required for Nfs1 to be stable in vivo.The manner in which the activities of these proteins, including their physical interactions, are coordinated to allow cluster formation on Isu in vivo is a matter of debate. Most relevant to this report, it has been recently argued that the relevant in vivo interaction between frataxin homologs and scaffolds is indirect, despite the fact th...