Soluble guanylate cyclase (sGC) catalyzes formation of cGMP, an important second messenger in mammalian cells. Nitric oxide (NO) activates sGC by binding to a ferrous heme in the regulatory domain of the enzyme, leading to proximal histidine release and a presumable conformation change in the catalytic domain. The mechanism of the sGC-NO reaction, and its regulatory properties, remain unclear despite extensive work. Manduca sexta (tobacco hornworm) sGC is structurally conserved and performs similar biological functions as the mammalian enzymes. We expressed and purified the MsGC regulatory domain as a functional heterodimer in E. coli. The kinetics of the NO-heme reaction, studied by stopped-flow spectroscopy with the fragment sGC, revealed a complicated histidine release process, displaying zero first kinetics at low NO concentration (1-30 µM), but second order kinetics at higher NO concentrations. sGC modulators, including ATP, GTP and YC-1 (3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole), affect the reaction in different fashions. With a stoichiometric amount of NO, GTP and YC-1 facilitate histidine release, but ATP restrains it. We also co-expressed and partially purified full-length MsGC as an active dimer in E. coli. In this protein, ATP inhibits MsGC activation by NO while YC-1 boosts it. We thus conclude that there must be a second nucleotide binding site away from catalytic domain, allosterically regulating NO activation of sGC. Investigation on affinity of this binding is under way.