We report the resonance Raman characterization of the heme domain of rat lung soluble guanylate cyclase (sGC) expressed in Escherichia coli. Like heterodimeric sGC isolated from bovine lung, the sGC heme domain [β1(1- 385)] and its heme ligand mutant H105G(Im) contain a stoichiometric amount of heme, which is five-coordinate, high-spin ferrous in both β1(1-385) and chemically reduced H105G(Im). In the presence of NO, both β1(1-385) and H105G(Im) form a five-coordinate nitrosyl heme complex with a υ(Fe-NO) value of 525 cm-1 and a υ(NO) value of 1676 cm-1. For the first time, the Fe- N-O bending mode near 400 cm-1 has been identified in a five-coordinate nitrosyl heme complex. Both β1(1-385) and H105G(Im) form a six-coordinate, low-spin complex with CO. We find evidence for two binding conformations of the Fe-CO unit. The conformation that is more prevalent in β1(1385) has a υ(Fe-CO) value of 478 cm-1 and a δ(Fe-C-O) value of 567 cm-1, whereas the dominant conformation in H105G(Im) is characterized by a υ(Fe-CO) value of 495 cm-1 and a δ(Fe-C-O) value of 572 cm-1. We propose that in the dominant conformation of H105G(Im)-CO the Fe-CO unit is hydrogen bonded to a distal residue, while this is not the case in β1(1-385). Reexamination of sGC isolated from bovine lung tissue indicates that it also has two binding conformations for CO; the more populated form is not hydrogen-bonded. We propose that the absence of hydrogen-bond formation between a distal residue and exogenous ligands is physiologically relevant in lowering the oxygen affinity of heterodimeric sGC and, therefore, stabilizing the ferrous, active form of the enzyme under aerobic conditions.