STUDIES OF THE HEME COORDINATION AND LIGAND-BINDING PROPERTIES OF SOLUBLE GUANYLYL CYCLASE (SGC) - CHARACTERIZATION OF FE(II)SGC AND FE(II)SGC(CO) BY ELECTRONIC ABSORPTION AND MAGNETIC CIRCULAR-DICHROISM SPECTROSCOPIES AND FAILURE OF CO TO ACTIVATE THE ENZYME

The mechanism of activation of soluble guanylyl cyclase by NO is poorl y 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 he me in the enzyme spectroscopically in the presence of known heme ligan ds and to correlate the spectral characteristics with other heme prote ins of known structure. Comparison of the electronic and magnetic circ ular dichroism (MCD) spectra for ferrous bovine soluble guanylyl cycla se (Fe(Il)sGC) in the absence and presence of the common heme ligand C O 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 si gnal intensity in the visible region to that in the Soret region is mo st 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 t o significantly activate the enzyme regardless of heme content or CO c oncentration. In contrast, the extent of activation of sGC by NO is de pendent 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 an d the proximal histidine must be broken for activation to take place.