Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase Joa ˜o Laranjinha* and Enrique Cadenas  *Faculty of Pharmacy and Center for Neurosciences, University of Coimbra, Portugal  Department of Molecular Pharmacology & Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California, USA Abstract This study aimed to characterize the redox interaction between 3,4-dihydroxyphenylacetic acid (DOPAC) and nitric oxide (ÆNO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and ÆNO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg 2+ , and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn-superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one-electron oxidation of DOPAC by ÆNO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to ÆNO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions. Keywords: DOPAC, dopamine, nitric oxide, Parkinson’s disease, superoxide dismutase. J. Neurochem. (2002) 81, 892–900. Nitric oxide (ÆNO) is a diffusible free radical and membrane permeant neurotransmitter in brain (Moncada et al. 1991; Dawson and Dawson 1998). Physiologically ÆNO is produced by a constitutive neuronal ÆNO synthase (nNOS) following glutamate stimulation of NMDA receptors and the subsequent cellular influx of calcium. Excessive stimu- lation of NMDA receptors may increase ÆNO concentration in brain, but expression of an inducible, Ca 2+ -independent NOS isoform (iNOS) may also occur with the consequent increase in ÆNO concentration (Dawson and Dawson 1998). ÆNO reacts at near diffusion-limited rates with other free radicals and with transition metals in proteins such as guanylate cyclase and haemoglobin, but despite its free radical character ÆNO is remarkably unreactive with most biological molecules at the low concentrations produced in vivo (Beckman 1996). However, excessive production of ÆNO is neurotoxic and has been implicated in a variety of neurological disorders (Dawson and Dawson 1998), inclu- ding Parkinson’s disease, which is characterized by the loss of dopaminergic neurones in the nigrostriatal system. Two salient features of Parkinson’s disease may be relevant to the context of this study. First, the toxic effects of ÆNO in Parkinson’s disease are highlighted by recent findings showing that induction of iNOS in glial cells contributestodegenerationofdopamine-containingneurones in a mice model of Parkinson’s disease (Liberatore et al. 1999), and that inhibition of nNOS prevents Parkinsonism in animal models (Hantraye et al. 1996; Przedborski et al. ReceivedNovember27,2001;revisedmanuscriptreceivedFebruary18, 2002; accepted February 20, 2002. Address correspondence and reprint requests to Joa ˜o Laranjinha Faculty of Pharmacy and Center for Neurosciences, University of Coimbra, Courac ¸a dos Apo ´stolos, 51, r/c, 3000 Coimbra, Portugal. E-mail: laranjin@ci.uc.pt Abbreviations used: DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DOPAC, 3,4-dihydroxyphenylacetic acid; DTPA, diethylenetriamine pentacetic acid; EPR, electron paramagnetic resonance; GSH, glutathi- one; GSÆ, glutathionyl radical, iNOS, NOS isoform; Mb–Fe III , metmyo- globin; Mb–Fe III –NO, nitrosylmyoglobin; nNOS, neuronal ÆNO synthase; SOD, superoxide dismutase. Journal of Neurochemistry , 2002, 81, 892–900 892 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 81, 892–900