1. Microsomes from ram seminal vesicles or purified prostaglandin H synthase supplemented with either arachidonic acid or prostaglandin G, formed an unstable spectral intermediate with maxima at 430 nm, 525 nm and 555 nm and minima at 410 nm, 490 nm and 630 nm. At -15 "C the band at 430 nm disappeared within 4 min whereas the trough at 410 nm increased three fold. At higher temperatures (10 -37 "C) spectral complex formation and decay were observed in less than 1 s.2. An apparent Ks-value of about 3 p M was determined for the titration of purified prostaglandin synthase with prostaglandin G2 at -20 C.3. Substrates for cooxidation reactions of prostaglandin synthase such as phenol, hydroquinone and reduced glutathione as well as the peroxidase inhibitors cyanide and azide inhibited the prostaglandin G,-induced spectral complex formation.4. The oxene donor iodosobenzene and hydrogen peroxide formed a spectral intermediate analogous to the complex observed with prostaglandin G , or arachidonic acid in ram seminal vesicle microsomes as well as with the purified prostaglandin synthase.5. These results are interpreted as the formation of a ferryl-oxo complex (FeO)3f of the heme of prostaglandin synthase with prostaglandin G, analogous to the formation of compound 1 of horseradish peroxidase.Prostaglandin H synthase is the key enzyme in prostaglandin synthesis. All prostaglandins are 'derived from the 15-hydroxy-9,lI -peroxidoprosta-5,13-dienoic acid (PGH,) which is formed from arachidonic acid by cyclization and hydroperoxidation (dioxygenase activity) and by subsequent reduction of the 1 5-hydroperoxy group (hydroperoxidase activity). Both activities are mediated by the prostaglandin H synthase, but it has not yet been established whether one bifunctional catalytic heme center or two active sites with only the hydroperoxidase being heme-dependent are involved Prostaglandin H synthase has received further attention because of its oxidizing action on a variety of organic compounds leading to reactive and therefore toxic intermediates. These oxidations require the presence of arachidonic acid and hence are characterized as cooxidations but the underlying mechanism is not clear. An intermediate free oxygen radical [3], a higher valence state of the iron porphyrin in hydroperoxidase [4] or singlet molecular oxygen [5,6] have been proposed as oxidant. The latter species was reported to cause in part the low-level chemiluminescence induced by PGG, or arachidonic acid with purified P G H synthase or microsomes from ram seminal vesicels [7]. For the [I, 21.Abbreviations. PGH synthase, prostaglandin H synthase; PGH,, 15-hydroxy-9,ll -peroxidoprosta-5,13-dienoic acid; PGG,, 15-hydroperoxy-9,11 -peroxidoprosta-5,13-dienoic acid; indomethacin, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetic acid; ascaridol, l-isopropyl-4-methyl-l,4-peroxido-2-cyclohexene; cumene hydroperoxide, benzeneisopropyl hydroperoxide; '0, singlet ('Ag) molecular oxygen ; EPR, electron paramagnetic resonance.

To characterize further the prosthetic group of PGH synthase (EC 1.14.99.1), titrations of the apoenzyme with hemin were investigated by EPR. The first hemin bound per polypeptide showed an EPR signal at g = 6.7 and 5.3 (rhombicity 9%) and was tentatively assigned to the hemin effective as prosthetic group of PGH synthase. Additional hemin bound showed a less rhombic signal (g = 6.3 and 5.8, rhombicity 3%) presumably due to nonspecific hydrophobic binding sites not effective in catalysis.

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono‐exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.