Cysteine 99 of Endothelial Nitric Oxide Synthase (NOS-III) Is Critical for Tetrahydrobiopterin-Dependent NOS-III Stability and Activity

Abstract

Tetrahydrobiopterin (BH 4) 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 BH 4 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 BH 4 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 BH 4 concentration-dependent citrulline formation between C99A and the wild type revealed that the BH 4 concentrations required for generating half-maximal citrulline were 10-fold higher for C99A. Purified C99A had no detectable BH 4 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 K m value for L-arginine (∼ 4 μM) as the wild type. These findings indicate that Cys-99 is critically involved in BH 4 binding. Mutation of this residue leads to reduced affinity for BH 4 and the resultant enzyme instability and irreversible heme loss.