Aspartate 102 in the heme domain of soluble guanylyl cyclase has a key role in NO activation.

Soluble guanylyl cyclase (sGC), a heme-containing heterodimeric enzyme, is the main receptor for nitric oxide (NO). Binding of NO to the heme of sGC increases the production of cGMP several hundred fold. The NO-cGMP pathway is involved in many physiological processes including cardiovascular homeostasis,1 inhibition of platelet aggregation,2 and synaptic plasticity.3 The heme is ligated to His 105 of the β subunit of sGC.4 Binding of NO to the heme induces the breakage of the heme iron–His bond leading to increased catalysis. We and others have solved structures of prokaryotic analogue heme domains (HNOX) in the presence of various ligands, allowing a better understanding of the initial events in NO-dependent stimulation of sGC,5–7 in addition to molecular dynamic simulation.8 Mutational analyses have in turn identified residues critical for the heme stability and binding.9,10 Furthermore, potential inhibitory interactions between the heme domain and the catalytic domain that would be released upon binding of NO were suggested as a mechanism of activation.11,12 In spite of all these studies, the major challenge remains to understand the mechanism of propagation of the NO signal, in particular, the conformational events following breakage of the iron–His bond. Through comparison between the inactive and active structures of the HNOX domains,13,14 we anticipate that one region undergoes major shift upon binding of NO, the αF helix.7 Our homology modeling showed that His 105 (H105), being liganded to the heme in the inactive state, is surrounded by residues conserved in both Nostoc H-NOX and sGC β1 heme domain pointing to a potentially critical role. These conserved residues include D102 which is the only other αF helix residue that is predicted to make a direct hydrogen-bonding interaction with the backbone nitrogen of F120 belonging to the β1 strand (Figure 1). Since the postulated shifting αF helix is only held in place to the rest of the H-NOX domain via the NO-labile H105–Fe bond and the D102/F120 interactions (in addition to other van der Waals interactions involving the αF helix), we investigated the activation importance of these residues. Furthermore, residues D102 and H105 are also in contact via a water-mediated interaction in the Nostoc structure that was also present in the sGC homology model. This water molecule likely provides bridging interactions with the backbone oxygen of P118, also a key conserved β1-strand residue found to be important for H-NOX function.15 We therefore speculate that these conserved interactions between the αF helix and β1 strand residues in the vicinity of H105 could be relaying key NO-dependent conformational activation changes. Residue D102 is partially solvent exposed as only one of its oxygens is involved in hydrogen bonding to F120, thereby offering the possibility that D102 is potentially involved in communicating conformational changes to other domains of sGC. Therefore, using structure-base mutational analysis combined with kinetics and spectral analysis of the full-length rat sGC, we tested the hypothesis that D102 and other residues in the vicinity are involved in the mechanism of NO propagation from the heme domain to the effector cyclase domain. Figure 1 Close-up view of proximal face of the heme in the homology model of the rat sGC β1 H-NOX domain. The helix αF and strand β1 (purple font) are labeled accordingly. The interaction network involving αF helix and β ...