EPR characterization of axial bond in metal center of native and cobalt- substituted guanylate cyclase

Abstract

The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g is perpendicular to component and a g is parallel with component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of α-subunits of Co2+- hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3/- elicited the activation of the ferric enzyme by yielding five- coordinate high spin N3/- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3/- binding.