Soluble guanylyl/guanylate cyclase (sGC), a heme-containing heterodimeric protein of similar to 150 kDa, is the primary receptor for nitric oxide, an endogenous molecule of immense physiological importance to animals. Recent studies have identified compounds such as YC-1 and BAY 41-2272 that stimulate sGC independently of NO binding, properties of importance for the treatment of endothelial dysfunction and other diseases linked to malfunctioning NO signaling pathways. We have developed a novel expression system for sGC from Manduca sexta (the tobacco hornworm) that retains the N-terminal two-thirds of both subunits, including heme, but is missing the catalytic domain. Here, we show that binding of compounds YC-1 or BAY 41-2272 to the truncated protein leads to a change in the heme pocket such that photolyzed CO cannot readily escape from the protein matrix. Geminate recombination of the trapped CO molecules with heme takes place with a measured rate of 6 x 10(7) s(-1). These findings provide strong support for an allosteric regulatory model in which YC-1 and related compounds can alter the sGC heme pocket conformation to retain diatomic ligands and thus activate the enzyme alone or in synergy with either NO or CO.