Aromatic residues and neighboring Arg414 in the (6R)-5,6,7, 8-tetrahydro-L-biopterin binding site of full-length neuronal nitric-oxide synthase are crucial in catalysis and heme reduction with NADPH.
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 Arg(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 EC(50) value for H4B (30-40-fold of wild type). The Tyr(706) phenol group interacts with the heme propionate and Arg(414) amine via hydrogen bonds. The mutation of Arg(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.
Sagami
I
I
Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan. ikuko@icrs.tohoku.ac.jp
Sato
Y
Y
Daff
S
S
Shimizu
T
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002384:Catalysis; D019281:Dimerization; D004330:Drosophila; D005973:Glutamine / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007930:Leucine / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism; D051381:Rats; D013054:Spectrophotometry, Atomic; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism
D005973:Glutamine; D001708:Biopterins; D006418:Heme; D010649:Phenylalanine; D009249:NADP; D014364:Tryptophan; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; C496322:Nos1 protein, rat; C003402:sapropterin; D007930:Leucine