Iron accumulation has been associated with several pathological conditions such as Friedreich ataxia. This human disorder is caused by decreased expression of frataxin. Iron-overload triggers oxidative stress, but the main targets of such stress are not known. In yeast cells lacking the frataxin ortholog YFH1, we have identified a set of 14 carbonylated proteins, which include mitochondrial ATP synthase, phosphoglycerate kinase, pyruvate kinase, and molecular chaperones. Interestingly, most of the target proteins are magnesium- and/or nucleotide-binding proteins. This key feature leads us to postulate that when iron accumulates, chelatable iron replaces magnesium at the corresponding metal-binding site, promoting selective damage to these proteins. Consistent with this hypothesis, in vitro experiments performed with pure pyruvate kinase and phosphoglycerate kinase showed that oxidation of these proteins can be prevented by magnesium and increased by the presence of ATP.