Versatile Regulation of Neuronal Nitric Oxide Synthase by Specific Regions of Its C-Terminal Tail
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- 作者：Mauro Tiso ; Jesú ; s Tejero ; Koustubh Panda ; Kulwant S. Aulak ; Dennis J. Stuehr
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：December 18, 2007
- 年：2007
- 卷：46
- 期：50
- 页码：14418 - 14428
- 全文大小：465K
- 年卷期：v.46,no.50(December 18, 2007)
- ISSN：1520-4995
文摘
The C-terminal tail (CT) of neuronal nitric oxide synthase (nNOS) is a regulatory elementthat suppresses nNOS activities in the absence of bound calmodulin (CaM). A crystal structure of thenNOS 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 detailedstudies. We generated truncation mutants in nNOS and nNOSr to test the importance of 3 different regionsof the CT. Eliminating the terminal half of the CT (all residues from Ile1413 to Ser1429), which is invisiblein 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 shieldingthat normally occurs in response to NADPH binding. Additional removal of the CT -helix (residues1401 to 1412) significantly increased the NADP+ release rate, flavin autoxidation, and NADPH oxidaseactivity, and caused hyper-deshielding of the FMN cofactor. These effects were associated with increasedreductase activity and slightly diminished heme reduction and NO synthesis. Further removal of residuesdownstream from Gly1396 (a full CT truncation) amplified the aforementioned effects and in additionaltered NADP+ interaction with FAD, relieved the kinetic suppression on flavin reduction, and furtherdiminished heme reduction and NO synthesis. Our results reveal that the CT exerts both multifaceted andregiospecific effects on catalytic activities and related behaviors, and thus provide new insights intomechanisms that regulate nNOS catalysis.