Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).

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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...