The function of the small insertion in the hinge subdomain in the control of constitutive mammalian nitric-oxide synthases.
Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a beta hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.
Jones
Rachel J
RJ
Department of Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA.
Smith
Susan M E
SM
Gao
Ying Tong
YT
DeMay
Bradley S
BS
Mann
Kevin J
KJ
Salerno
Kathleen M
KM
Salerno
John C
JC
D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry; D002417:Cattle; D002845:Chromatography; D003579:Cytochrome Reductases / Q000737:chemistry; D045304:Cytochromes c / Q000378:metabolism; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006418:Heme / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D017434:Protein Structure, Tertiary; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014466:Ultraviolet Rays
D002147:Calmodulin; D009569:Nitric Oxide; D006418:Heme; D000244:Adenosine Diphosphate; D045304:Cytochromes c; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; C496316:Nos3 protein, rat; D003579:Cytochrome Reductases; D009251:NADPH-Ferrihemoprotein Reductase; D010450:Endopeptidases