identification and characterization of mitochondrial mia40 as an ironsulfur protein mia40 is a highly conserved mitochondrial protein that plays an essential role in the import and oxidative folding of many proteins of the mitochondrial intermembrane space mia40 uses its redox active cpc motif to shuttle disulfides between its client proteins newly imported proteins and the thiol oxidase erv1 as a thiol oxidoreductase no cofactor was found in mia40 nor is a cofactor required for this function in the present study we for the first time based on both in vitro and in vivo studies show that yeast mia40 can exist as an fes ironsulfur protein as well we show that mia40 binds a 2fe2s cluster in a dimer form with the cluster coordinated by the cysteine residues of the cpc motifs the biological relevance of the cofactor binding was confirmed in vivo by cysteine redox state and iron uptake analyses which showed that a significant amount of cellular mia40 binds iron in vivo furthermore our oxygen consumption results suggested that the fescontaining mia40 is not an electron donor for erv1 thus we conclude that mia40 is a novel fes protein with a new clusterbinding motif cpc and apart from the thiol oxidoreductase activity mia40 may have another important as yet undefined function in cells