The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (IrrAt) were investigated. Several IrrAt mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of IrrAt. Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of IrrAt. H94 is essential for the iron responsiveness of IrrAt. Furthermore, the IrrAt mutant proteins showed differential abilities to complement the H2O2-hyper-resistant phenotype of an irr mutant.