Neuronal NOS, Nitroarenes, and Neurotoxicity

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DESCRIPTION (provided by applicant): Unraveling the structural secrets of neuronal nitric oxide synthase (nNOS) has become an important goal for the purpose of understanding how nNOS can be differentially regulated and/or modulated by specific chemicals. The interaction of nNOS with environmental pollutants such as nitroarenes, resulting in the production of reactive intermediates and toxicity, is the subject of this proposal. In order to investigate the mechanisms of 1,3-dinitrobenzene (1,3-DNB)-mediated neurotoxicity, we hypothesize that in the presence of nitroarenes, nNOS is converted from a purely nitric oxide (NO*) and L-citrulline synthase to a peroxynitrite (ONOO-) and L-citrulline synthase. ONOO- is a very potent and reactive oxidant formed when nNOS simultaneously produces NO* and superoxide anion radical (O2-). O2- is formed by the nNOS-mediated reduction and subsequent reoxidation of nitroarenes. Concomitantly, nNOS maintains adequate electron flow to the heme to produce its normal products, NO* and L-citrulline. The simultaneous production of both NO* and O2- in close proximity leads immediately to ONOO- formation via the combination of these two radicals at a near diffusion-controlled reaction rate. The ONOO- that is produced, along with partially-reduced intermediates of the nitroarene, are proposed to play a role in the neurotoxicity associated with exposure to 1,3-DNB. The long-term objective of this project is to determine how metabolism of nitroarenes, resulting in the production of reactive intermediates (such as ONOO-, O2-, H2O2, NOx), and active reduced metabolites, mediate toxicity within the central nervous system. Further, we will determine how enzymatic activity of nNOS can be regulated by nitroarenes, O2-, and active reduced metabolites of nitroarenes such as the nitroso- and N-hydroxy-species. Toward this goal, our immediate specific aims are: Aim #1: To dissect electron transfer from nNOS to nitroarenes such as 1,3-DNB by using recombinantly-expressed and purified nNOS and nNOS constructs. Aim #2: To test the hypothesis that interaction of NOS with neurotoxic nitroarenes, including 1,3- DNB, results in modulation of NOS activity, stimulation of O2- production, and a gain of function by becoming a ONOO-generating enzyme.