Characterization of mouse nNOS2, a natural variant of neuronal nitric-oxide synthase produced in the central nervous system by selective alternative splicing

Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expresse d in the central nervous system having a 105-amino acid deletion in the hem e-binding domain as a result of in-frame mutation by specific alternative s plicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escheric hia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no L-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-pr omoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) s ignal at g = 6.0 indicating the presence of a high spin ferriheme as isolat ed and showed a peak at around 420 nm in the CO difference spectrum, instea d of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Th us, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreov er, negligible spectral perturbation of the nNOS2 ferriheme was detected up on addition of either L-arginine or imidazole. These provide a possible rat ional explanation for the inability of nNOS2 to catalyze the cytochrome P45 0-type monooxygenase reaction.