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Nitric oxide is the main physiologic ligand of soluble gua-nylyl cyclase (sGC), a hemoprotein with high affinity forNO. Purified sGC binds NO with an estimated picomolarKd for NO, which corresponds to only a few molecules ofNO per cell. The His105 residue of the β subunit is theproximal ligand of the heme (His105-heme complex witha 430 nm Soret band). Binding of NO to the sGC hemeforms a six coordinate (6C) complex (His105-heme-NO),which is then converted into a five coordinate (5C) com-plex (His105 and heme-NO) [1]. This transformationcoincides with several hundred fold activation of thecyclase activity. The enzyme displays NO-concentrationdependence not only in the NO binding, but also in the6C sGC-NO →5C sGC-NO complex conversion [1]. Previ-ous studies showed that the sGC-NO complex obtainedunder stoichiometric NO amounts is not fully activated,while addition of GTP substrate or additional NO resultsin a fully activated sGC [2].In this current report we investigated the molecular basisof this NO- and GTP-dependence of enzyme activation.Stop-flow techniques were used to monitor the kinetics ofNO-sGC complex formation and its fate under stoichio-metric and excess NO conditions or in the presence ofGTP. Under both stoichiometric and excess NO condi-tions 6C His105-heme-NO complex (~420 nm Soretband) is formed at a diffusion limited rate independentlyof the presence or absence of GTP. Although in all testedconditions the His105-heme-NO complex is convertedinto five coordinate complex (free His105 and heme-NOcomplex; 399 nm Soret band), the fate of this complexdepends on the amount of NO applied or the presence ofGTP substrate.Under stoichiometric conditions, the heme-NO revertsback to a 430 nm species with sub-second kinetics, pre-sumably due to the loss of NO. The newly formed His105-heme complex (sGC*) may rebind NO and forms a 5CsGC-NO complex (399 nm species), but with muchslower kinetics (minutes vs. sub-millisecond kinetics forintial sGC). This secondary sGC-NO* complex is, mostprobably, the weakly activated sGC-NO complexdescribed previously [2]. However, even a two-fold molarexcess NO or the presence of GTP generates a 5C NO-heme complex with no sign of NO loss.Spectral properties of the sGC* state generated after theloss of NO (430 nm Soret peak) indicate that the hememoiety remains coordinated by a histidine residue. Differ-ent NO-binding properties, however, suggest that signifi-cant changes in the properties of the enzyme occurred. Weinvestigated whether a change of proximal ligands mayexplain such transformation during the conversion of 5CsGC-NO to sGC*. We substituted the βHis107 position,which is conserved in all mammalian β1 subunits, to leu-cine. Similar to wild type sGC mutant αβHis107→Leuenzyme also contains heme with a Soret peak at 430 nm.The mutant has different NO-binding kinetics, since itforms a stable sGC-NO complex, which does not looseNO even under sub-stoichiometric NO conditions.

Dynamic ligand exchange as mechanism for sGC selectivity, NO activation and its reversal