title: Role of heme-protein covalent bonds in mammalian peroxidases. Protection of the heme by a single engineered heme-protein link in horseradish peroxidase.
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.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.
authors.forename: Liusheng
authors.initials: L
authors.lastname: Huang
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Wojciechowski
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D000818:Animals; D001966:Bromine / Q000737:chemistry; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D007313:Insecta; D007784:Lactoperoxidase / Q000737:chemistry; D008956:Models, Chemical; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods
chemical_list: D003432:Cross-Linking Reagents; D006420:Hemeproteins; D006418:Heme; D006735:Horseradish Peroxidase; D007784:Lactoperoxidase; D010544:Peroxidases; D010100:Oxygen; D001966:Bromine