Abstract
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 radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra 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 MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) 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 an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. 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 support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..
Highlights
The tyrosyl radical mechanism nicely explains the heme dependence of both enzyme activities and the requirement of the cyclooxygenase for a hydroperoxide activator [3, 7]
The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36 –38G, but was able to convert arachidonic acid (AA) to an AA1⁄7 with an EPR spectrum similar to that found with prostaglandin H synthase-2 (PGHS-2). These results indicate that the peroxide-generated radicals in Prostaglandin H synthase (PGHS)-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide
Oxidation of AA by Peroxide-induced PGHS Radicals—Oxidation of arachidonate to a fatty acid radical is a key step in the proposed branched-chain mechanism for PGHS cyclooxygenase catalysis (Step 3 in Scheme 1)
Summary
The tyrosyl radical mechanism nicely explains the heme dependence of both enzyme activities and the requirement of the cyclooxygenase for a hydroperoxide activator [3, 7]. It is consistent with x-ray crystallographic data that show a tyrosine residue (Tyr-385 in PGHS-1 and Tyr-371 in PGHS-2) positioned between the heme and the arachidonic acid binding channel (8 –10). Droperoxide; PGG2, prostaglandin G2; PGH2, prostaglandin H2; d8-AA, 5,6,8,9,11,12,14,15-octadeuterated arachidonate; WS2, wide-singlet radical found in PGHS-2; MnPGHS-1, PGHS-1 reconstituted with mangano protoporphyrin IX; HPETE, hydroperoxyeicosatetraenoic acid. Analysis of the EPR spectrum of the arachidonyl radicals provides insight into the structure of the enzyme-bound fatty acid radicals in both PGHS isoforms
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