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 Glu41 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.
Highlights
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)
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 horseradish peroxidase (HRP) in which the heme is attached to the protein via a covalent bond between Glu[41] and the heme 3-methyl
ABTS assay (2.5 Ϯ 0.5) ϫ 104 (1.8 Ϯ 0.1) ϫ 103 (3.6 Ϯ 0.7) ϫ 103 noncovalently bound heme, but exposure of this protein to several equivalents of H2O2 results in autocatalytic cross-linking of the heme 3-methyl group to Glu[41] via an ester link essentially identical to those found in the mammalian peroxidases
Summary
Materials and General Methods—Sf9 cells (Invitrogen) were grown in Excell 420TM (JRH Biosciences), and High FiveTM cells were grown in Express FiveTM (Invitrogen) supplemented with glutamine (2.7 g/liter of medium). The supernatant was diluted with an equal volume of deionized water, followed by adding phenylmethylsulfonyl fluoride (as a protease inhibitor) at a final concentration of 200 mg/liter To this solution Amberlite CG50 cation exchange resin (pre-equilibrated in 50 mM Tris1⁄7HCl, pH 7.4) was added in three portions over 2 h The fractions with (0.1 M, pH 8.2), followed by exchange of the sample into citrate absorbance at 412 nm were pooled, concentrated to ϳ2 ml, and buffer (0.5 M, pH 4.4) with a Millipore ultrafree centrifugal filter diluted 10-fold with deionized water. The final volume was 100 l of ϳ18 M protein (based on heme absorption) To this were added KBr (60 l, 2 M) and H2O2 (40 l, 1.81 mM, 40 equivalents), and the solution was incubated for 20 min before it was exchanged into bis-tris propane buffer (0.1 M, pH 8.2). The activity reflects the amount of HOBr generated per min by the enzyme
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