The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO- independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 μM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm-1. Similarly, YC-1 has no effect on the RR spectrum of ferrous β1(1-385), the isolated sGC heme-binding domain, but shifts the v(Fe-CO) of CO-β1(1-385) from 478 to 491 cm-1, indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and β1(1-385) in the presence of YC-1 lie on the v(Fe-CO) vs v(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift v(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of β1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.