To pursue structure−function relationships of heme enzymes in the activation of peroxides, we have chosen to use myoglobin as the framework for our molecular engineering studies. Comparison of the crystal structures of myoglobin and peroxidases reveals differences in the arrangement of amino acid residues in heme active sites. On the basis of these structural differences and the reaction mechanisms of peroxidases, we have converted myoglobin into a peroxidase-like enzyme by alternation of the heme distal pocket via site-directed mutagenesis. The replacement of the proximal histidine with cysteine and the exogenous substituted imidazoles slightly accelerates the peroxide O−O bond cleavage due to the electron donor characteristics. However, we have not observed an enhancement in the activation of peroxide by the proximal mutant with tyrosine, the exogenous phenolate, and benzoate. A clear understanding of the absolute role of the proximal ligand remains elusive.