Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and-2

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cycloo xygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2, Rapid-freez e EPR and stopped-flow were used to characterize tyrosyl radical kinetics i n PGHS-1 and -2 reacted with ethyl hydrogen peroxide, In PGHS-1, a wide dou blet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition t o a wide singlet (33-34 G); changes in total radical intensity paralleled t hose of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but t ransition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS -1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the I ntermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesul ide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the ind omethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical f ormed in concert with Intermediate II, but later persisted while Intermedia te II relaxed. These results substantiate the kinetic competence of a tyros yl radical as the catalytic intermediate for both PGHS isoforms and also in dicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.