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 been expressed in an insect cell system. Kineti c 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, Com pounds I and II are the two- and one-electron oxidized intermediates, respectively, of hHRP. In peroxygenative catalysis, the F41A and H42A mutants catalyze thioanisole sulfoxidation 100 and 10 times faster, re spectively, than hHRP. Styrene epoxidation is catalyzed by both the Ph e-41 and His-42 mutants but not by wild type hHRP. The higher peroxyge nase activity of the mutants reflects increased accessibility of the f erryl 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 wi th 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 ke y catalytic role in the formation of Compound I. The peroxygenase acti vities of the Phe-41 and His-42 mutants approach those of cytochrome P 450.