In parallel, it is clear that large shifts in regions of the heme domain occur during activation. How these shifts lead to increased catalytic activity is similarly unresolved. To address these fundamental questions, we first carried out homology modeling of the rat sGC heme domain to identify solvent-exposed residues in the regions that exhibit the largest shifts, the aF helix-b1 strand and the flanking aB-aC loop, as recently studied in our structure of the sGC activator BAY 58– 2667 bound to Ns HNOX and in our structure-function analysis of residues involved in NO propagation downstream of the His-iron breakage. As such, these two regions could be the missing link to propagate activation, i.e. be directly involved in interaction between the heme domain and the catalytic domains as proposed by others or induce conformational changes, transmitted by the intermediary domains. In the aF helix region, replacement of residue R116 with Ala led to decreased activation by YC-1, DEA-NO and YC-1+DEANO, as expected if this region is involved in propagation of activation. Interestingly, the full UV-Vis spectrum of the semipurified R116A revealed that the Soret band absorption maximum was blue-shifted from 431 nm to 410 nm, which could indicate a weakening and breakage of the His-iron bond in this mutant. In addition, the reconstitution with hemin was only partial compared to WT or T110A of the aF helix. These data suggest that the replacement of residue R116 with Ala not only affect the spectral properties of the heme group but also affects heme stability and affinity. However, in the same region the replacement of T110 with Ala only slightly reduced the Vmax of the DEA-NO response curves. Interestingly, T110A also displayed a 2-fold increase in basal activity compared to WT, which partially supports the idea that under basal conditions, the heme domain has an inhibitory effect on the catalytic domain, as replacement with Ala could partially relieve the inhibition. Furthermore, considering the low heme content of T110A, the response of T110A to DEA-NO was high compared to WT while EC50 was similar. We speculate that T110 is a key residue for interactions between the heme and catalytic domains. This supports our most recent structural analysis with the compound BAY58–2667 that predicted the largest shift resided in the aF carboxy-terminus, which includes T110. Those results suggest that T110 is a critical residue for basal activity and NO activation by “loosening” a potential inhibitory interaction between the catalytic and heme domains. This will also explain the significant LY2157299 abmole higher activation of T110A by PPIX over WT despite a lower PPIX reconstitution, in addition to the fact that the higher response to PPIX is also partially due to the T110A apo form, as observed previously with the R139 mutant. Thus, T110 and to a lesser extent R116 appear to be crucial residues for heme stability/affinity as shown by the lower heme content and increased PPIX response when replaced with Ala. The function of these residues in sGC is different from other residues of the same aF helix, in particular D102 and F120. D102 and F120 are also involved in propagation of the NO signal yet unlike T110 or R116, D102 is predicted to interact with the backbone of F120.