Characterization of mouse nNOS2, a natural variant of neuronal nitric-oxide synthase produced in the central nervous system by selective alternative splicing.
Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no L-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either L-arginine or imidazole. These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction.
Iwasaki
T
T
Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan.
Hori
H
H
Hayashi
Y
Y
Nishino
T
T
Tamura
K
K
Oue
S
S
Iizuka
T
T
Ogura
T
T
Esumi
H
H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002490:Central Nervous System / Q000201:enzymology*; D006418:Heme / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D006418:Heme; D009249:NADP; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496321:Nos1 protein, mouse; C496317:Nos2 protein, mouse