Soluble guanylate cyclase (sGC) is highly activated by NO, whereas CO, a competing ligand, only weakly activates the enzyme. The fact that NO, but not CO, breaks the sGC heme Fe-proximal histidine bond, has been assumed to be the key step in the NO activation of sGC. In this paper, we investigate the response of the heme pocket of three forms of sGC - native sGC, the homodimeric heme domain fragment [β1(1-385)], and the proximal heme-ligand mutant of β1(1-385) [H105G(Im)] - to CO photolysis by using time-resolved resonance Raman spectroscopy to obtain better insight into the interaction of CO with sGC. Our results show that the heme pocket of native sGC assumes its equilibrium conformation within 10 ps after CO photolysis, while in β1(1- 385) a 7 cm-1 upshift in ν(Fe-His) indicates a non-equilibrium conformation of the heme pocket, which relaxes with a time constant of 20 ns. In H105G(Im), a frequency downshift of 6 cm-1 is observed for ν(Fe-Im), and heme pocket relaxation has not fully occurred at 1 μs after CO photolysis. These differences can be explained by strain in the proximal heme pocket, which is large in sGC, smaller in β1(1-385), and greatly diminished in H105G(Im). We propose that the strain in the proximal heme pocket plays an important role in the regulation of sGC activation. A model for the activation of sGC is presented.