STRUCTURAL CHARACTERIZATION OF ARACHIDONYL RADICALS FORMED BY PROSTAGLANDIN-H SYNTHASE-2 AND PROSTAGLANDIN-H SYNTHASE-1 RECONSTITUTED WITH MANGANO PROTOPORPHYRIN-IX

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction, Peroxides also induce radica l formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 recon stituted with mangano, protoporphyrin IX (MnPGHS-1), but the EPR spect ra of these radicals are distinct from the initial tyrosyl radical in PGHS-1, We have examined the ability of the radicals in PGHS-2 and MnP GHS-1 to oxidize AA, using single-turnover EPR studies. One wide singl et tyrosyl radical with an overall EPR line width of 29-31 gauss (G) w as generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic additions of AA to PGHS-2, immediately after formation of this radica l led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Sub sequent addition of oxygen resulted in regeneration of the tyrosyl rad ical, In contrast, the peroxide-generated radical (a 21G narrow single t) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with a n EPR spectrum similar to that found with PGHS-2. These results indica te that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can eac h serve as immediate oxidants of AA to form the same carbon-centered f atty acid radical that subsequently reacts with oxygen to form a hydro peroxide, The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results supp ort a functional role for peroxide-generated radicals in cyclooxygenas e catalysis by both PGHS isoforms and provide important structural cha racterization of the carbon-centered AA..