Horseradish peroxidase mutants that autocatalytically modify their prosthetic heme group: insights into mammalian peroxidase heme-protein covalent bonds.

Abstract

The mammalian peroxidases, including myeloperoxidase and lactoperoxidase, bind their prosthetic heme covalently through ester bonds to two of the heme methyl groups. These bonds are autocatalytically formed. No other peroxidase is known to form such bonds. To determine whether features other than an appropriately placed carboxylic acid residue are important for covalent heme binding, we have introduced aspartate and/or glutamic acid residues into horseradish peroxidase, a plant enzyme that exhibits essentially no sequence identity with the mammalian peroxidases. Based on superposition of the horseradish peroxidase and myeloperoxidase structures, the mutated residues were Leu(37), Phe(41), Gly(69), and Ser(73). The F41E mutant was isolated with no covalently bound heme, but the heme was completely covalently bound upon incubation with H(2)O(2). As predicted, the modified heme released from the protein was 3-hydroxymethylheme. The S73E mutant did not covalently bind its heme but oxidized it to the 8-hydroxymethyl derivative. The hydroxyl group in this modified heme derived from the medium. The other mutations gave unstable proteins. The rate of compound I formation for the F41E mutant was 100 times faster after covalent bond formation, but the reduction of compound I to compound II was similar with and without the covalent bond. The results clearly establish that an appropriately situated carboxylic acid group is sufficient for covalent heme attachment, strengthen the proposed mechanism, and suggest that covalent heme attachment in the mammalian peroxidases relates to peroxidase biology or stability rather than to intrinsic catalytic properties.