Horseradish peroxidase His-42 --> Ala, His-42 --> Val, and Phe-41 --> Ala mutants. Histidine catalysis and control of substrate access to the heme iron.

Polyhistidine-tagged horseradish peroxidase (hHRP) and its F41A, H42A, and H42V mutants have expressed in an insect cell system. Kinetic studies show that the rates of Compound I formation and peroxidative catalysis are greatly decreased by the His-42 mutation. Furthermore, Compound II is not detected during turnover of the His-42 mutants. Compounds I and II are the two- and one-electron oxidized intermediates, respectively, of hHRP. In peroxygenative catalysis, the F41A and H42A mutants catalyze thioanisole sufoxidation 100 and 10 times faster, respectively, than hHRP. Styrene epoxidation is catalyzed by both the Phe-41 and His-42 mutants but not by wild-type hHRP. The higher peroxygenase activity of the mutants reflects increased accessibility of the ferryl species. This is indicated by the finding that, contrary to the reaction with wild-type hHRP, reaction of phenyldiazene with the F41A mutant yields a new and unidentified product, and the same reaction with the His-42 mutants yields phenyl-iron complexes. Phe-41 and His-42 thus shield the iron-centered catalytic species, and His-42 plays a key catalytic role in the formation of Compound I. The peroxygenase activities of the Phe-41 and His-42 mutants approach those of cytochrome P450.