Peroxidase catalyzes one-electron oxidation of various substrates by utilizing hydrogen peroxide (H 2 O 2 ) as an oxidant. Horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP), while sharing the same active species, compound I, in the catalytic reaction, have a different radical center. The compound I of HRP contains a porphyrin π cation radical (por+ ), whereas, in CcP, the radical center is on the tryptophan (Trp191) residue located near the heme active site. On the basis of sequence alignment of HRP and CcP, the phenylalanine (Phe221) residue in HRP corresponds to Trp191 in CcP. Thus, in order to elucidate the role of the Trp residue near the heme and the control mechanism for the position of the radical center in peroxidase compound I, we prepared a CcP-like HRP mutant, F221W, by replacing the Phe221 with a Trp residue.In the reaction of the F221W mutant with H 2 O 2 , rapid spectral scanning experiments showed the characteristic decrease in the absorbance of the Soret band for the por+ formation, followed by the increase in the absorbance. The increase in the absorbance corresponds to the formation of a ferryl (Fe I V =O) species. These absorbance changes indicates the succesive formation of the ferryl species after the radical center is generated on the porphyrin by addition of H 2 O 2 (Shceme 1). To estimate the rate for the formation of porphyrin radical (k 1 ) and the ferryl species (k 2 ), stopped-flow studies were performed. The transient por+ was initially formed in the reaction with H 2 O 2 for F221W mutant at the same rate (k 1 = 1.3 10 7 M - 1 s - 1 ) as for the wild type enzyme and the produced por+ decayed rapidly at a rate (k 2 = 1.7 10 5 M - 1 s - 1 ) to the ferryl species. To confirm the position of the radical center, we followed EPR spectra for the mutant HRP in the presence of H 2 O 2 . After addition of H 2 O 2 to the mutant HRP, a broad signal appeared and its g value was 2.041, which was characteristic of the Trp radical in CcP. These spectral changes suggest that the ferryl species contains the Trp radical in F221W mutant, contrary to wild-type HRP. Thus, we can conclude that por+ is initially produced in the reaction of F221W mutant with H 2 O 2 , subsequently one electron is transferred from Trp221 to the por+ , resulting in the formation of the Trp radical as found for CcP. The Trp residue near the active site is a key residue to control the position of the radical center in peroxidase.