title: Detection of an Fe2+-protoporphyrin-IX intermediate during aspirin-treated prostaglandin H2 synthase II catalysis of arachidonic acid to 15-HETE.
abstract: Spectral intermediates associated with the dioxygenase and peroxidase activities of prostaglandin H2 (PGH2) synthase I and II were monitored by stopped-flow spectrometry. During reactions of PGH2 synthase I with arachidonic acid (AA) and ethyl hydrogen peroxide (EtOOH), compound I (Fe5+; formally (protoporphyrin-IX) x +Fe4+=O) and compound II (Fe4+; formally (protoporphyrin-IX)Fe4+=O) were detected. These intermediates were observed sooner with EtOOH (within 50 ms) than with AA (within 200 ms). Compound I and compound II were found to be kinetically competent with respect to AA-dependent O2 uptake. These findings are consistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. During reactions with PGH2 synthase II with AA, compound I and compound II were again observed within 200 ms and were kinetically competent to participate in dioxygenation. However, during reactions of PGH2 synthase II with EtOOH, compound I and compound II were detected much later (after 10 s). These findings would be inconsistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. When aspirin-treated PGH2 synthase II was reacted with EtOOH, a normal peroxidase cycle occurred with compound I and compound II formation occurring over 10 s. However, when aspirin-treated PGH2 synthase II was reacted with AA, a unique spectral intermediate with lambda(max) at 446 nm was detected within 3 ms and was strikingly similar to ferrous (Fe2+) protoporphyrin-IX. Aspirin-treated PGH2 synthase II was found to produce 15-HETE, and the appearance of the Fe2+ intermediate (within 3 ms) indicated that it was kinetically competent to participate in the 15-dioxygenation event. The detection of this Fe2+ intermediate and the slow formation of compound I and compound II observed with EtOOH in PGH2 synthase II suggest that peroxidative cleavage is not the initiating event in dioxygenation. Instead, it is proposed that the reduction of Fe3+ in heme to Fe2+ oxidizes a peroxide to yield an initiating peroxy radical. Since it is unlikely that 11- and 15-dioxygenation occurs via different mechanisms, our findings question mechanisms of catalysis in both PGH2 synthases.
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Tang
authors.forename: R A
authors.initials: RA
authors.lastname: Copeland
authors.forename: T M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism*; D001241:Aspirin / Q000494:pharmacology*; D002384:Catalysis; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D005296:Ferrous Compounds / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D010101:Oxygen Consumption; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis
chemical_list: D016861:Cyclooxygenase Inhibitors; D005296:Ferrous Compounds; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D011524:Protoporphyrins; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D006861:Hydrogen Peroxide; C028025:protoporphyrin IX; D011451:Prostaglandin-Endoperoxide Synthases; D001241:Aspirin