title: Role of the covalent glutamic acid 242-heme linkage in the formation and reactivity of redox intermediates of human myeloperoxidase.
abstract: In human myeloperoxidase the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu242 and Asp94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met243 and the beta-carbon of the vinyl group on pyrrole ring A. In the present study, wild-type recombinant myeloperoxidase (recMPO) and the variant Glu242Gln were produced in Chinese hamster ovary cells and investigated in a comparative sequential-mixing stopped-flow study in order to elucidate the role of the Glu242-heme ester linkage in the individual reaction steps of both the halogenation and peroxidase cycle. Disruption of the ester bond increased heme flexibility, blue shifted the UV-vis spectrum, and, compared with recMPO, decelerated cyanide binding (1.25 x 10(4) versus 1.6 x 10(6) M(-)(1) s(-)(1) at pH 7 and 25 degrees C) as well as compound I formation mediated by either hydrogen peroxide (7.8 x 10(5) versus 1.9 x 10(7) M(-)(1) s(-)(1)) or hypochlorous acid (7.5 x 10(5) versus 2.3 x 10(7) M(-)(1) s(-)(1)). The overall chlorination and bromination activity of Glu242Gln was 2.0% and 24% of recMPO. The apparent bimolecular rate constants of compound I reduction by chloride (65 M(-)(1) s(-)(1)), bromide (5.4 x 10(4) M(-)(1) s(-)(1)), iodide (6.4 x 10(5) M(-)(1) s(-)(1)), and thiocyanate (2.2 x10(5) M(-)(1) s(-)(1)) were 500, 25, 21, and 63 times decreased compared with recMPO. By contrast, Glu242Gln compound I reduction by tyrosine was only 5.4 times decreased, whereas tyrosine-mediated compound II reduction was 60 times slower compared with recMPO. The effects of exchange of Glu242 on electron transfer reactions are discussed.
authors.affiliation: Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Zederbauer
authors.forename: Walter
authors.initials: W
authors.lastname: Jantschko
authors.forename: Karin
authors.initials: K
authors.lastname: Neugschwandtner
authors.forename: Christa
authors.initials: C
authors.lastname: Jakopitsch
authors.forename: Nicole
authors.initials: N
authors.lastname: Moguilevsky
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
authors.forename: Paul Georg
authors.initials: PG
authors.lastname: Furtmüller
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001965:Bromides / Q000378:metabolism; D016466:CHO Cells; D002712:Chlorides / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D003486:Cyanides / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics; D047209:Eosinophil Peroxidase / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008715:Methionine / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001965:Bromides; D002712:Chlorides; D003486:Cyanides; D005290:Ferric Compounds; D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D006418:Heme; D008715:Methionine; D047209:Eosinophil Peroxidase; D009195:Peroxidase