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J. Biol. Chem., Vol. 279, Issue 18, 18323-18333, April 30, 2004

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A Conserved Aspartate (Asp-1393) Regulates NADPH Reduction of Neuronal Nitric-oxide Synthase

IMPLICATIONS FOR CATALYSIS^*

Koustubh Panda, Subrata Adak, David Konas, Manisha Sharma, and Dennis J. Stuehr¶

From the Department of Immunology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195

Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP⁺ reductase (FNR)-like module that contains NADPH- and FAD-binding sites. FNR-like modules in related flavoproteins have three conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM. All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated with slower rates of CaM-dependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s) in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPH-dependent reduction of the nNOS flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently fast to enable NO synthesis by nNOS.

Received for publication, September 21, 2003 , and in revised form, February 2, 2004.

^* This work was supported by National Institutes of Health Grant GM51491 (to D. J. S.) and a grant from The American Heart Association (to S. A.). 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.

Both authors contributed equally to this work.

Present address: Dept. of Infectious Disease, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Rd., Calcutta-700032, India.

^¶ To whom correspondence should be addressed: Immunology NB-30, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-6950; Fax: 216-444-9329; E-mail: stuehrd{at}ccf.org.

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