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J. Biol. Chem., Vol. 278, Issue 39, 37122-37131, September 26, 2003

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Distinct Influence of N-terminal Elements on Neuronal Nitric-oxide Synthase Structure and Catalysis^*

Koustubh Panda, Subrata Adak, Kulwant S. Aulak, Jerome Santolini, John F. McDonald and Dennis J. Stuehr 

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

Nitric oxide (NO) is a signal molecule produced in animals by three different NO synthases. Of these, only NOS I (neuronal nitric-oxide synthase; nNOS) is expressed as catalytically active N-terminally truncated forms that are missing either an N-terminal leader sequence required for protein-protein interactions or are missing the leader sequence plus three core structural motifs that in other NOS are required for dimer assembly and catalysis. To understand how the N-terminal elements impact nNOS structure-function, we generated, purified, and extensively characterized variants that were missing the N-terminal leader sequence (296nNOS) or missing the leader sequence plus the three core motifs (349nNOS). Eliminating the leader sequence had no impact on nNOS structure or catalysis. In contrast, additional removal of the core elements weakened but did not destroy the dimer interaction, slowed ferric heme reduction and reactivity of a hemedioxy intermediate, and caused a 10-fold poorer affinity toward substrate L-arginine. This created an nNOS variant with slower and less coupled NO synthesis that is predisposed to generate reactive oxygen species along with NO. Our findings help justify the existence of nNOS N-terminal splice variants and identify specific catalytic changes that create functional differences among them.

Received for publication, April 29, 2003 , and in revised form, June 25, 2003.

^* Supported by National Institutes of Health Grants CA53914 and GM51491 (to D. J. S.). 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.

To whom correspondence should be addressed: Immunology NB3, 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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