Soluble guanylyl cyclase (sGC), the target enzyme of the signalling molecule NO, contains one prosthetic haem group and consists of an alpha and a beta subunit. So far, only the alpha1beta1 heterodimer has been shown to exist in different cells and tissues, and most biochemical studies of sGC have been performed with the alpha1 beta1 heterodimer. Here we demonstrate for the first time the natural occurrence of the alpha2 subunit on the protein level. The alpha2 subunit co-precipitated with the beta1 subunit from human placenta, showing the existence of the alpha2 beta1 isoform in vivo. The new enzyme was expressed in and purified from cells from the Spodoptera frugiperda ovary cell line Sf 9. Spectral analysis showed that the alpha2 beta1 heterodimer contains a prosthetic haem group revealing the same characteristics as the haem in the alpha1 beta1 form. The kinetic properties of both isoforms and sensitivity towards NO were indistinguishable. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC, abolished NO-stimulated activity of both heterodimers. The new NO-independent activator, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), increased the maximal NO-stimulated activity of the new isoform, caused a leftward-shift in the NO concentration-response curve and turned CO into an effective activator, as it did for the alpha1 beta1 heterodimer (200-fold activation). In summary, the differences in primary structure of both alpha subunits are contrasted by their functional similarity. Further studies will be needed to elucidate the physiological purpose of the new isoform.

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, α1 and β1. NO binds to the heme cofactor in the β1 subunit, forming a 5-coordinate NO complex that activates the enzyme several hundred-fold. In this report the heme domain has been localized to the N-terminal 194 residues of the β1 subunit. This fragment represents the smallest construct of the β1 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 β2 subunit has been localized to the first 217 amino acids β2(1-217). These proteins are ∼40% identical to the rat β1 heme domain, form 5-coordinate, low-spin NO complexes and 6-coordinate, low-spin CO complexes. Like sGC these constructs have a weak Fe-His stretch (208 and 207 cm −1 for β1 and β2(1-217), respectively). β2(1-217) forms a CO complex that is very similar to sGCand has a high ν(CO) stretching frequency at 1994 cm −1 . The autoxidation rate of β1 (1-194) was 0.073 per minute, while the β2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003 per minute at 37 °C. This report has identified and characterized the minimum functional ligand binding heme domain derived from sGC providing key details towards a comprehensive characterization.Soluble guanylate cyclase (sGC 1 ) is a well-characterized NO-sensor, containing a hemebound, N-terminal domain that binds NO, regulating a C-terminal guanylate cyclase. sGC catalyzes the conversion of GTP to cGMP, and it is a heterodimer, composed of α1 and β1 subunits. The ferrous heme is ligated to the β1 subunit via H105 (1), and when NO is bound, the activity of the enzyme is 300-fold elevated over basal activity (2). sGC has evolved to selectively bind NO and not O 2 . It does not form a stable, isolable complex with O 2 , permitting NO to bind selectively to the Fe +2 heme in an aerobic environment. This selectivity is essential Δ This work was supported in part by LDRD fund of the Lawrence Berkeley National Laboratory ‡ Present address: Pacific Northwest National Laboratory, K8-88 Richland, WA 99352. * To whom correspondence should be addressed: University of California, Berkeley, Department of Chemistry, Berkeley, CA, 94720-1460. Telephone: 510-643-9325. Fax: 510-643-9388. E-mail: marletta@berkeley.edu 1 Abbreviations: CO, carbon monoxide; sGC, soluble guanylate cyclase; NO, nitric oxide; TtTar4H, N-terminal 188 amino acids of Thermoanaerobacter tengcongensis Tar4 (heme domain); MCP, methyl-accepting chemotaxis protein; AxPDEA1H, Acetobacter xylinum phosphodiesterase A1 heme domain; Mb, myoglobin; H-NOX domain, Heme-Nitric oxide/OXygen binding domain. sGC has been characterized in animals ranging from Drosophila to humans and, until recently, there had been no reports of any other NO sensors except for sGC. Notably, no heme-containing NO sensors had been found in prokaryotes, which could be exposed to both exogenous and endogenou...