title: Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site.
abstract: Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.
authors.affiliation: Division of Cardiology, Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas 77030, United States. emil.martin@uth.tmc.edu
authors.forename: Emil
authors.initials: E
authors.lastname: Martin
authors.forename: Vladimir
authors.initials: V
authors.lastname: Berka
authors.forename: Iraida
authors.initials: I
authors.lastname: Sharina
authors.forename: Ah-Lim
authors.initials: AL
authors.lastname: Tsai
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D018411:Spodoptera
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase