Direct Observation of Polarization in Short Hydrogen Bonds due to Proton Delocalization

Abstract

Based on their relatively shallow potential energy surfaces (PESs), short hydrogen bonds with heavy atom distances about or less than 2.5 Å are believed to exhibit proton delocalization, which has been argued to be beneficial to biological catalysis1. While the degree of delocalization is usually characterized by neutron diffraction or inferred from broad bands in the IR spectra, these approaches can result in ambiguities2 and information on energetics cannot be directly obtained. Here we apply an external electric field to perturb the proton equilibrium position of a short hydrogen bond in previously characterized GFP S65T/H148D mutants with various degrees of proton affinity mismatch (ΔpKα) between the chromophore and aspartate3. The chromophore serves as a spectral probe of the proton position, allowing us to perform electronic Stark spectroscopy. The local curvature of the PES is reflected by the ease of proton polarization, and the resulting proton displacement effectively amplifies the electric field experienced by the chromophore, leading to anomalously large Stark tuning rates as readouts. These Stark tuning rates are sensitive to the presence of the short hydrogen bond, proton position, ΔpKα and deuteration, in accordance with large hydrogen bond polarizations predicted by Zundel4. The results reinforce our previous arguments on the absence of low barrier hydrogen bonds in these variants3: the general shape of PESs is double-welled with a rather high barrier in between and the proton is delocalized within each well. [1] Perrin, C. L.; Nielson, J. B. Annu. Rev. Phys. Chem. 1997, 48, 511-544. [2] Graen, T. et al. J. Am. Chem. Soc. 2016, 138, 16620-16631. [3] Oltrogge, L. M.; Boxer, S. G. ACS Cent. Sci. 2015, 1, 148-156. [4] Janoshek, R. et al. J. Am. Chem. Soc. 1972, 94, 2387-2396.