DINITROBENZENES STIMULATE ELECTRON FLUX WITHIN NEURONAL NITRIC OXIDE SYNTHASE IN THE ABSENCE OF CALMODULIN

[摘要] Efficient electron transfer and conversion of L-arginine to L-citrulline and nitric oxide (NO●) by neuronal nitric oxide synthase (nNOS) requires calmodulin (CaM) binding.  The present study focused on electron transfer ability of resting state CaM-free nNOS in presence of dinitrobenzene isomers (DNBs).  NADPH oxidation (NADPHox) and acetylated cytochrome-c reduction (AcCyt-cred) catalyzed by nNOS and the CaM binding sequence-deficient nNOS reductase construct (nNOS-FP) were estimates of total electron flux and O2● production, respectively.  All the DNBs (o-, m-, p-) independently stimulated rates of NADPHox by CaM-free nNOS and by nNOS-FP in isomer- and concentration-dependent manner.  Blocking nNOS heme by imidazole or L-arginine did not affect CaM-free nNOS-catalyzed NADPHox stimulated by DNBs.  This stimulated electron flux by DNBs did not support NO● formation by CaM-free nNOS.  The DNBs, like FeCN, extract electrons from both FMN and FAD of the nNOS reductase domain.  All three DNBs greatly stimulated nNOS and nNOS-FP catalyzed AcCyt-cred that was significantly inhibited by SOD demonstrating O2● formation.  Thus, in presence of DNBs, resting-state CaM-deficient nNOS efficiently transfers electrons generating O2●, inferring that additional metabolic roles for nNOS exist that are not yet explored.

[学科分类] 基础医学

[关键词] nNOS;NADPH;redox;superoxide