Distinct influence of N-terminal elements on neuronal nitric-oxide synthase structure and catalysis.
Nitric oxide (NO) is a signal molecule produced in animals by three different NO synthases. Of these, only NOS I (neuronal nitric-oxide synthase; nNOS) is expressed as catalytically active N-terminally truncated forms that are missing either an N-terminal leader sequence required for protein-protein interactions or are missing the leader sequence plus three core structural motifs that in other NOS are required for dimer assembly and catalysis. To understand how the N-terminal elements impact nNOS structure-function, we generated, purified, and extensively characterized variants that were missing the N-terminal leader sequence (Delta296nNOS) or missing the leader sequence plus the three core motifs (Delta349nNOS). Eliminating the leader sequence had no impact on nNOS structure or catalysis. In contrast, additional removal of the core elements weakened but did not destroy the dimer interaction, slowed ferric heme reduction and reactivity of a hemedioxy intermediate, and caused a 10-fold poorer affinity toward substrate l-arginine. This created an nNOS variant with slower and less coupled NO synthesis that is predisposed to generate reactive oxygen species along with NO. Our findings help justify the existence of nNOS N-terminal splice variants and identify specific catalytic changes that create functional differences among them.
Panda
Koustubh
K
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Adak
Subrata
S
Aulak
Kulwant S
KS
Santolini
Jerome
J
McDonald
John F
JF
Stuehr
Dennis J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D019281:Dimerization; D005415:Flavins / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D051381:Rats
D005415:Flavins; D009569:Nitric Oxide; D006418:Heme; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat