Nitric-oxide synthase (NOS) requires the cofactor, (6R)-5,6,7,8-tetrahydrobiopterin (H4B), for catalytic activity. The crystal structures of NOSs indicate that H4B is surrounded by aromatic residues. We have mutated the conserved aromatic acids, Trp(676) Trp(678), Phe(691) His(692), and Tyr(706), together with the neighboring Ar-414 residue within the H4B binding region of full-length neuronal NOS. The W676L, W678L, and F691L mutants had no NO formation activity and had very low heme reduction rates (<0.02 min(-1)) with NADPH, Thus, it appears that Trp(676), Trp(678), and Phe(691) are important to retain the appropriate active site conformation for H4B/L-Arg binding and/or electron transfer to the heme from NADPH, The mutation of Tyr(706) to Leu and Phe decreased the activity down to 13 and 29%, respectively, of that of the wild type together with a dramatically increased EC50 value for H4B (30-40-fold of wild type). The Tyr706 phenol group interacts with the heme propionate and Arg(414) amine via hydrogen bonds. The mutation of Ar-414 to Leu and Glu resulted in the total loss of NO formation activity and of the heme reduction with NADPH, Thus, hydrogen bond networks consisting of the heme carboxylate, Tyr(706), and Arg(414) are crucial in stabilizing the appropriate conformation(s) of the heme active site for H4B/L-Arg binding and/or efficient electron transfer to occur.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 25 Aug 2000|
- FERRIC CYTOCHROME P-450-CAM
- NO SYNTHASE
- PTERIN COFACTOR
- LIGAND TRANS