Insights into the distal heme pocket of H-NOX using fluoride as a probe for H-bonding interactions.

Nitric oxide (NO) and dioxygen (O2) are gases of similar size, shape, and electrostatic potential, but different physiological function. In aerobic organisms, the cellular concentration of O2 far exceeds that of NO; instead NO relies heavily on the ability of its receptor to discriminate against O2. In mammals, soluble guanylate cyclase (sGC) serves this role, binding NO with picomolar sensitivity and excluding O2 binding. Interestingly, some bacterial homologs of sGC, including the H-NOX (heme-nitric oxide/oxygen) domain from Thermoanaerobacter tengcongensis, tightly bind O2. Three distal pocket residues (Trp9, Asn74, and Tyr140) form a hydrogen-bonding network that stabilizes O2 binding to TtH-NOX. Therefore, a current hypothesis to explain sGC ligand specificity is that sGC lacks H-bond donors that preferentially stabilize O2 binding. The wavelength maximum of the charge-transfer band (CT1) in the electronic spectrum of the fluoride complex of ferric hemoproteins is a sensitive probe of H-bonding. Here, in order to gain further understanding of the distal pocket H-bonding network in TtH-NOX, we employ fluoride as a spectroscopic probe. As expected, our results indicate that Y140 donates a strong H-bond to the heme-bound ligand. We find that an H-bond from Asn74 as well as distal pocket crowding contributes to positioning Tyr140 for a strong and directed H-bond to iron-bound ligands; indeed crowding may be the primary role for Trp9. We clarify the role of H-bonding in sGC ligand discrimination and suggest that sterics also regulate ligand binding in the H-NOX family.