Heme spin states and peroxide-induced radical species in prostaglandin H synthase.
We have examined the optical, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectra of pure ovine prostaglandin H synthase in its resting (ferric) and ferrous states and after addition of hydrogen peroxide or 15-hydroperoxyeicosatetraenoic acid. In resting synthase, the distribution of heme between high- and low-spin forms was temperature-dependent: 20% of the heme was low-spin at room temperature whereas 50% was low-spin at 12 K. Two histidine residues were coordinated to the heme iron in the low-spin species. Anaerobic reduction of the synthase with dithionite produced a high-spin ferrous species that had no EPR signals. Upon reaction with the resting synthase, both hydroperoxides quickly generated intense (20-40% of the synthase heme) and complex EPR signals around g = 2 that were accompanied by corresponding decreases in the intensity of the signals from ferric heme at g = 3 and g = 6. The signal generated by HOOH had a doublet at g = 2.003, split by 22 G, superimposed on a broad component with a peak at g = 2.085 and a trough at g = 1.95. The lipid hydroperoxide generated a singlet at g = 2.003, with a linewidth of 25 G, superimposed on a broad background with a peak at g = 2.095 and a trough around g = 1.9. These EPR signals induced by hydroperoxide may reflect synthase heme in the ferryl state complexed with a free radical derived from hydroperoxide or fragments of hydroperoxide.
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds; D005296:Ferrous Compounds; D005609:Free Radicals; D006418:Heme; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015289:Leukotrienes; D008054:Lipid Peroxides / Q000494:pharmacology; D008297:Male; D010084:Oxidation-Reduction; D011451:Prostaglandin-Endoperoxide Synthases; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013053:Spectrophotometry
D001095:Arachidonic Acids; D005290:Ferric Compounds; D005296:Ferrous Compounds; D005609:Free Radicals; D015289:Leukotrienes; D008054:Lipid Peroxides; D006418:Heme; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D006861:Hydrogen Peroxide; D011451:Prostaglandin-Endoperoxide Synthases