An Examination of the Source of the Tyrosyl Radical in Ovine Prostaglandin Endoperoxide Synthase-1

Abstract

A tyrosyl radical, which may initiate the cyclooxygenase reaction, has been detected in prostaglandin H synthase by electron paramagnetic resonance spectroscopy. In the crystal structure of ovine prostaglandin H synthase-1, Tyr348 and Tyr385 are in close proximity to the heme. We mutated these residues to phenylalanine to test for their involvement in tyrosyl radical formation. Native enzyme formed a tyrosyl radical centered at g=2.0036 with a width of 28 gauss. The Y348F mutant formed a singlet signal similar to that of native enzyme with a width of 28 gauss (g=2.0039). In contrast, the radical signals seen with the Y385F and Y348F/Y385F mutants were 23 gauss (g=2.004) and 22 gauss (g=2.0037). In short, tyrosyl radicals are formed even in the absence of both Tyr348 and Tyr385. In Y345F containing mutants, a cluster of aromatic amino acids which surrounds the heme group may provide an alternate pathway for electron abstraction from a more distant tyrosine, yielding a narrow tyrosyl radical signal.