Apdl, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxin-like ferredoxin (TLF) domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi, and unicellular pathogenic eukaryotes but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X-8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apdl and Aim32 proteins are essential for viability of yeast cells upon treatment with the redox mediators gallobenzophenone or pyrogallol, respectively. UV-vis, EPR, and Mossbauer spectroscopy of purified wild-type Apdl and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion, and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S](2+/1+) cluster. The [2Fe-2S] center of Apdl (E-m,E-7 = -164 +/- 5 mV, pK(ox1,2) = 7.9 +/- 0.1 and 9.7 +/- 0.1) differs from both dioxygenase (E-m,E-7 approximate to -150 mV, pK(ox1,2) = 9.8 and 11.5) and cytochrome bc(1)/b(6)f Rieske clusters (E-m,E-7 approximate to +300 mV, pK(ox1,2)= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands, or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of the [2Fe-2S] cluster containing proteins with bishistidinyl coordination.