Friedreich's ataxia is a neurodegenerative disorder caused by the low expression of a mitochondrial protein called frataxin. Studies in the yeast Saccharomyces cerevisiae have unraveled a role for the frataxin homologue (Yfh1p) in iron-sulfur cluster (Fe/S) biosynthesis, probably by interacting with the scaffold protein, Isu1p, and providing iron to the machinery. Yfh1p possesses a large â-sheet platform that may be involved in the interaction with other proteins through conserved residues at its surface. We have used directed mutagenesis associated with polymerase chain reaction (PCR) to study conserved residues localizing either at the surface of the protein, Thr110, Thr118, Val120, Asn122, Gln124, Gln129, Trp131, Ser137 and Arg141, or buried in the core of the protein, Ile130 and Leu132. Mutants T110A, T118A, V120A, N122A, Q124A, Q129A, I130A, W131A, L132A, S137A and R141A were generated in yeast. Growth on iron- or copper-containing medium was severely impaired for mutants Q129A, I130A, W131A and R141A. Others were roughly growing as well as the wild-type strain. We assessed the efficiency of Fe/S biosynthesis by measuring aconitase activity. The results confirmed those obtained on metal-containing medium: mutants Q129A, I130A, W131A and R141A showed a high decrease in their aconitase activity that dropped to the deleted strain level. Moreover, S137A showed also a decreased aconitase activity. We monitored the interaction between Yfh1p and Isu1p by co-immunoprecipitation and it turned out that only the W131A mutation affects directly this interaction. Even if the amount of Yfh1p determined by western blot analysis was highly decreased for several mutants, it is not sufficient to explain the phenotypes as they were poorly restored by overexpression of the mutant proteins to wild-type levels, except for W131F. We have concluded that Gln129, Trp131, and Arg141 are important for Yfh1p function, while Ile130 and Ser137 are required for the folding of the protein. All these residues cluster to the 4th and 5th â-strand of the protein. Our work has demonstrated for the first time the importance of this area for Yfh1p function and shows that Trp131 is involved in the interaction with Isu1p.