Chimeric analysis links increased NO sensitivity to GC-2 domains and higher maximal activity to GC-1

Soluble guanylyl cyclase (sGC) is the primary receptor for nitric oxide (NO) and catalyzes cGMP formation. Mammalian sGC exists as two heterodimeric isoforms, α₁/β₁ and α₂/β₁, but intrinsic differences in NO sensitivity and catalytic output remain poorly defined. Here, we directly compared NO-dependent activation of both isoforms in HEK293 and Sf9 cell homogenates and purified enzyme preparations. In cellular homogenates, α₁/β₁ displayed higher maximal NO-stimulated activity, while α₂/β₁ showed greater NO sensitivity. These differences were markedly attenuated after purification and remained apparent only as non-significant trends, indicating that NO-dependent activation is strongly influenced by the cellular environment or cofactors. To identify underlying structural determinants, we generated α-subunit chimeras in which H-NOX, PAS, coiled-coil, and catalytic domains were exchanged between α₁ and α₂. The α₂ H-NOX domain increased α-subunit protein levels but decreased maximal activity, while the combined α₂ H-NOX/PAS module led to an additional increase in NO sensitivity. Isolated introduction of the α₂ coiled-coil region into the α₁ subunit also increased NO sensitivity. Catalytic domain exchange showed that the α₁ catalytic domain promotes higher catalytic output, whereas the α₂ catalytic domain limits it. Collectively, the data indicate that isoform-specific NO-dependent activation profiles arise from contributions of multiple α-subunit domains rather than a single regulatory element. Our findings support a model in which α₂/β₁ functions as a highly sensitive NO detector, whereas α₁/β₁ acts as a lower-sensitivity, higher-capacity signaling amplifier. The chimera panel established here provides a basis for future studies of sGC allosteric regulation and isoform-selective pharmacology.