The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding

It has been proposed that Cys(99) of human endothelial nitric oxide synthas e (eNOS) is responsible for tetrahydrobiopterin (BH,) binding. To examine t his possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escheri chia coli, with the homologous Cys(331) to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme, C331A -nNOS, as isolated, was catalytically incompetent. Upon prolonged incubatio n with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine ad dition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited a n absorbance maximum at 420 nm, C331A-nNOS, as isolated, did not bind detec table levels of either [H-3]N-omega-nitro-L-arginine or [H-3]BH4, but [H-3] BH4 binding was reinstated after extended incubation with excess L-arginine , On the other hand, C331A-nNOS and WT-NOS were identical with regard to im idazole binding affinity, CaM binding affinity, and rates of cytochrome c a nd 2,6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conver sion of low to high spin heme after extended incubation with high concentra tions of L-arginine (0.1-10 mM). The estimated K-d for L-arginine binding t o C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 mu M ve rsus 2-3 mu M). Here we propose that Cys(331) plays an important role in st abilizing L-arginine binding to nNOS, Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for no rmalized BH4 binding and nitric oxide synthetic activity.