| Abstract: | 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 alpha-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 alpha-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. |
