Apd1 and Aim32 Are Prototypes of Bishistidinyl-Coordinated Non-Rieske [2Fe–2S] Proteins

Apd1, 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–X8–C–X24–75–H–X–G–G–H motif of the TLF domain of Apd1 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 Apd1 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 Apd1 (Em,7 = −164 ± 5 mV, pKox1,2 = 7.9 ± 0.1 and 9.7 ± 0.1) differs from both dioxygenase (Em,7 ≈ −150 mV, pKox1,2 = 9...