Prostaglandin H synthase (PGHS) is a hemoprotein with both cyclooxygenase and peroxidase activities. Several aspects of the peroxidase and cyclooxygenase activities and the formation of substrate-induced free radical species were characterized with ovine seminal vesicle PGHS reconstituted with manganese protoporphyrin IX (Mn-PGHS) for comparison with the enzyme-containing heme (Fe-PGHS). Compared to Fe-PGHS, the Km of Mn-PGHS peroxidase for ethyl hydroperoxide was much higher, but that for 15-hydroperoxyeicosatetraenoic acid (15-HPETE) was little changed. The Mn-PGHS peroxidase Vmax value with 15-HPETE was about 4% that with Fe-PGHS. Mn-PGHS oxidized 0.95 +/- 0.05 and Fe-PGHS oxidized 2.06 +/- 0.09 mol of TMPD/mol of 15-HPETE. Reaction of 15-HPETE with Mn-PGHS resulted in approximately equal proportions of two lipid products: 15-hydroxyeicosatetraenoic acid (15-HETE) and a compound identified as 15-ketoeicosatetraenoic acid. Fe-PGHS produced only 15-HETE. Thus, 15-HETE can serve as an efficient two-electron reductant of oxidized Mn-PGHS intermediates. The rate of accumulation of oxidized Mn-PGHS intermediate was dependent on the substrate, decreasing in the following order: 15-HPETE > 11,14-eicosadienoate > arachidonate > EtOOH. The cyclooxygenase specific activity increased in a saturable fashion as the concentration of Mn-PGHS was increased, reaching a value higher than that for Fe-PGHS. Computer simulations of the reaction kinetics indicated that this dependence on the Mn-PGHS level was a consequence of the low rate of formation of oxidized peroxidase intermediate. Incubation of Mn-PGHS with either 15-HPETE or arachidonate resulted in the rapid production of a free radical species. The initial accumulation of radical coincided with the synthesis of PGG2 and PGH2, indicating that the radical was kinetically competent to participate in cyclooxygenase catalysis. Reaction with tetranitromethane, a reagent that selectively nitrates tyrosyl residues, destroyed the cyclooxygenase activity of Mn-PGHS, resulting in a much narrower free radical EPR signal. These effects indicate that tyrosine residues are essential for cyclooxygenase activity and that they influence the radical structure in Mn-PGHS.