Characterization of functional heme domains from soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha 1 and beta 1. NO binds to the heme cofactor in the 1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta 1 subunit. This fragment represents the smallest construct of the PI subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta 2 subunit has been localized to the first 217 amino acids beta 2(1-217). These proteins are similar to 40% identical to the rat beta 1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-1) for 1(1194) and beta 2(1-217), respectively]. beta 2(1-217) forms a CO complex that is very similar to sGC and has a high v(CO) stretching frequency at 1994 cm(-1). The autoxidation rate of beta 1(1-194) was 0.073/min, while the beta 2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.