The soluble form of guanylate cyclase (sGC) is the only definitive receptor for the signaling agent nitric oxide (.NO). The enzyme is a homologous heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin heme. .NO increases the Vmax of sGC up to 400-fold, probably by binding directly to the heme. Carbon monoxide (CO) forms a 6-coordinate complex with the heme and weakly activates the enzyme. Using stopped-flow spectrophotometry, the on-rate and off-rate for the binding of CO to the heme have been determined to be (3.58 +/- 0.15) x 10(4) M-1 s-1 and 3.5 +/- 0.5 s-1, respectively, at 10 degrees C. The equilibrium dissociation constant (Kd) has been independently determined to be 97 +/- 9 microM. Comparison of this Kd with that calculated from the rate constants indicates that the binding of CO to sGC is a simple one-step process, in which the off-rate of CO from the hexacoordinate complex is much faster than typically found in hemoproteins. The Kd of CO for activating the enzyme was also determined and compared to that for binding to the heme. Nitrosomethane forms irreversible complexes with typical ferrous hemoproteins but was observed to bind reversibly to the heme in sGC, with an off-rate > or = (7.6 +/- 0.2) x 10(-3) s-1. In general, the ferrous heme in sGC has a low affinity for ligands that form 6-coordinate complexes due primarily to fast ligand off-rates.