Proton Transfer Kinetics in Histidine Side Chains Determined by pH-Dependent Multi-Nuclear NMR Relaxation

Histidine is a key amino-acid residue in proteins with unique properties engendered by its imidazole side chain that can exist in three different states: two different neutral tautomeric forms and a protonated, positively charged one with a pK_a value close to physiological pH. Commonly, two or all three states coexist and interchange rapidly, enabling histidine to act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Understanding the exchange dynamics among the three states is critical for assessing histidine's mechanistic role in catalysis, where the rate of proton exchange and interconversion among tautomers might be rate limiting for turnover. Here, we determine the exchange kinetics of histidine residues with pK_a values representative of the accessible range from 5 to 9 by measuring pH-dependent ¹⁵N, ¹³C, and ¹H transverse relaxation rate constants for 5 nuclei in each imidazole. Proton exchange between the imidazole and the solvent is mediated by hydronium ions at acidic and neutral pH, whereas hydroxide mediated exchange becomes the dominant mechanism at basic pH. Proton transfer is very fast and reaches the diffusion limit for pK_a values near neutral pH. We identify a direct pathway between the two tautomeric forms, likely mediated by a bridging water molecule or, in the case of high pH, hydroxide ion. For histidines with pK_a 7, we determine all rate constants (lifetimes) involving protonation over the entire pH range. Our approach should enable critical insights into enzymatic acid/base catalyzed reactions involving histidines in proteins.