The Heme Prosthetic Group of Lactoperoxidase

Rae, Tracey; Goff, Harold M.

doi:10.1074/jbc.273.43.27968

Cited by 52 publications

(53 citation statements)

References 33 publications

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“…This haem peak now coincided with the protein peak (monitored at 280 nm) ( Figure 3B). Co-elution of haem and protein is clear evidence for the presence of a covalent haem-protein interaction and has been used previously for characterization of other covalently linked haem proteins [3,10,[18][19][20][21]. There was some protein degradation observed upon reduction of ferric S160C, resulting in the appearance of the broad peak eluted at 27 min in the 280 nm chromatogram ( Figure 3B).…”

Section: Properties Of Ferrous S160cmentioning

confidence: 89%

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

Metcalfe

Daltrop

Ferguson

et al. 2007

Biochemical Journal

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Raven (2004) J. Am. Chem. Soc. 126, [16242][16243][16244][16245][16246][16247][16248] has shown that the introduction of a methionine residue (S160M variant) close to the 2-vinyl group of the haem in ascorbate peroxidase leads to the formation of a covalent haem-methionine linkage under oxidative conditions (i.e. on reaction with H 2 O 2 ). In the present study, spectroscopic, HPLC and mass spectrometric evidence is presented to show that covalent attachment of the haem to an engineered cysteine residue can also occur in the S160C variant, but, in this case, under reducing conditions analogous to those used in the formation of covalent links in cytochrome c. The data add an extra dimension to our understanding of haem to protein covalent bond formation because they show that different types of covalent attachment (one requiring an oxidative mechanism, the other a reductive pathway) are both accessible within same protein architecture.

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Section: Properties Of Ferrous S160cmentioning

confidence: 89%

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

Metcalfe

Daltrop

Ferguson

et al. 2007

Biochemical Journal

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“…The heme prosthetic group in lactoperoxidase, the only one that has been fully characterized, is iron 1,5-bis(hydroxymethyl)-3,8-dimethyl-2,4-divinylporphyrin-6,7-dipropionic acid. The prosthetic group is covalently attached to the protein via ester bonds between the 1-and 5-hydroxymethylene groups and the carboxylic acid side chains of Glu-275 and Asp-125, respectively (34). Proteolytic release of the heme from lactoperoxidase yields a heme species 32 atomic mass units heavier than heme itself, as expected from the presence of two additional hydroxy groups at the 1-and 5-methyl positions.…”

Section: Fig 5 Comparison Of Lc Analysis Of Cyp4a1 Before (A) and Amentioning

confidence: 99%

“…As reported here, proteolysis of the CYP4A enzymes releases a mono-rather than dihydroxy heme species, so that the autocatalytic process is only required to make one cross-link. Cross-linking of the heme via one of its methyl groups, as in lactoperoxidase (34), is a strong possibility in view of the observation that the hydroxyl group is added without significantly perturbing the spectrum of the heme in the bound or unbound state.…”

Section: Fig 5 Comparison Of Lc Analysis Of Cyp4a1 Before (A) and Amentioning

confidence: 99%

Covalently Linked Heme in Cytochrome P4504A Fatty Acid Hydroxylases

Hoch

Montellano

2001

Journal of Biological Chemistry

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Three independent experimental methods, liquid chromatography, denaturing gel electrophoresis with heme staining, and mass spectrometry, establish that the CYP4A class of enzymes has a covalently bound heme group even though the heme is not cross-linked to the protein in other P450 enzymes. Covalent binding has been demonstrated for CYP4A1, -4A2, -4A3, -4A8, and -4A11 heterologously expressed in Escherichia coli. However, the covalent link is also present in CYP4A1 isolated from rat liver and is not an artifact of heterologous expression. The extent of heme covalent binding in the proteins as isolated varies and is substoichiometric. In CYP4A3, the heme is attached to the protein via an ester link to glutamic acid residue 318, which is on the I-helix, and is predicted to be within the active site. This is the first demonstration that a class of cytochrome P450 enzymes covalently binds their prosthetic heme group. Heme1 is the essential prosthetic group of many important proteins, including the hemoglobins, catalases, peroxidases, and cytochromes. In cytochrome P450 enzymes, the heme sits deep inside the protein with its iron ligated to a cysteine thiolate group and the sixth iron coordination site occupied by a water molecule in the substrate-free state (1). This coordination arrangement gives cytochrome P450 enzymes their unique spectral and catalytic properties. These enzymes are involved in many vital processes, including the biosynthesis of steroids and other lipophilic physiological effectors, the metabolism of drugs, and the degradation of xenobiotics. A unique feature of the cytochrome P450 enzymes is their ability to catalyze the hydroxylation of unactivated hydrocarbons under physiological conditions rather than the extreme conditions required for the reaction to occur under uncatalyzed conditions. Sequence homologies within the heme-binding region of the protein suggest that, despite the existence of numerous P450 forms with different substrate specificities, individual P450 enzymes share a common catalytic mechanism of oxygen activation.Work in this laboratory has focused on the P450 enzymes of family 4 (CYP4), especially the four known rat isoforms (CYP4A1, -4A2, -4A3, and -4A8) and the single characterized human isoform (CYP4A11) (2-5). The first three rat enzymes are present in liver and are induced by clofibrate, whereas all four enzymes are constitutively expressed in the rat kidney. The enzymes of the CYP4 family are distinguished by their unique ability to hydroxylate preferentially the thermodynamically disfavored terminal methyl group of medium and long chain fatty acids, prostaglandins, and other eicosanoids. Although other P450 enzymes hydroxylate fatty acids at internal positions of the hydrocarbon chain (6 -8), only the CYP4 enzymes preferentially hydroxylate the energetically disfavored terminal methyl group. Site-directed mutagenesis studies have begun to shed some light on the mechanism by which these enzymes achieve this specificity (4, 5, 9), but the detailed mechanism remains obscur...

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“…Preparation of the Mature E375D Mutant of LPO-Mature LPO has two covalent bonds, the first between Asp 225 and the 5-methyl and the second between Glu 375 and the 1-methyl (8,9). Previous work has shown that mutation of Glu 375 to an Asp prevents formation of the covalent bond to that residue, although the 1-methyl is still oxidized to some extent to the 1-hydroxymethyl derivative (8).…”

Section: Preparation Of the Hrp F41e Mutant With A Heme-proteinmentioning

confidence: 99%

“…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)(8)(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).…”

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confidence: 99%

Role of Heme-Protein Covalent Bonds in Mammalian Peroxidases

Huang

Wojciechowski

Montellano

2006

Journal of Biological Chemistry

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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...

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The Heme Prosthetic Group of Lactoperoxidase

Cited by 52 publications

References 33 publications

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

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

Covalently Linked Heme in Cytochrome P4504A Fatty Acid Hydroxylases

Role of Heme-Protein Covalent Bonds in Mammalian Peroxidases

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