Although a high-resolution crystal structure is not available for horseradish peroxidase isozyme c (HRP), sequence alignments with peroxidases for which crystal structures are available indicate that Phe-41 is adjacent to the catalytic histidine in the active site of HRP. The replacement of Phe-41 by smaller amino acids has been investigated to determine if limited access to the ferryl species is responsible for the poor peroxygenase activity of HRP. The leucine (F41L) and threonine (F41T) mutants have been expressed in a baculovirus/insect cell system and have been purified and characterized. The spectroscopic properties of the two mutants are nearly identical to those of native HRP, and their kinetic constants for the oxidation of guaiacol, ABTS, and iodide differ by no more than a factor of 3 from those of the native enzyme. The F41L and F41T mutants, as expected from earlier work, have higher V-max, values for the sulfoxidation of phenyl alkyl thioethers. Furthermore, F41L yields sulfoxides with higher enantioselectivity than native HRP. More significantly, they catalyze the epoxidation of styene and cis-beta-methylstyrene, reactions not catalyzed by native HRP. These reactions proceed with low to high enantioselectivity and, in the case of-cis-beta-methylstyrene, yield both the cis- and trans-epoxides. Studies with O-18-labeled H2O2 and O-2 indicate that the oxygen incorporated into the cis-beta-methylstyrene epoxide derives exclusively from the peroxide, whereas a substantial fraction of the oxygen in the styrene and trans-beta-methylstyrene products derives from O-2. Surprisingly, trans-beta-methylstyrene is oxidized by both the native and mutant enzymes, and a large fraction of the oxygen incorporated into the epoxide derives from O-2. The results indicate that the F41L and F41T mutations greatly enhance the peroxygenase activity of HRP and show that the epoxidation reactions catalyzed by the mutants occur in part by mechanisms other than ferryl oxygen transfer.