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Dinitrobenzene Isomers Structure‐ Dependent Stimulation of Neuronal Nitric Oxide Synthase Catalyzed Electron Flux in the Absence of Calmodulin
Author(s) -
Joshi Chintamani N,
Miller R Timothy
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.502.18
Subject(s) - chemistry , calmodulin , nitric oxide , nitric oxide synthase , electron transport chain , citrulline , electron transfer , stimulation , reductase , biochemistry , biophysics , arginine , stereochemistry , enzyme , photochemistry , organic chemistry , amino acid , endocrinology , biology
Neuronal nitric oxide synthase (nNOS) requires calmodulin (CaM) ‐ binding for efficient electron transfer converting L ‐arginine to L ‐citrulline and nitric oxide. The present studies focus on electron transfer to and through CaM‐free nNOS. Turnover rates (min −1 ) of nNOS‐mediated NADPH oxidation (NADPH ox ) and acetylated cytochrome‐ c reduction (AcCyt‐ c red ) were used to estimate total electron flux and O 2 . production. Dinitrobenzenes (DNBs) stimulated the rate of NADPH ox by CaM‐free nNOS in an isomer‐ and concentration‐ dependent manner. Rates for NADPH ox by CaM‐free nNOS at the limit of solubility of the DNBs (160 ?M) were 189 ± 5 min −1 , 94 ± 3 min −1 and 38 ± 2 min −1 for 1,4‐, 1,2‐ and 1,3‐DNB, respectively, compared to the solvent control (7 ± 1 min −1 ). Highly stimulated rates of AcCyt‐ c red were observed in presence of all the three DNBs. The nNOS reductase construct without the CaM binding domain (aa 745‐1729) behaved in a manner similar to holo‐nNOS in the presence of DNBs, in the NADPH ox and AcCyt‐ c red assays. These results indicate that CaM‐free nNOS generates ROS at environmentally‐relevant levels of DNBs (ASTDR). Considering the absolute amount of constitutive NOS expressed in organisms; chronic exposure to low levels of DNBs may lead to continuous oxidative stress. Supported by ES011982 and RR008124 to RTM and UTEP respectively.