Topology of the chloroperoxidase active site: regiospecificity of heme modification by phenylhydrazine and sodium azide.

Abstract

Chloroperoxidase (CLP) from Caldariomyces fumago is rapidly and irreversibly inactivated by phenylhydrazine and H2O2 but not by H2O2 alone. Inactivation is characterized by a phenylhydrazine-to-CLP partition ratio of approximately 15, formation of trans-azobenzene, and formation of a sigma-bonded phenyl-iron heme complex with a characteristic absorption maximum of 472 nm. Anaerobic extraction of the heme complex from the protein, followed by exposure to dioxygen under acidic conditions, shifts the phenyl group from the iron to the porphyrin nitrogens and yields the four possible N-phenylprotoporphyrin IX regioisomers. Oxidation of the iron-phenyl complex within the intact protein by ferricyanide or high peroxide concentrations results in protein-directed phenyl migration to give exclusively the N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A and C. CLP also catalyzes the H2O2-dependent oxidation of azide to the azidyl radical and is inactivated by azide in the presence of H2O2. Inactivation of CLP by azide and H2O2 results in loss of heme Soret absorbance and formation of delta-meso-azidoheme. These results suggest a topological model for the CLP active site and indicate that the tertiary structure of the enzyme permits substrates to interact with both the delta-meso heme edge and catalytic ferryl (FeIV = O) species, in agreement with the fact that CLP catalyzes both H2O2-dependent peroxidation and monooxygenation reactions.