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J. Biol. Chem., Vol. 282, Issue 2, 897-907, January 12, 2007

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Dissociation of Nitric Oxide from Soluble Guanylate Cyclase and Heme-Nitric Oxide/Oxygen Binding Domain Constructs^*

Jonathan A. Winger¹², Emily R. Derbyshire¹, and Michael A. Marletta^¶||3

From the Department of Medicinal Chemistry, the University of Michigan, Ann Arbor, Michigan 48109 and the ^¶Department of Chemistry, the Department of Molecular and Cell Biology, and the ^||Division of Physical Biosciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length 11 sGC, and the constructs 1(1–194), 1(1–385), and 2(1–217), at 37 and 10 °C with and without the substrate analogue guanosine-5'-[(,-methylene]triphosphate (GMPCPP) or the activator 3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and 2(1–217) at 37 °C and dramatically accelerated NO dissociation from sGC at 10 °C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 °C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.

Received for publication, July 3, 2006 , and in revised form, November 8, 2006.

^* This work was supported by Laboratory Directed Research and Development funding from Lawrence Berkeley National Laboratory (to M. A. M.), by an Eli Lilly fellowship, a Pharmaceutical Sciences Training Program Fellowship Grant NIGMS GM07767 from the National Institutes of Health, and an American Foundation for Pharmaceutical Education Fellowship (to J. A. W.). 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 Both authors contributed equally to this paper.

² Present address: Dept. of Molecular and Cell Biology, 16 Barker Hall MC 3202, University of California, Berkeley, CA 94720-3202.

³ To whom correspondence should be addressed: Dept. of Chemistry, 211 Lewis Hall, University of California, Berkeley, CA 94720-1460. Tel.: 510-643-9325; Fax: 510-643-9388; E-mail: marletta{at}berkeley.edu.

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