Low Barrier Hydrogen Bonds: Getting Close, but Not Sharing...

Hydrogen bonding represents one of the primary interactions controlling chemical structure and reactivity. The properties of H-bonds are predicted to vary with the distance between the H-donor and H-acceptor, Figure 1, from a canonical structure (>2.5 Å, Figure 1A), in which the proton resides within a double welled structure, to one in which the proton has become fully delocalized within a single well (≪2.5 Å, Figure 1C). The low barrier hydrogen bond (LBHB), Figure 1B, lies between these extreme states, with a predicted donor− acceptor distance in the range of 2.5 Å and a hydrogen transfer just above the top of the reaction barrier. Analogous to Figure 1C, the hydrogen is equally shared between its donor and acceptor. Over a period of decades, the presence and role of such LBHBs has been a topic of ongoing controversy. At the heart of the controversy has been the question of whether enzymes are capable of accelerating rates for C−H activation by matching active site pKas for the hydrogen donor and acceptor, producing a greatly reduced reaction barrier height, and a resulting highly accelerated rate for bond cleavage. In a new investigation, Oltrogge and Boxer turn to the well-characterized green fluorescent protein (GFP) to pursue the properties of short hydrogen bonds within a biological context. The results from their elegant experiments, which depend on the incorporation of a series of unnatural amino acids to produce modified protein chromophores with pKas that vary by 3.5 units (Figure 2), indicate that the active site hydrogen bond within GFP is short but not delocalized. Historically, evidence for LBHBs in proteins has come from a compelling and repeated observation of chemical shifts in H NMR spectra that are significantly downfield shifted (to ca. 17−21 ppm), together with inverse fractionation factors (of ca. 0.3) for the behavior of such H-bonds in D2O vs H2O. 1 The latter is attributed to a decrease in bond order at the transferred hydrogen that increases discrimination against deuterium in favor of an accumulation of protium. The new studies by Oltrogge and Boxer are multifaceted. They start by solving the X-ray crystal structures for each of the proteins with modified chromophores, showing a consistently short donor−acceptor distance of ca. 2.45 Å. The experimental findings become especially intriguing when UV/vis spectra are interrogated at low pH where large differences in absorbance spectra emerge correlating with the pKa of the modified chromophore. (By contrast, high pH shows essentially identical properties among the variants.) Oltrogge and Boxer proceed to simulate spectra for the low pH spectra, using methods developed by McKenzie for the analysis of the extent of coupling between H-bonded diabatic states. Focusing on the magnitude of the offdiagonal coupling factor, ΔDA, they are able to simulate the low pH trends in both spectral position and line width; although a first approximation fit of the data suggested a Figure 1. Categorization of hydrogen bonds based on donor−acceptor distances, from >2.5 Å (A) to ≪2.5 Å (C). The horizontal lines within the wells represent zero point energies for protium and deuterium. The low barrier hydrogen bond, LBHB (B), has the unique property of a barrier height for H-transfer that has fallen below the ground state vibrational levels of the donor and acceptor. Reprinted with permission from ref 1. Copyright 1998 American Society for Biochemistry and Molecular Biology, Inc.