PubMed2001
The quenching of the Y(D)(.) tyrosyl radical in photosystem II by nitric oxide was reported to result from the formation of a weak tyrosyl radical-nitric oxide complex (Petrouleas, V., and Diner, B. A. (1990) Biochim. Biophys. Acta 1015, 131-140). This radical/radical reaction is expected to generate an electron spin resonance (ESR)-silent 3-nitrosocyclohexadienone species that can reversibly regenerate the tyrosyl radical and nitric oxide or undergo rearrangement to form 3-nitrosotyrosine. It has been proposed that 3-nitrosotyrosine can be oxidized by one electron to form the tyrosine iminoxyl radical (>C=N-O*). This proposal was put forth as a result of ESR detection of the iminoxyl radical intermediate when photosystem II was exposed to nitric oxide (Sanakis, Y., Goussias, C., Mason, R. P., and Petrouleas, V. (1997) Biochemistry 36, 1411-1417). A similar iminoxyl radical was detected in prostaglandin H synthase-2 (Gunther, M. R., Hsi, L. C., Curtis, J. F., Gierse, J. K., Marnett, L. J., Eling, T. E., and Mason, R. P. (1997) J. Biol. Chem., 272, 17086-17090). Although the iminoxyl radicals detected in the photosystem II and prostaglandin H synthase-2 systems strongly suggest a mechanism involving 3-nitrosotyrosine, the iminoxyl radical ESR spectrum was not unequivocally identified as originating from tyrosine. We report here the detection of the non-protein L-tyrosine iminoxyl radical generated by two methods: 1) peroxidase oxidation of synthetic 3-nitroso-N-acetyl-L-tyrosine and 2) peroxidase oxidation of free L-tyrosine in the presence of nitric oxide. A newly developed ESR technique that uses immobilized enzyme was used to perform the ESR experiments. Analysis of the high resolution ESR spectrum of the tyrosine iminoxyl radical generated from free tyrosine and nitric oxide reveals a 28.4-G isotropic nitrogen hyperfine coupling and a 2.2-G proton hyperfine coupling assigned to the proton originally ortho to the phenoxyl oxygen.