Tetrahydrobiopterin (BH4) is an essential cofactor for all three isoforms of nitric oxide synthase (NOS). However, its binding sites and functional roles remain elusive. Here, we demonstrated that cys-99 of human endothelial NOS (ecNOS) is critical for BH4 involvement in NOS catalytic activity and stability. Mutation of cys-99 to alanine in ecNOS resulted in loss of catalytic activity which could be restored to the level of wild type by adding a high concentration of exogenous BH4 to the crude extract. Purified C99A mutant was unstable and its maximal activity was only about 20% of the purified wild type activity. Comparison of BH4 concentration-dependent citrulline formation between C99A and the wild type revealed that the BH4 concentrations required for generating half-maximal citrulline were 10-fold higher for C99A. Purified C99A had no detectable BH4 and had a reduced heme content when compared to the purified wild type, but retained the ability of forming CO-ferrous heme complex and had the same Km value for L-arginine (~ 4 µM) as the wild type. These findings indicate that Cys-99 is critically involved in BH4 binding. Mutation of this residue leads to reduced affinity for BH4 and the resultant enzyme instability and irreversible heme loss.Copyright 1995, 1999 Academic Press, Inc.