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J. Biol. Chem., Vol. 280, Issue 47, 39208-39219, November 25, 2005

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C-terminal Tail Residue Arg¹⁴⁰⁰ Enables NADPH to Regulate Electron Transfer in Neuronal Nitric-oxide Synthase^*

Mauro Tiso¶, David W. Konas, Koustubh Panda, Elsa D. Garcin||, Manisha Sharma, Elizabeth D. Getzoff||, and Dennis J. Stuehr1

From the Departments of Immunology and Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the ^¶Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, and the ^||Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg¹⁴⁰⁰ in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg¹⁴⁰⁰ in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K_m for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg¹⁴⁰⁰-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.

Received for publication, July 18, 2005 , and in revised form, August 29, 2005.

^* This work was supported by National Institutes of Health Grants GM51491 (to D. J. S.) and R01HL5883 (to E. D. Getzoff) and by American Heart Association fellowship 0415154B (to M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Immunology, NB-30, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-6950; Fax: 216-444-9329; E-mail: stuehrd{at}ccf.org.

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