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

It has been proposed that Cys⁹⁹ of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH₄) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys³³¹ to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (r-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 r-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [³H]N(ω)-nitro-L- arginine or [³H]BH₄, but [³H]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 imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2,6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated K(d) for t-arginine binding to C331A-nNOS was two orders of magnitude greater than WTnNOS (>100 μm versus 2-3 μM). Here we propose that Cys³³¹ plays an important role in stabilizing 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 normalized BH₄ binding and nitric oxide synthetic activity.