It was reported by Chen et al. [(1995) Biochem. Biophys. Res. Commun. 210, 1119-1129] that the C99A mutant of endothelial NOS, corresponding to C331 in rat nNOS, is critical for tetrahydrobio-pterin (BH4) binding. To examine this possibility, we expressed rat nNOS holoenzyme (C331A) in E. co/i. The mutated protein is able to bind L-arg only after prolonged incubation (3-6 hrs) and its estimated Ks for L-arg is 500 times higher than that of wild-type (wt) nNOS (1 \M). The Kd for imidazole is 75±5 uM for wt nNOS and 71±6 u.M for C331A nNOS. We found that BH4 binds to C331A nNOS but only after L-arg binding (Kd for [3HJ-BH4 binding = 166 nM). Incubation with L-arg fully restores activity to the level of wt nNOS (560 nmol NO fomned/min/mg/@25°C). Soret CD of C331A nNOS, as isolated, exhibits a decreased rotational strength while retaining a similar spectral shape as wt nNOS, suggesting that the asymmetric protein environment around the heme chromophore may be altered before prolonged L-arg supplementation. The ability of C331A nNOS protein to bind [ 125I]-Calmodulin and to reduce cytochrome c was similar to that of wt nNOS. We speculate, based on the analysis of the C331A mutant, that conformational changes after L-arg binding allow BH4 to be bound.