Shielding of Peroxidase Heme Vinyl Groups from Autocatalytically Generated Electrophilic Metabolites
Grzegorz Wojciechowski and Paul R. Ortiz de Montellano
Department of Pharmaceutical Chemistry, University of California, 600 16th
Street, San Francisco,
* Corresponding author: E-mail: firstname.lastname@example.org
The heme vinyl substituents of horseradish peroxidase (HRP) are converted to halohydrin and vinyl halide substituents by the HOX generated on halide (X–) oxidation. To determine the role of active site residues in shielding the vinyl groups, we have investigated the L37M, F41M, S151M, and F152M mutants. A methionine was employed to simultaneously test for possible crosslinking to the vinyl group, but crosslinking was not observed. Kinetic analysis reveals that the F41M mutant is particularly altered, its compound I forming more slowly but decaying more rapidly than that of native HRP. The F41M mutant also has the highest halide oxidation activity. Modification of the heme vinyl groups of all the mutants by catalytically-generated HOBr and HOCl gives epoxides in addition to the previously observed halohydrins and vinyl halides. Individual replacement of residues near the vinyl groups with a methionine increases the formation of bromohydrins at the expense of vinyl halides. The results indicate that the normal active site residues partially protect the vinyl groups and restrict water access. The lower polarity of the heme cavity in HRP than myeloperoxidase is consistent with the formation of heme vinyl halides and may help explain the absence of heme vinyl-methionine crosslinking.
Keywords: Horseradish peroxidase, myeloperoxidase, heme modification, hypobromide, hypochloride, heme crosslinking