Frataxin (Yfh1 in yeast) is a conserved protein and deficiency leads to the neurodegenerative disease Friedreich's ataxia. Frataxin is a critical protein for Fe-S cluster assembly in mitochondria, interacting with other components of the Fe-S cluster machinery, including cysteine desulfurase Nfs1, Isd11 and the Isu1 scaffold protein. Yeast Isu1 with the methionine to isoleucine substitution (M141I), in which the E. coli amino acid is inserted at this position, corrected most of the phenotypes that result from lack of Yfh1 in yeast. This suppressor Isu1 behaved as a genetic dominant. Furthermore frataxin-bypass activity required a completely functional Nfs1 and correlated with the presence of efficient scaffold function. A screen of random Isu1 mutations for frataxin-bypass activity identified only M141 substitutions, including Ile, Cys, Leu, or Val. In each case, mitochondrial Nfs1 persulfide formation was enhanced, and mitochondrial Fe-S cluster assembly was improved in the absence of frataxin. Direct targeting of the entire E. coli IscU to ∆yfh1 mitochondria also ameliorated the mutant phenotypes. In contrast, expression of IscU with the reverse substitution i.e. IscU with Ile to Met change led to worsening of the ∆yfh1 phenotypes, including severely compromised growth, increased sensitivity to oxygen, deficiency in Fe-S clusters and heme, and impaired iron homeostasis. A bioinformatic survey of eukaryotic Isu1/prokaryotic IscU database entries sorted on the amino acid utilized at the M141 position identified unique groupings, with virtually all of the eukaryotic scaffolds using Met, and the preponderance of prokaryotic scaffolds using other amino acids. The frataxin-bypassing amino acids Cys, Ile, Leu, or Val, were found predominantly in prokaryotes. This amino acid position 141 is unique in Isu1, and the frataxin-bypass effect likely mimics a conserved and ancient feature of the prokaryotic Fe-S cluster assembly machinery.