title: Mechanism of hydroperoxide reduction by mangano-prostaglandin endoperoxide synthase.
abstract: Reaction of manganese-reconstituted prostaglandin endoperoxide synthase (Mn-PGHS) with 15-hydroperoxyeicosatetraenoic acid (15-HPETE) generates two products in nearly equal amounts: 15-hydroxyeicosatetraenoic acid (15-HETE) and 15-ketoeicosatetraenoic acid (15-KETE) [Kulmacz et al. (1994) Biochemistry 33, 5428-5439]. Their proposed mechanism to explain 15-KETE formation, namely oxidation of 15-HETE by the peroxidase activity of MnPGHS, was tested and found not to occur. Instead, 15-KETE may arise by one-electron reduction of 15-HPETE followed by oxidation of an intermediate alkoxyl radical. The mechanism of hydroperoxide reduction by Mn-PGHS was investigated using 10-hydroperoxyoctadeca-8,12-dienoic acid (10-OOH-18:2), a diagnostic probe of hydroperoxide reduction pathways. Reaction of Mn-PGHS with 10-OOH-18:2 generated the two-electron reduced product, 10-hydroxyoctadeca-8,12-dienoic acid (10-OH-18:2), as well as the one-electron reduction products, 10-oxooctadeca-8,12 dienoic acid (10-oxo-18:2) and 10-oxodec-8-enoic acid (10-oxo-10:1) in relative yields of 82, 10, and 7%, respectively. The identity of the one-electron reduction products was confirmed by electrospray ionization mass spectrometry. The detection of 10-oxo-10:1 provides strong evidence for the production of an alkoxyl radical during 10-OOH-18:2 reduction by Mn-PGHS. Like 15-HPETE, reaction of Mn-PGHS with 13-hydroperoxyoctadeca-8,12-dienoic acid (13-OOH-18:2) generated two products in equal amounts: 13-hydroxyoctadeca-8,12-dienoic acid (13-OH-18:2) and the keto fatty acid 13-oxooctadeca-8,12-dienoic acid (13-oxo-18:2). Comparison of the three hydroperoxides demonstrates that 15-HPETE is a much better substrate for Mn-PGHS than 10-OOH-18:2 or 13-OOH-18:2 with 10-fold greater turnovers. The results show that Mn-PGHS catalyzes both one- and two-electron hydroperoxide reduction and that the pathway of alkoxyl radical decomposition is influenced by the protein component of Mn-PGHS and the structure of the alkoxyl radical intermediate.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: L M
authors.initials: LM
authors.lastname: Landino
authors.forename: L J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism; D004583:Electrons; D005609:Free Radicals; D006893:Hydroxyeicosatetraenoic Acids / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D015289:Leukotrienes / Q000737:chemistry / Q000378:metabolism*; D008054:Lipid Peroxides / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008345:Manganese / Q000737:chemistry; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep; D013379:Substrate Specificity
chemical_list: D001095:Arachidonic Acids; D005609:Free Radicals; D006893:Hydroxyeicosatetraenoic Acids; D015289:Leukotrienes; D008054:Lipid Peroxides; D008345:Manganese; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C054992:15-keto-5,8,11,13-eicosatetraenoic acid; D011451:Prostaglandin-Endoperoxide Synthases