Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated withcyclooxygenase inhibitors or substituted with mangano protoporphyrin IX

Self-inactivation imposes an upper limit on bioactive prostanoid synthesis by prostaglandin H synthase (PGHS). Inactivation of PGHS peroxidase activit y has been found to begin with Intermediate II, which contains a tyrosyl ra dical. The structure of this radical is altered by cyclooxygenase inhibitor s, such as indomethacin and flurbiprofen, and by replacement of heme by man ganese protoporphyrin IX (forming MnPGHS-1). Peroxidase self-inactivation i n inhibitor-treated PGHS-1 and MnPGHS-1 was characterized by stopped-flow s pectroscopic techniques and by chromatographic and mass spectrometric analy sis of the metalloporphyrin. The rate of peroxidase inactivation was about 0.3 s(-1) in inhibitor-treated PGHS-1 and much slower in MnPGHS-1 (0.05 s(- 1)); as with PGHS-1 itself, the peroxidase inactivation rates were independ ent of peroxide concentration and structure, consistent with an inactivatio n process beginning with Intermediate II. The changes in metalloporphyrin a bsorbance spectra during inactivation of inhibitor-treated PGHS-1 were simi lar to those observed with PGHS-1 but were rather distinct in MnPGHS-1; the kinetics of the spectral transition from Intermediate II to the next speci es were comparable to the inactivation kinetics in each case. In contrast t o the situation with PGHS-1 itself, significant amounts of heme degradation occurred during inactivation of inhibitor-treated PGHS-1, producing iron c hlorin and heme-protein adduct species. Structural perturbations at the per oxidase site (MnPGHS-1) or at the cyclooxygenase site (inhibitor-treated PG HS-1) thus can influence markedly the kinetics and the chemistry of PGHS-1 peroxidase inactivation.