Crucial role of Lys(423) in the electron transfer of neuronal nitric-oxide synthase.
Nitric-oxide synthase (NOS) is composed of an oxygenase domain having cytochrome P450-type heme active site and a reductase domain having FAD- and FMN-binding sites. To investigate the route of electron transfer from the reductase domain to the heme, we generated mutants at Lys(423) in the heme proximal site of neuronal NOS and examined the catalytic activities, electron transfer rates, and NADPH oxidation rates. A K423E mutant showed no NO formation activity (<0.1 nmol/min/nmol heme), in contrast with that (72 nmol/min/nmol heme) of the wild type enzyme. The electron transfer rate (0.01 min(-1)) of the K423E on addition of excess NADPH was much slower than that (>10 min(-1)) of the wild type enzyme. From the crystal structure of the oxygenase domain of endothelial NOS, Lys(423) of neuronal NOS is likely to interact with Trp(409) which lies in contact with the heme plane and with Cys(415), the axial ligand. It is also exposed to solvent and lies in the region where the heme is closest to the protein surface. Thus, it seems likely that ionic interactions between Lys(423) and the reductase domain may help to form a flavin to heme electron transfer pathway.
Shimanuki
T
T
Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan.
Sato
H
H
Daff
S
S
Sagami
I
I
Shimizu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D004579:Electron Transport; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000502:physiology*; D052248:Nitric Oxide Synthase Type I; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic
D009419:Nerve Tissue Proteins; D006418:Heme; D011189:Potassium Chloride; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D008239:Lysine