Involvement of the perferryl complex of nitric oxide synthase in the catalysis of secondary free radical formation

• Published in: Biochimica et biophysica acta Vol. 1526; no. 1; pp. 95 - 104
• Main Authors: Porasuphatana, Supatra, Tsai, Pei, Pou, Sovitj, Rosen, Gerald M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 03.04.2001
• Subjects: Arginine - metabolism; Catalysis; Cell Line; Electron Spin Resonance Spectroscopy; Ethanol - metabolism; Free Radicals - metabolism; Iron - chemistry; Models, Chemical; Nitric oxide; Nitric oxide synthase; Nitric Oxide Synthase - antagonists & inhibitors; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type I; omega-N-Methylarginine - pharmacology; Perferryl complex; Signal Transduction; Spin Trapping; Superoxide; Superoxides - metabolism; Transfection; l-NAME, N G-nitro- l-arginine methyl ester; O ⋅− 2, superoxide; Perferryl complex; NOS I, neuronal nitric oxide synthase; DEPMPO-OOH, 2-(diethoxyphosphoryl)-5-hydroperoxy-2-methyl-1-pyrrolidinyoxyl; ONOO −, peroxynitrite; EtOH, ethanol; EPR, electron paramagnetic resonance; Superoxide; NO ⋅, nitric oxide; HO ⋅, hydroxyl radical; 4-POBN, α-(4-pyridyl-1-oxide)- N- tert-butylnitrone; SOD, superoxide dismutase; l-NMA, N G-methyl- l-arginine; l-NMMA, N G-monomethyl- l-arginine; Nitric oxide; H 2O 2, hydrogen peroxide; Nitric oxide synthase; (NH 4) 2DTCS, ammonium N-(dithiocarboxy)sarcosine; DMPO, 5,5-dimethyl-1-pyrroline- N-oxide; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline- N-oxide
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(01)00114-3

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO ⋅) and superoxide (O ⋅− 2) during enzymatic cycling, and the ratio of each free radical is dependent upon the concentration of l-arginine. Using spin trapping and electron paramagnetic resonance spectroscopy, we detected α-hydroxyethyl radical (CH 3 ⋅CHOH), produced during the NOS I metabolism of ethanol (EtOH). The generation of CH 3 ⋅CHOH by NOS I was found to be Ca 2+/calmodulin dependent. Superoxide dismutase prevented CH 3 ⋅CHOH formation in the absence of l-arginine. However, in the presence of l-arginine, the production of CH 3 ⋅CHOH was independent of O ⋅− 2 but dependent upon the concentration of l-arginine. Formation of CH 3 ⋅CHOH was inhibited by substituting d-arginine for l-arginine, or inclusion of the NOS inhibitors N G-nitro- l-arginine methyl ester, N G-monomethyl- l-arginine and the heme blocker, sodium cyanide. The addition of potassium hydrogen persulfate to NOS I, generating the perferryl complex (NOS-[Fe 5+=O] 3+) in the absence of oxygen and Ca 2+/calmodulin, and EtOH resulted in the formation of CH 3 ⋅CHOH. NOS I was found to produce the corresponding α-hydroxyalkyl radical from 1-propanol and 2-propanol, but not from 2-methyl-2-propanol. Data demonstrated that the perferryl complex of NOS I in the presence of l-arginine was responsible for catalyses of these secondary reactions.