Probing the hydrogen bonding environment in the heme pocket of a hemoprotein can greatly aid in understanding ligand specificity in hemoproteins. For example, soluble guanylate cyclase (sGC), a eukaryotic nitric oxide (NO) sensor, has picomolar sensitivity to NO, while completely excluding molecular oxygen (O2) as a ligand. The current hypothesis to explain this ligand specificity is that sGC lacks distal pocket hydrogen bond donors that would preferentially stabilize O2 as a ligand. Interestingly, a closely related bacterial homolog of sGC, the H-NOX (Heme-Nitric oxide/OXygen binding) domain from Thermoanaerobacter tengcongensis (Tt) binds O2 with a dissociation constant of 90nm. Structural studies of Tt H-NOX indicate that there are three distal pocket residues that form a hydrogen bonding network that may stabilize O2 binding. This work focuses on characterizing the distal heme pocket hydrogen-bonding network in Tt H-NOX. Previous studies have shown that the wavelength maximum of the charge-transfer band (CT1) in the electronic spectrum of a fluoride-heme complex is a sensitive probe of distal pocket hydrogen-bonding. Using this assay, we found that tyrosine 140 (Y140) in the distal pocket of Tt H-NOX donates the strongest hydrogen bonding to bound fluoride. Additionally, we found that mutation of phenylalanine 78 (F78) to tyrosine (F78Y) donates an additional hydrogen bond to bound fluoride. We also observe that tryptophan 9 (W9) and asparagine 74 (N74) play a role in the stabilization of Y140. In the W9F, N74A and W9F/N74A mutants, Y140 has a weaker hydrogen-bond with fluoride. However, the stabilization of Y140 by W9 appears to be very low. N74 provides the majority of hydrogen-bond stabilization to Y140, indicating that W9 may have evolved to fulfill some other evolutionary need. The structure of the heme cofactor is critical for hemoprotein function. These functions include transportation and storage of diatomic gases, chemical catalysis, diatomic gas detection, and electron transfer. In Tt H-NOX WT, the heme has been shown to be distorted, due to a proline in the 115 position. In order to investigate the nature of the non-planar heme in H-NOX, we created a panel of mutant Tt H-NOX proteins, in which the proline in position 115 was mutated to various residues (P115G, P115A, P115L, P115V, P115W and P115F). At the conclusion of this work we were unable to find a link between heme planarity and redox potential or ligand stabilization by hydrogen-bonding. However, we are undertaking further studies to reach this goal.