NITRIC-OXIDE TRAPPING OF THE TYROSYL RADICAL OF PROSTAGLANDIN-H SYNTHASE-2 LEADS TO TYROSINE IMINOXYL RADICAL AND NITROTYROSINE FORMATION

The determination of protein nitrotyrosine content has become a freque ntly used technique for the detection of oxidative tissue damage, Prot ein nitration has been suggested to be a final product of the producti on of highly reactive nitrogen oxide intermediates (e.g. peroxynitrite ) formed in reactions between nitric oxide (NO.) and oxygen-derived sp ecies such as superoxide. The enzyme prostaglandin H synthase-2 (PHS-2 ) forms one or more tyrosyl radicals duping its enzymatic catalysis of prostaglandin formation, In the presence of She NO.-generator diethyl amine nonoate, the electron spin resonance spectrum of the PHS-a-deriv ed tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom. The magnitude of the nitrogen hyperfine c oupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitros otyrosine, a stable product of the addition of NO. to tyrosyl radical. Addition of superoxide dismutase did not alter the spectra, indicatin g that peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to the NO.-generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine ni tration that does not require formation of more highly reactive nitrog en oxide intermediates such as peroxynitrite or nitrogen dioxide.