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 frequently used technique for the detection of oxidative tissue damage, Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e.g. peroxynitrite) formed in reactions between nitric oxide (NO.) and oxygen-derived species 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 diethylamine nonoate, the electron spin resonance spectrum of the PHS-a-derived tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom. The magnitude of the nitrogen hyperfine coupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitrosotyrosine, a stable product of the addition of NO. to tyrosyl radical. Addition of superoxide dismutase did not alter the spectra, indicating 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 nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.