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

• Published in: The Journal of biological chemistry 2007-01, Vol.282 (2), p.897-907
• Main Authors: Winger, Jonathan A., Derbyshire, Emily R., Marletta, Michael A.
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites - physiology; Cells, Cultured; Enzyme Activation - physiology; Guanylate Cyclase - chemistry; Guanylate Cyclase - metabolism; Heme - metabolism; Kinetics; Models, Chemical; Nitric Oxide - metabolism; Oxygen - metabolism; Protein Structure, Tertiary; Rats; Receptors, Cytoplasmic and Nuclear - chemistry; Receptors, Cytoplasmic and Nuclear - metabolism; Soluble Guanylyl Cyclase; Spodoptera; Temperature
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M606327200

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 α1β1 sGC, and the constructs β1(1–194), β1(1–385), and β2(1–217), at 37 and 10 °C with and without the substrate analogue guanosine-5′-[(α,β-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 β2(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.