The mechanism of activation of soluble guanylyl cyclase by NO is poorly understood although it is clear that NO interacts with a heme group in the protein via formation of a heme-nitrosyl adduct. The objective of this study is to investigate the coordination environment of the heme in the enzyme spectroscopically in the presence of known heme ligands and to correlate the spectral characteristics with other heme proteins of known structure. Comparison of the electronic and magnetic circular dichroism (MCD) spectra for ferrous bovine soluble guanylyl cyclase (Fe(II)sGC) in the absence and presence of the common heme ligand CO with those of other hemoproteins suggests that histidine is an axial ligand to the heme iron in Fe(II)sGC. Further analysis indicates that Fe(II)sGC is predominantly bis-histidine ligated; the ratio of MCD signal intensity in the visible region to that in the Soret region is most consistent with an admixture of pentacoordinate and hexacoordinate ferrous heme in Fe(II)sGC at pH 7.8. Spectral changes upon CO binding have been correlated with the activity of the enzyme to determine the relationship between coordination structure and activity. Although CO clearly binds to Fe(II)sGC to form a six-coordinate adduct, it fails to significantly activate the enzyme regardless of heme content or CO concentration. In contrast, the extent of activation of sGC by NO is dependent on the heme content in the enzyme and on the concentration of NO. These observations are consistent with a mechanism for activation of soluble guanylyl cyclase in which the bond between the heme iron and the proximal histidine must be broken for activation to take place.

Activation of soluble guanylyl cyclase (sGC) by NO correlates with scission of the proximal iron-histidine bond, as demonstrated by the application of electronic absorption and resonance Raman spectroscopy to the study of metalloporphyrin-substituted enzymes. The non-native metalloporphyrins, Mn(II)PPIX and Co(II)PPIX, can be introduced into heme-deficient sGC forming five-coordinate complexes. The similarity among Mn(II)sGC, Co(II)-sGC, and the corresponding metalloporphyrin-substituted derivatives of Mb and Hb provides confirming evidence for the presence of an axial histidine ligand in sGC. Upon addition of NO, Mn(II)sGC forms a six-coordinate species with the histidine ligand still bound to the Mn, and the enzyme is not activated. In contrast, the Co(II)-sGC(NO) adduct is five-coordinate and the enzyme is activated. These data imply that the activated state of sGC is attained when the proximal histidine-metal bond is broken.Soluble guanylyl cyclase (sGC), the cytosolic enzyme that catalyzes the conversion of guanosine 5′-triphosphate (GTP) to guanosine 3′,5′-monophosphate (cGMP), is the only proven biological receptor for nitric oxide. 1-3 Nitric oxide (NO) is an unique messenger molecule being both freely diffusible and highly labile; consequently, its action is limited to the immediate environment in which it is released. NO is synthesized from L-arginine by the enzyme NO-synthase and regulates vascular smooth muscle relaxation, inhibition of platelet aggregation, and neuronal communication. 4,5 NO controls these functions through activation of sGC. 6 sGC is a 150 kDa heterodimer with subunits of similar size 7,8 and was believed to contain 1 mol of heme (Fe(II)PPIX) per mol of heterodimer. 9 Recent results suggest, however, that there may be one heme in each subunit. 10 Enzyme activity is dependent on the presence of a divalent metal ion cofactor Mg 2+ (the natural cofactor) or Mn 2+ , in excess of the substrate GTP.

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Soluble guanylyl cyclase (sGC) is known to be activated by NO binding to the heme moiety; previous studies have shown that CO does not activate sGC to the same extent as NO. Resonance Raman spectroscopy reveals different heme pocket structures for soluble guanylyl cyclase prepared by alternate methods, all of which display activation by NO. In our preparation, and in the expressed protein sGC1, the resting Fe(II) state is mainly 6-coordinate and low-spin, and the CO adduct has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment. In contrast, the protein sGC2 is 5-coordinate, high-spin in the resting state, and the CO adduct has perturbed vibrational frequencies indicative of a negatively polarizing residue in the binding pocket. The differences may result from the need to reconstitute sGC1 or different isolation procedures for sGC1 versus sGC2. However, both sGC1 and sGC2 are activated by the same mechanism, namely displacement of the proximal histidine ligand upon NO binding, and neither one is activated by CO. If CO is an activator in vivo, some additional molecular component is required.