The Iro protein is a member of HiPIP family with the [Fe(4)S(4)] cluster for electron transfer. Many reports proposed that the conserved aromatic residues might be responsible for the stability of the iron-sulfur cluster in HiPIP. In this study, Tyr10 was found to be a critical residue for the stability of the [Fe(4)S(4)] cluster according to site-directed mutagenesis results. Tyr10, Phe26 and Phe48 were essential for the stability of the [Fe(4)S(4)] cluster under acidic condition. Trp44 were not involved in the stability of the [Fe(4)S(4)] cluster. Molecular structure modeling for the mutant Tyr10 proteins revealed that the aromatic group of Tyr10 may form a hydrophobic barrier to protect the [Fe(4)S(4)] cluster from solvent.