SPECTRAL AND LIGAND-BINDING PROPERTIES OF AN UNUSUAL HEMOPROTEIN, THEFERRIC FORM OF SOLUBLE GUANYLATE-CYCLASE

The soluble form of guanylate cyclase (sGC) is a hemoprotein which ser ves as the only known receptor for the signaling agent nitric oxide (( NO)-N-.), The enzyme is a heterodimer in which each subunit binds 1 eq uiv of 5-coordinate high-spin type b heme. (NO)-N-. increases the V-ma x of sGC up to 400-fold by binding to the heme to form a 5-coordinate ferrous nitrosyl complex. The electron paramagnetic resonance spectrum of the ferric form of the enzyme has been obtained. The spectrum disp lays rhombic symmetry and is indicative of a high-spin heme. Computer simulation of the EPR spectrum yields g values of 6.36, 5.16, and 2.0 with linewidths of 3.3, 4.1, and 3.3 mT, respectively, Using electroni c absorption spectroscopy, it was observed that the ferric heme binds cyanide to farm a 6-coordinate low-spin complex. The rate constants fo r association (k(on)) and dissociation (k(off)) of cyanide at 10 degre es C have been determined to be (7.8 +/- 0.3) x 10(-2) M(-1) s(-1) and (7.2 +/- 0.2) x 10(-5) s(-1). respectively. Unlike the ferrous form o f the enzyme, which has a low affinity for ligands that form 6-coordin ate complexes due to an unusually fast off-rate, the ferric form of th e enzyme appears to have a low affinity for ligands due to a slow on-r ate. The ferric heme binds azide with a K-d of 26 +/- 4 mM to form a h igh-spin complex. The ferric form of the enzyme has a specific activit y of similar to 57% that of the nonactivated ferrous farm of the enzym e, However, in contrast to the mild activation of the ferrous enzyme b y carbon monoxide, the ferric enzyme is not activated by cyanide. Thes e results indicate that there may be a significant structural change i n the protein upon the oxidation of the heme iron.