We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K-12. The FhuF protein was labeled with a His-tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and Mössbauer and EPR spectroscopy, FhuF was identified as a [2Fe-2S] protein. The g values (gx = 1.886, gy = 1.961, gz = 1.994) and some of the Mössbauer parameters of FhuF obtained [oxidized protein as isolated: delta EQ,4.2K = 0.474 mm s-1; Fe3+ (reduced protein): delta EQ = 0.978 mm s-1] are not typical of common [2Fe-2S] proteins and indicate that FhuF has unusual structural properties. The primary sequence of FhuF does not show any sequence similarities to known [2Fe-2S] proteins. By site-directed mutagenesis, each of the six cysteines of FhuF was replaced by serine. EPR of the six reduced mutant proteins revealed that the terminal cysteine residues 244, 245, 256, and 259 form the [2Fe-2S]Cys4 cluster. Mutants having the Cys-to-Ser replacement at positions 244, 245, 256, or 259 did not complement a fhuF mutant. The motif Cys-Cys-Xaa10-Cys-Xaa2-Cys in FhuF differs considerably from the motif Cys-Xaa2-Cys-Xaa9-15-Cys-Xaa2-Cys found in other [2Fe-2S] proteins. The unusual Cys-Cys terminal group of the cluster may explain the atypical EPR and Mössbauer spectroscopic properties of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is distorted. The phenotype of fhuF mutants and the structural features of the FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B.