A gene coding for the F172Y mutant of horseradish peroxidase isozyme C (HRP) has been constructed and expressed in both Spodoptera frugiperda (SF-9) and Trichoplusia ni egg cell homogenate (HighFive) cells. Homology modeling with respect to three peroxidases for which crystal structures are available places Phe172 on the proximal side of the heme in the vicinity of porphyrin pyrrole ring C. The pH optimum and spectroscopic properties of the F172Y mutant are essentially identical to those of wild type HRP. Vmax values show that the mutant protein retains most of the guaiacol oxidizing activity. Stopped flow studies indicate that Compound I is formed with H2O2 at the same rate (kappa 1 = 1.6 x 10(7) M-1 s-1) at both pH 6.0 and 8.0 as it is with the wild type enzyme. This Compound I species decays rapidly at a rate kappa 2 = 1.01 s-1, pH 7.0, to a second two-electron oxidized species that retains the ferryl (FeIV = O) absorption. EPR studies establish that a ferryl porphyrin radical cation is present in the initial Compound I, but electron transfer from the protein results in formation of a second Compound I species with an unpaired electron on the protein (presumably on Tyr172). The presence or absence of oxidizable amino acids adjacent to the heme is thus a key determinant of whether the second oxidation equivalent in Compound I is found as a porphyrin or protein radical cation.