title: Dissociation of nitric oxide from soluble guanylate cyclase and heme-nitric oxide/oxygen binding domain constructs.
abstract: 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 alpha1beta1 sGC, and the constructs beta1(1-194), beta1(1-385), and beta2(1-217), at 37 and 10 degrees C with and without the substrate analogue guanosine-5'-[(alpha,beta-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 beta2(1-217) at 37 degrees C and dramatically accelerated NO dissociation from sGC at 10 degrees C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 degrees 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.
authors.affiliation: Department of Medicinal Chemistry, the University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Winger
authors.forename: Emily R
authors.initials: ER
authors.lastname: Derbyshire
authors.forename: Michael A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009569:Nitric Oxide / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D018411:Spodoptera; D013696:Temperature
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D010100:Oxygen