Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H(2), an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo-ferryl (Fe(IV)O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P(1/2), of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g(x), g(y), g(z)) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P(1/2) value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration ( approximately 0.2 s(-1)) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.