Probing soluble guanylate cyclase activation by CO and YC-1 using resonance Raman spectroscopy.

Soluble guanylate cyclase (sGC) is weakly activated by carbon monoxide (CO) but is significantly activated by the binding of YC-1 to the sGC-CO complex. In this report, resonance Raman (RR) spectroscopy was used to study selected sGC variants. Addition of YC-1 to the sGC-CO complex alters the intensity pattern of RR bands assigned to the vinyl and propionate heme substituents, suggesting changes in the tilting of the pyrrole rings to which they are attached. YC-1 also shifts the RR intensity of the nu(FeC) and nu(CO) bands from 473 and 1985 cm(-1) to 487 and 1969 cm(-1), respectively, and induces an additional nu(FeC) band, at 521 cm(-1), assigned to five-coordinate heme-CO. Site-directed variants in the proximal heme pocket (P118A) or in the distal heme pocket (V5Y and I149Y) reduce the extent of YC-1 activation, along with the 473 cm(-1) band intensity. These lower-activity sGC variants display another nu(FeC) band at 493 cm(-1) which is insensitive to YC-1 addition and is attributed to protein that cannot be activated by the allosteric activator. The results are consistent with a model in which YC-1 binding to the sGC-CO complex results in a conformational change that activates the protein. Specifically, YC-1 binding alters the heme geometry via peripheral nonbonded contacts and also relieves an intrinsic electronic effect that weakens FeCO backbonding in the native, YC-1 responsive protein. This electronic effect might involve neutralization of the heme propionates via H-bond contacts or negative polarization by a distal cysteine residue. YC-1 binding also strains the Fe-histidine bond, leading to a population of the five-coordinate sGC-CO complex in addition to a conformationally distinct population of the six-coordinate sGC-CO complex. The loss of YC-1 activation in the sGC variants might involve a weakening of the heme-protein contacts that are thought to be critical to a YC-1-induced conformational change.

Duke Scholars

Author Emily R. Derbyshire Chemistry