Activation and deactivation of soluble guanylate cyclase: Domain organization and the requirement for non-heme equivalents of nitric oxide.

The accepted model for activation and deactivation of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) involves the formation and decay of a 5-coordinate ferrous-nitrosyl heme species. Using electronic absorption spectroscopy and kinetic analysis, the rate of dissociation of NO from the sGC heme and the rate of deactivation of NO-stimulated sGC were measured, and a discrepancy was found indicating that the 5-coordinate ferrous-nitrosyl species is not responsible for full activation of sGC. Therefore, the accepted model for activation and deactivation of sGC cannot be correct. It was shown that sGC could be isolated as a ferrous-nitrosyl complex with diminished activity relative to the fully stimulated enzyme, and that full activity could be recovered by addition of excess NO. This demonstrates that activation of the enzyme happens via the binding of NO to a non-heme site on the enzyme, indicating that the paradigm for activation and deactivation of sGC must be revised. Additionally, the catalytic domains of sGC were successfully expressed and purified for the first time. They are active as heterodimers, as is the full-length sGC, and are activated 300-fold by the presence of Mn2+ as compared to the physiological cofactor Mg2+. This degree of activation is reminiscent of activation of the full-length enzyme by NO, suggesting mechanistic similarities. The catalytic domains are inhibited by interaction with the N-terminal regulatory domain of sGC in trans , suggesting a domain-scale mechanism of activation by NO in which NO binding causes relief of an autoinhibitory interaction between the regulatory domain and the catalytic domain.Ph.D.BiochemistryBiological SciencesMolecular biologyPharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124593/2/3150119.pd