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J. Biol. Chem., Vol. 279, Issue 23, 24131-24140, June 4, 2004

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Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

INSIGHTS INTO MAMMALIAN PEROXIDASE HEME-PROTEIN COVALENT BONDS^*

Christophe Colas and Paul R. Ortiz de Montellano

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280

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 Leu³⁷, Phe⁴¹, Gly⁶⁹, and Ser⁷³. The F41E mutant was isolated with no covalently bound heme, but the heme was completely covalently bound upon incubation with H₂O₂. 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.

Received for publication, February 16, 2004 , and in revised form, March 23, 2004.

^* This work was supported by Grant GM32488 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Fax: 415-502-4728; E-mail: ortiz{at}cgl.ucsf.edu.

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