15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and ethyl-hydroperoxide

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..

Topics: Arachidonic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Isoenzymes; Leukotrienes; Lipid Peroxides; Molecular Conformation; Mutation; Oxygen; Peroxidases; Peroxides; Prostaglandin-Endoperoxide Synthases; Protoporphyrins; Tyrosine

Topics: Animals; Cyclooxygenase Inhibitors; Electron Spin Resonance Spectroscopy; Free Radicals; Indomethacin; Leukotrienes; Lipid Peroxides; Peroxides; Prostaglandin-Endoperoxide Synthases; Sheep; Tetranitromethane

Prostaglandin H synthase has both a heme-dependent peroxidase activity and a cyclooxygenase activity. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by an interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical [Stubbe, J. A. (1989) Annu. Rev. Biochem. 58, 257-285]. We have examined the kinetics of radical formation with both ethyl hydroperoxide (EtOOH) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and have analyzed the effects of indomethacin (a selective cyclooxygenase inhibitor) and tetranitromethane (TNM; a selective agent for nitration of tyrosyl residues) on the synthase. At -14 degrees C both EtOOH and 15-HPETE generated within 5 s a free radical species whose electron paramagnetic resonance spectrum was dominated by a doublet centered at g = 2.005 (splitting of approximately 16 G; overall peak-to-trough width of 35 G) that has been attributed to tyrosyl radical. The doublet subsequently gave way to a singlet with a similar peak-to-trough width; the doublet-to-singlet transition was complete in 20-60 s. The intensity of the doublet/singlet combination peaked at 0.6 spins/heme after 120 s with EtOOH and at about 0.3 spins/heme after 20 s with 15-HPETE; the radical intensity declined slowly with EtOOH but more rapidly with 15-HPETE. Reaction of the indomethacin-synthase complex with EtOOH resulted in a narrower (peak-to-trough width of 24 G) singlet free radical signal, with no evidence of an earlier doublet; the intensity of the singlet peaked at 0.45 spins/heme after about 300 s. Reaction of TNM-treated synthase with EtOOH resulted in a singlet almost identical with that seen for the indomethacin-synthase complex. Reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both cyclooxygenase and peroxidase activity, with the former being lost rapidly and completely while the latter was lost slowly and to about 50%. Ibuprofen, a competitive cyclooxygenase inhibitor, slowed the rate of inactivation of the cyclooxygenase by about 20-fold. The rate of inactivation of the cyclooxygenase activity in synthase apoenzyme by TNM was also about 20-fold less than that observed with the holoenzyme. Amino acid analyses revealed that TNM-reacted holoenzyme with less than 10% residual activity contained 1.8 nitrotyrosines/subunit; apoenzyme reacted under the same conditions had greater than 80% of the original activity and conta

Topics: Cyclooxygenase Inhibitors; Electron Spin Resonance Spectroscopy; Free Radicals; Indomethacin; Leukotrienes; Lipid Peroxides; Peroxides; Prostaglandin-Endoperoxide Synthases; Tetranitromethane; Tyrosine