Abstract
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.
Highlights
Nitric-oxide synthase (NOS)1 produces nitric oxide (NO) for a range of important biological functions
NOS consists of an oxygenase domain with a thiol-coordinated heme active site similar to that of cytochrome P450 (P450), and an electron-transfer domain related to NADPH-cytochrome P450 reductase which binds FMN and FAD
H4B is bound to a site distant from the L-Arg-binding site located on the heme distal side within the oxygenase domain, based on the x-ray crystal structure of the dimeric oxygenase domain of inducible NOS and endothelial NOS (9 –11)
Summary
Materials—H4B was purchased from Schircks Laboratories (Jona, Switzerland). Other reagents, which were from Wako Pure Chemicals (Osaka, Japan), were of the highest guaranteed grade and were used without further purification. The concentration of nNOS was determined optically from the [CO-reduced] Ϫ [reduced] difference spectrum using ⌬⑀444–467 nm ϭ 55 mMϪ1 cmϪ1. This ⌬⑀ value was estimated by the pyridine hemochromogen method [28] assuming that one heme is bound to one subunit of this enzyme. The NADPH oxidation rate was determined spectrophotometrically as an absorbance decrease at 340 nm, using an extinction coefficient of 6.22 mMϪ1 cmϪ1. Cytochrome c reductase activity was determined by monitoring the absorbance at 550 nm using an extinction coefficient ϭ 21 mMϪ1 cmϪ1. Potassium ferricyanide reductase activity was determined by monitoring the absorbance at 420 nm using an extinction coefficient ϭ 1.2 mMϪ1 cmϪ1. RasMac 2.6-ucb1.0 software was used to determine the distance between Lys194 and Trp180 in eNOS
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