Oxygen Binding and Redox Properties of the Heme in Soluble Guanylate Cyclase IMPLICATIONS FOR THE MECHANISM OF LIGAND DISCRIMINATION

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O-2, thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O-2 has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe2+-O-2) when frozen at 77 K in the presence of O-2. The ligation of O-2 was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at -7 degrees C, with the extremely low affinity for O-2. The low O-2 affinity was not caused by a distal steric protein effect and by rupture of the Fe2+-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe2+-proximal His bond. Based on our results, we propose that the weak Fe2+-proximal His bond is a key determinant for the low O-2 affinity of the heme moiety of soluble guanylate cyclase.