Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase

Eight polar amino acid residues in the putative substrate-binding region fr om Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Th r-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) w ere individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system, Mutant eNOS ha d a very low L-citrulline formation activity with the exception of D369E an d R372K, which retained 27% and 44% of the wild-type enzyme activity, respe ctively, Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm), All the Asp-369 mutants h ad higher K-d values for L-arginine (1-10 mM) than wild-type eNOS (0.4 mu M ) and an unstable heme-CO complex, and except for D369E, had a very low (6R )5,6,7,8-Letrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidaz ole did not bind to wild-type eNOS home, but bound to all Asp-369 and Arg-3 72 mutants (K-d ranged from 10 to 65 mu M) except R372K, Heme spin-state ch anges caused by binding of 3,5-lutidine appeared to depend on both charge a nd size of the side chains of residues 369 and 372, Furthermore, all Asp-36 9 and Arg-372 mutants were defective in dimer formation. These results sugg est that residues Asp-369 and Arp-372 in eNOS play a critical role in oxyge nase domain active-site structure and activity.