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Low-Spin Cyanide Complexes of 3‑Mercaptopropionic Acid Dioxygenase (MDO) Reveal the Impact of Outer-Sphere SHY-Motif Residues

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journal contribution
posted on 09.12.2021, 22:15 by Nicholas J. York, Molly M. Lockart, Brad S. Pierce
3-Mercaptopropionic acid (3MPA) dioxygenase (MDO) is a non-heme Fe­(II)/O2-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 3MPA was titrated with cyanide to yield a low-spin (S = 1/2) (3MPA/CN)-bound ternary complex (referred to as 1C). UV–visible and electron paramagnetic resonance (EPR) spectroscopy were used to monitor the binding of 3MPA 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 3MPA- 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 g-values for the low-spin ferric site are diagnostic of the directionality of Tyr159 H-bond donation. Computational models coupled with CASSCF/NEVPT2-calculated g-values were used to verify that a major shift in the central g-value (g2) 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.

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