Dinitrobenzene-mediated production of peroxynitrite by neuronal nitric oxide synthase

Neuronal nitric oxide synthase (nNOS) is a modular enzyme that consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, fused by a calmodulin (CaM)-binding sequence. Within the central nervous system, nNOS is localized in the cerebellum. CaM binding to nNOS activates both intradomain as well as interdomain electron transfer, and thus activity. The nNOS reductase shares many characteristics with NADPH-cytochrome P450 reductase (CPR), such as catalyzing the reduction of exogenous electron acceptors such as quinones and nitroarenes. The nitroarene 1,3-dinitrobenzene (1,3-DNB) is a cerebellar neurotoxicant in rats. 1,3-DNB is metabolized by CPR in liver, and it was proposed that metabolism of 1,3-DNB to reactive intermediates is involved in mediating the cerebellar neurotoxicity. We have found that, in a manner similar to CPR, nNOS can interact with 1,3-DNB and generate superoxide anion radical (O2*-). Electron transfer through the nNOS reductase is not limiting for nitric oxide (NO.) and L-citrulline production, even in the presence of certain exogenous electron acceptors such as 1,3-DNB. Therefore, NO., L-citrulline, and O2*- are simultaneously produced by nNOS in the presence of 1,3-DNB and other nitroarenes. The simultaneous production of NO. and O2*- leads to peroxynitrite (ONOO-) formation via the combination of these two radicals at a near-diffusion-controlled reaction rate. We present convincing data supporting the hypothesis that in the presence of 1,3-DNB, nNOS is converted from a purely NO. and L-citrulline synthase to a ONOO- and L-citrulline synthase, and propose that the resulting nitosative stress plays a role in the cerebellar neurotoxicity of 1,3-DNB. This paper introduces a new and novel enzymatic mechanism with direct toxicological implications whereby nNOS is converted into a ONOO- synthase by certain nitroarenes.