The optical interconversion of the P-450 and P-420 forms of neuronal nitric oxide synthase: effects of sodium cholate, mercury chloride and urea

We investigated whether or not neuronal nitric oxide synthase (nNOS) (EC 1. 14.13.39) was converted to the P-420 form on exposure to sodium cholate, me rcury chloride or urea, and the reconversion of the P-420 to the P-450 form . Sodium cholate and mercury chloride induced the conversion of nNOS from t he P-450 to the P-420 form in concentration- and incubation time-dependent manners, and the nNOS activity decreased. In the presence of glycerol, L-ar ginine and/or tetrahydrobiopterin, the sodium cholate-treated P-420 form co uld be reconverted to the P-450 form under constant experimental conditions , and the nNOS activity could also be restored. The mercury chloride-treate d P-420 form of nNOS could be reconverted to the P-450 form on incubation w ith reduced glutathione (GSH) or L-cysteine, and the nNOS activity was reco vered. However, no reconversion of the mercury chloride-treated P-420 form to the P-450 form was observed in the presence of glycerol, L-arginine, or tetrahydrobiopterin. Urea (4.0 M) dissociated nNOS into its subunits, but n NOS remained in the P-450 form. The nNOS monomer was more susceptible to so dium cholate. After removing the urea by dialysis, and supplementation of t he nNOS solution with glycerol, L-arginine or BH,, the P-420 was reconverte d to the P-450 form, and the reassociation of nNOS monomers was also observ ed. These results suggested that nNOS was more stable as to exposure to sod ium cholate: mercury chloride or urea in comparison to microsomal cytochrom e P-450, which may be due to the different heme environment and protein str ucture. (C) 2001 Elsevier Science Ltd. All rights reserved.