title: Versatile regulation of neuronal nitric oxide synthase by specific regions of its C-terminal tail.
abstract: The C-terminal tail (CT) of neuronal nitric oxide synthase (nNOS) is a regulatory element that suppresses nNOS activities in the absence of bound calmodulin (CaM). A crystal structure of the nNOS reductase domain (nNOSr) (Garcin, E. D., Bruns, C. M., Lloyd, S. J., Hosfield, D. J., Tiso, M., Gachhui, R., Stuehr, D. J., Tainer, J. A., and Getzoff, E. D. (2004) J. Biol. Chem. 279, 37918-37927) revealed how the first half of the CT interacts with nNOSr and thus provided a template for detailed studies. We generated truncation mutants in nNOS and nNOSr to test the importance of 3 different regions of the CT. Eliminating the terminal half of the CT (all residues from Ile1413 to Ser1429), which is invisible in the crystal structure, had almost no impact on NADP+ release, flavin reduction, flavin autoxidation, heme reduction, reductase activity, or NO synthesis activity, but did prevent an increase in FMN shielding that normally occurs in response to NADPH binding. Additional removal of the CT alpha-helix (residues 1401 to 1412) significantly increased the NADP+ release rate, flavin autoxidation, and NADPH oxidase activity, and caused hyper-deshielding of the FMN cofactor. These effects were associated with increased reductase activity and slightly diminished heme reduction and NO synthesis. Further removal of residues downstream from Gly1396 (a full CT truncation) amplified the aforementioned effects and in addition altered NADP+ interaction with FAD, relieved the kinetic suppression on flavin reduction, and further diminished heme reduction and NO synthesis. Our results reveal that the CT exerts both multifaceted and regiospecific effects on catalytic activities and related behaviors, and thus provide new insights into mechanisms that regulate nNOS catalysis.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. m_tiso@yahoo.com
authors.forename: Mauro
authors.initials: M
authors.lastname: Tiso
authors.forename: Jesús
authors.initials: J
authors.lastname: Tejero
authors.forename: Koustubh
authors.initials: K
authors.lastname: Panda
authors.forename: Kulwant S
authors.initials: KS
authors.lastname: Aulak
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D003579:Cytochrome Reductases / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D009249:NADP; D005486:Flavin Mononucleotide; D052248:Nitric Oxide Synthase Type I; D003579:Cytochrome Reductases