3-Mercaptopropionic acid () dioxygenase (MDO) is a non-heme Fe(II)/O-dependent oxygenase that catalyzes the oxidation of thiol-substrates to yield the corresponding sulfinic acid. Hydrogen-bonding interactions between the Fe-site and a conserved set of three outer-sphere residues (Ser-His-Tyr) play an important catalytic role in the mechanism of this enzyme. Collectively referred to as the SHY-motif, the functional role of these residues remains poorly understood. Here, catalytically inactive Fe(III)-MDO precomplexed with was titrated with cyanide to yield a low-spin ( = 1/2) (/CN)-bound ternary complex (referred to as ). UV-visible and electron paramagnetic resonance (EPR) spectroscopy were used to monitor the binding of and cyanide. Comparisons of results obtained from SHY-motif variants (H157N and Y159F) were performed to investigate specific H-bonding interactions. For the wild-type enzyme, the binding of - and cyanide to the enzymatic Fe-site is selective and results in a homogeneous ternary complex. However, this selectivity is lost for the Y159F variant, suggesting that H-bonding interactions contributed from Tyr159 gate ligand coordination at the Fe-site. Significantly, the -values for the low-spin ferric site are diagnostic of the directionality of Tyr159 H-bond donation. Computational models coupled with CASSCF/NEVPT2-calculated -values were used to verify that a major shift in the central -value () displayed between wild-type and SHY variants could be attributed to the loss of Tyr159 H-bond donation to the Fe-bound cyanide. Applied to native cosubstrate, this H-bond donation provides a means to stabilize Fe-bound dioxygen and potentially explains the attenuated (∼15-fold) rate of catalytic turnover previously reported for MDO SHY-motif variants.