Oxyferryl heme and not tyrosyl radical is the likely culprit in prostaglandin H synthase-1 peroxidase inactivation.

Prostagladin H synthases (PGHSs) catalyze the first two steps in biosynthesis of prostaglandins, the cyclooxygenase reaction that converts arachidonic acid (AA)1 to prostaglandin (PG)G2 and the peroxidase reaction that reduces PGG2 to PGH2 (1). The cyclooxygenase reaction is dependent upon formation of the Tyr385 radical during the peroxidase reaction (2). A simplified mechanistic model is shown in Figure 1. According to this model, resting PGHS first reacts with peroxide and generates Intermediate I, an oxidized species that contains oxyferryl heme and a porphyrin cation radical in the peroxidase active site. An intramolecular electron transfer converts Intermediate I to Intermediate II, which contains two oxidants: oxyferryl heme, or Compound II, in the peroxidase site and a tyrosyl radical on residue 385 in the cyclooxygenase channel (3–5). The Tyr385 radical abstracts the pro-S hydrogen atom from C13 of AA, initiating cyclooxygenase catalysis and PGG2 formation (6). FIGURE 1 Peroxidase mechanism of FePGHS-1 and MnPGHS-1 with hypothetical self-inactivation pathways, adapted from Dietz et al. (4) and Wu et al. (9). Fe4+=O/P+•, oxyferryl heme with a porphyrin cation radical, or Mn5+ (Intermediate I); Fe4+=O/P or Mn4+ ... Reaction with peroxide drives the redox events that lead to strong feedback amplification of cyclooxygenase catalysis, and it also drives the irreversible self-inactivation of both peroxidase and cyclooxygenase activities that imposes an upper limit on prostanoid synthesis (7, 8). Cyclooxygenase inactivation induced by peroxide occurs on the same time scale as peroxidase inactivation, although the former seems more sensitive to the peroxide structure (9, 10). Intermediate II is the starting point for peroxide-induced peroxidase inactivation (9). However, it remains unclear which of the two oxidants generated after reaction with peroxide, the oxyferryl heme or the tyrosyl radical, is the damaging species. Earlier analyses of PGHS-1 peroxidase kinetics relied primarily on rapid-scan stopped-flow measurements (3, 4, 11). Tyrosyl radical kinetic studies using rapid-freeze quench and EPR methods were conducted primarily with convenient, water soluble peroxides such as EtOOH (12), and more physiologically relevant lipid peroxides have not been investigated in detail. The kinetic relationship between the oxyferryl heme and the tyrosyl radical present in Intermediate II has not been thoroughly tested by parallel stopped-flow and RFQ EPR kinetic measurements in PGHS-1, although there are indications that kinetic divergence of oxyferryl heme and tyrosyl radical can occur in PGHS-2 (12). In this study, the kinetics of oxyferryl heme and tyrosyl radical formation and decay and of peroxidase inactivation were examined in reactions of PGHS-1 with a lipid peroxide, 15-(S)-HPETE, at different heme to peroxide ratios. This long-chain fatty acid peroxide is a cyclooxygenase side product (13) and is much closer in physical characteristics and peroxidase kinetic parameters to PGG2 than is EtOOH (11, 14). To improve the discriminating power, the kinetic analyses were conducted with native PGHS-1, with Mn-PGHS-1, which has impaired peroxidase catalysis (15–17), and with native PGHS-1 complexed with indomethacin, which selectively impairs cyclooxygenase catalysis (18). Correlated kinetic data for both the heme group and the tyrosyl radical in this study strongly support their independent decay kinetics and suggest that it is the oxyferryl heme, and not the tyrosyl radical, that is the dominant oxidant in peroxidase inactivation. This conclusion is consistent with computer fitting to a minimal model for PGHS peroxidase catalysis and self-inactivation.