15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and manganese-protoporphyrin

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..

Topics: Arachidonic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Isoenzymes; Leukotrienes; Lipid Peroxides; Molecular Conformation; Mutation; Oxygen; Peroxidases; Peroxides; Prostaglandin-Endoperoxide Synthases; Protoporphyrins; Tyrosine

Prostaglandin H synthase (PGHS) is a heme protein that catalyzes both the cyclooxygenase and peroxidase reactions needed to produce prostaglandins G2 and H2 from arachidonic acid. Replacement of the heme group by mangano protoporphyrin IX largely preserves the cyclooxygenase activity, but lowers the steady-state peroxidase activity by 25-fold. Thus, mangano protoporphyrin IX serves as a useful tool to evaluate the function of the heme in PGHS. A detailed kinetic analysis of the peroxidase reaction using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), EtOOH, and other peroxides as substrates has been carried out to compare the characteristics of PGHS reconstituted with mangano protoporphyrin IX (Mn-PGHS) to those of the native heme enzyme (Fe-PGHS). The rate constant describing the reaction of Mn-PGHS with 15-HPETE to form the oxidized, Mn(IV) intermediate with absorption at 420 nm, exhibits saturable behavior as the 15-HPETE concentration is raised from 10 to 400 microM. This is most likely due to the presence of a second, earlier intermediate between the resting enzyme and the Mn(IV) species. Measurements at high substrate concentrations permitted resolution of the absorbance spectra of the two oxidized Mn-PGHS intermediates. The spectrum of the initial intermediate, assigned to a Mn(V) species, had a line shape similar to that of the later intermediate, assigned to a Mn(IV) species, suggesting that a porphyrin pi-cation radical is not generated in the peroxidase reaction of Mn-PGHS. The rate constant estimated for the formation of the earlier intermediate with 15-HPETE is 1.0 x 10(6) M-1 s-1 (20 degrees C, pH 7.3). A rate constant of 400 +/- 100 s-1 was estimated for the second step in the reaction. Thus, Mn-PGHS reacts considerably more slowly than Fe-PGHS with 15-HPETE to form the first high-valent intermediate, but the two enzymes appear to follow a similar overall reaction mechanism for generation of oxidized intermediates. The difference in rate constants explains the observed lower steady-state peroxidase activity of Mn-PGHS compared with Fe-PGHS.

Topics: Animals; Aspirin; Cyclooxygenase Inhibitors; Heme; Hydrogen Peroxide; Indomethacin; Leukotrienes; Lipid Peroxides; Lipoxygenase Inhibitors; Male; Peroxidase; Photosensitizing Agents; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins G; Prostaglandins H; Protoporphyrins; Sheep; Spectrophotometry, Atomic