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J. Biol. Chem., Vol. 279, Issue 35, 36876-36883, August 27, 2004

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The Function of the Small Insertion in the Hinge Subdomain in the Control of Constitutive Mammalian Nitric-oxide Synthases^*

Rachel J. Jones, Susan M. E. Smith, Ying Tong Gao, Bradley S. DeMay, Kevin J. Mann, Kathleen M. Salerno, and John C. Salerno

From the Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.

Received for publication, March 12, 2004 , and in revised form, June 14, 2004.

^* This work was supported by the American Diabetes Association and the American Heart Association. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) X59949 (rat nNOS; 11), M89952 (bovine eNOS; 15), and G198407 (murine iNOS; 11, 18).

The NADPH P450 reductase nucleotide sequence reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession no. P16435 (37).

To whom correspondence should be addressed: Dept. of Biology, JR Science Center, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180. Tel.: 518-276-6392; Fax: 518-276-2344; E-mail: salerj{at}rpi.edu.

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