Soluble guanylate cyclase is a heterodimeric hemoprotein composed of ␣-and ␤-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for ␣,␤-methylene guanosine 5-triphosphate (GMP-CPP). In contrast, 2-dADP occupied both sites with equivalent affinities. Adenosine-5-␤,␥-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2-dADP, probably by competing for the same site as 2-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO-and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272. Soluble guanylate cyclase (sGC)2 is a well characterized NO receptor involved in cell-cell signal transduction pathways associated with neuronal communication and vasodilation (1-7). Mammalian sGC is a heterodimeric (␣␤) hemoprotein (8 -10) in which the ␤ subunit binds a stoichiometric amount of heme via a weak His-iron bond (11-13). The binding of NO to the heme markedly stimulates the enzymatic production of cGMP (9, 14 -16). The NO-mediated stimulation of enzyme activity occurs in two steps: a six-coordinate NO complex is initially formed in the reaction with NO, which is then converted to an active five-coordinate NO complex, resulting in rupture of the weak His-iron bond (17, 18).Guanylate cyclases, including soluble and particulate forms, have several functional and structural features common to those of adenylate cyclases. Both classes of enzyme catalyze the cyclization of chemically related compounds (i.e. GTP and ATP). Mammalian ACs contain two cytoplasmic catalytic domains, designated C1 and C2, that share 35% sequence identity (19 -21). When mixed together, the isolated C1 and C2 domains form an active heterodimer, which is stimulated by forskolin, a specific activator of AC (22, 23). In contrast, neither the C2 nor the C1 homodimer display detectable enzyme activity. The crystal structure of the C1-C2 heterodimer reveals a shallow trough at the interfacial region created by the head-totail association of the C1 and C2 domains (24). ATP-binding site and pseudosymmetric site are formed within the cleft of the interfacial region: the former s...