Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

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.Mammalian peroxidases share a unique feature, the formation of two or three covalent bonds to their prosthetic heme 1 group, not found in the peroxidases of other organisms. The two common bonds in all mammalian peroxidases are ester links between a conserved glutamate and aspartate and the heme 1-methyl and 5-methyl substituents, respectively. In MPO a third bond is forged between a methionine and the ␤ carbon of the 2-vinyl substituent (1). Mutagenesis studies with LPO have demonstrated that at least one of the two ester bonds is required for catalytic activity (2). Interestingly, a single covalent link between the heme and protein is also found in most members of the CYP4A, -4B, and -4F classes of cytochrome P450 enzymes (3-7). In contrast to LPO, however, mutagenesis studies show that the heme-protein covalent bond is not essential for the catalytic activity of at least some of these P450 enzymes (8, 6). A long known example of heme covalent binding is provided by cytochrome c, in which a cysteine residue is covalently bound to each of the two porphyrin vinyl groups. Although many hypotheses have been formulated concerning the functional advantages of the links in cytochrome c, including bending of the heme, increasing the heme affinity (9), and increasing the stability of the methionyl-Fe(II) coordination (10), the question has yet to be definitively answered.Despite m...

Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase

“…In a separate work, Colas and Ortiz de Montellano [27] duplicated the ester link in horseradish peroxidase by incorporation of a glutamate residue close to the 3-methyl group (F41E variant of horseradish peroxidase) and also showed that the formation of the link was H 2 O 2 -dependent. A view has therefore emerged that any peroxidase, or perhaps any haem protein, that can react with H 2 O 2 and that has a suitable amino acid side chain poised in the right place will, under the right conditions, form a covalent link to the haem.…”

Caenorhabditis elegans and Human Dual Oxidase 1 (DUOX1) “Peroxidase” Domains

“…The result of enzymatic digestion and LC-MS analysis of the classic peroxidases with two covalent bonds (LPO, TPO, and EPO) is a 1,5-dihydroxyheme derivative. Mutation studies of heme proteins (horseradish peroxidase F41E, horseradish peroxidase S73E, LPO D225E, and LPO E375D) to investigate the autocatalytic nature and sequence of covalent bond formation have also led to non-native linkages at the 3-and 8-methyl positions (33,34). Because of single covalent bond formation in each mutant enzyme, once digested (trypsin/Pronase or proteinase K), each liberated heme co-factor was found as a monohydroxyheme (1-OH, 3-OH, 5-OH, or 8-OH) derivative.…”

Role of Heme-Protein Covalent Bonds in Mammalian Peroxidases

“…Expression and Purification of the HRP F41E Mutant-The HRP F41E mutant was expressed and purified following modifications of the previously reported procedure (14). Viral stock was generated and amplified from single virus populations.…”

“…Here we report the first direct and unambiguous evidence that the covalent links between the protein and the heme do, in fact, protect the heme from modification by HOX. HRP was engineered to introduce a covalent ester bond between the protein and the 3-methyl of the heme by mutating Phe 41 to a glutamic acid and pretreating the enzyme with low amounts of peroxide (14). The heme covalent bond is formed in essentially quantitative yield by this procedure.…”