SPECTROSCOPIC EVIDENCE FOR REACTION OF PROSTAGLANDIN-H SYNTHASE-1 TYROSYL RADICAL WITH ARACHIDONIC-ACID

The coupling between the peroxidase and cyclooxygenase activities of prostaglandin H synthase (PGHS) has been proposed to be mediated by a critical tyrosyl radical through a branched chain mechanism (Dietz, R,, Nastainczyk, W,, and Ruf, H. H, (1988) fur. J, Biochem, 171, 321-328), In this study, we have examined the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with arachidonate, Anaerobic addition of arachidonate following formation of the peroxide-induced wide doublet or wide singlet tyrosyl radical led to disappearance of the tyrosyl radicals and emergence of a new EPR signal, which is distinct from known PGHS-1 tyrosyl radicals, The new radical was clearly derived from arachidonate because its EPR line shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was used, Subsequent addition of oxygen to samples containing the fatty acyl radical resulted in regeneration of tyrosyl radical EPR, In contrast, the peroxide-generated tyrosyl radical in indomethacin-treated PGHS-1 (a narrow singlet) failed to react with arachidonate, consistent with the cyclooxygenase inhibition by indomethacin, These results indicate that the peroxide-generated wide doublet and wide singlet tyrosyl radicals serve as immediate oxidants of arachidonate bound at the cyclooxygenase active site to form a carbon-centered fatty acyl radical, which reacts with oxygen to form a hydroperoxide, These observations represent the first direct evidence of chemical coupling between the peroxidase reaction and arachidonate oxygenation in PGHS-1 and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis.