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 °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 ~16 G; overall peak-to-trough width of 35 G) that has been attributed to a 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 <10% residual activity contained 1.8 nitrotyrosines/subunit; apoenzyme reacted under the same conditions had >80% of the original activity and contained 0.7 nitrotyrosine/subunit. Reaction of the synthase with hydroperoxides thus appears to lead to the sequential generation of two tyrosyl radicals, with one or both of the radicals required for cyclooxygenase catalysis. The electronic absorbance and magnetic circular dichroism spectra of the synthase holoenzyme before and after addition of heme ligands (cyanide, azide, and fluoride) and the electron paramagnetic spectrum of the resting synthase were little changed from the corresponding spectra of the indomethacin-synthase complex. Indomethacin thus does not appear to greatly alter the heme environment in the synthase; inhibition of the cyclooxygenase by this agent is more likely due to a perturbation of the hydroperoxide-induced radical species. © 1990, American Chemical Society. All rights reserved.
