Role of Heme-Protein Covalent Bonds in Mammalian Peroxidases

Abstract

Oxidation of SCN؊ , Br ؊ , and Cl ؊ (X ؊ ) by horseradish peroxidase (HRP) and other plant and fungal peroxidases results in the addition of HOX to the heme vinyl group. This reaction is not observed with lactoperoxidase (LPO), in which the heme is covalently bound to the protein via two ester bonds between carboxylic side chains and heme methyl groups. To test the hypothesis that the heme of LPO and other mammalian peroxidases is protected from vinyl group modification by the hemeprotein covalent bonds, we prepared the F41E mutant of HRP in which the heme is attached to the protein via a covalent bond between Glu 41 and the heme 3-methyl. We also examined the E375D mutant of LPO in which only one of the two normal covalent heme links is retained. The prosthetic heme groups of F41E HRP and E375D LPO are essentially not modified by the HOBr produced by these enzymes. The double E375D/D225E mutant of LPO that can form no covalent bonds is inactive and could not be examined. These results unambiguously demonstrate that a single heme-protein link is sufficient to protect the heme from vinyl group modification even in a protein (HRP) that is normally highly susceptible to this reaction. The results directly establish that one function of the covalent heme-protein bonds in mammalian peroxidases is to protect their prosthetic group from their highly reactive metabolic products.The mammalian enzymes lactoperoxidase (LPO), 2 myeloperoxidase (MPO), and eosinophil peroxidase efficiently oxidize iodide, bromide, thiocyanide, and, at least in the case of myeloperoxidase, chloride ions (1-3). Indeed, the antimicrobial and other roles of the mammalian peroxidases depend on their oxidation of halide and/or pseudohalide ions. In contrast, the substrates of most plant and fungal peroxidases are low oxidation potential compounds such as phenols, but the enzymes can also oxidize iodide, thiocyanide, bromide, and, albeit very poorly, chloride ions (4 -6). Apart from these differences in their normal substrates, the most notable difference between the mammalian and plant/fungal peroxidases is the presence, in the mammalian enzymes, of two (or in MPO three) covalent bonds between the heme group and active site residues. In LPO, Glu 375 and Asp 225 form covalent ester bonds with the 1-and 5-methyl groups, respectively, of the heme (7-9). In MPO, in addition to the two ester bonds common to all the mammalian peroxidases, the 2-vinyl is attached via an unusual vinyl sulfonium link to Met 243 (10,11). No such covalent links have been detected in native plant or fungal peroxidases.We have demonstrated that HRP can oxidize thiocyanide, bromide, and chloride ions and that these reactions result in the addition of HOX (where X ϭ SCN, Br, or Cl) to the prosthetic heme 2-and/or 4-vinyl groups (6, 12). These results were recently extended to the Arthromyces ramosus and soybean peroxidases (13), confirming that modification of the heme vinyl groups is a common property in the oxidation of halides and pseudohalides by plant and fungal peroxidases...

Impact of Two Novel Mutations on the Structure and Function of Human Myeloperoxidase

“…Although at least one covalent bond is required for peroxidase activity and the presence of covalent bonds with heme is a feature shared by all animal peroxidases, the functional or biological advantage of covalently bound heme is not known (73). Recent studies indicate that the presence of a single covalent bond between the heme group and its protein is sufficient to protect the heme vinyl group from modification by hypohalous acid generated by peroxidases (77)(78)(79). In this way, peroxidases such as MPO can generate large amounts of HOCl and its by-products within the neutrophil phagosome without incurring autoinactivation by modification of the heme vinyl groups and subsequent inactivation.…”

Structural Basis of Peroxide-mediated Changes in Human Hemoglobin

“…It is difficult at this stage to speculate as to the origin of this heme/porphyrin-like structure and the basis of its transfer to the ␣1 and ␣2 interfaces. One interesting possibility is that a protective mechanism exists in Hb such as in mammalian peroxidases where covalent cross-links between heme and the active site of the protein are established to prevent oxidation from auto-catalytically derived peroxides and reactive metabolic products (40). Another example of naturally occurring protective covalent heme-protein linkages stems from the recent revelation that ␣-Hb-stabilizing protein in complexing with the ferrous ␣ subunit functions to stabilize it and inhibit its ability to generate reactive oxygen species during red blood cell development.…”

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.…”