Imidazole C‑2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine C^ε1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second-order with respect to the concentrations of the imidazolium cation and OD^– in D₂O. The second-order rate constant (<i>k</i>₂) of this reaction was calculated from the pH dependency of the pseudo-first-order rate constant (<i>k</i>_φ) obtained from the change in average mass [Δ<i>M</i>_r (0 ≤ Δ<i>M</i>_r < 1)] of a peptide fragment containing a defined histidine residue at incubation time (<i>t</i>) such that <i>k</i>_φ = −[ln­(1 – Δ<i>M</i>_r)]/<i>t</i>. We preferred using <i>k</i>₂ rather than <i>k</i>_φ because <i>k</i>₂^max (maximal value of <i>k</i>₂) was empirically related to p<i>K</i>_a as illustrated with a Brønsted plot [log <i>k</i>₂^max = −0.7p<i>K</i>_a + α (α is an arbitrary constant)], so that we could analyze the effect of structure on the H/D exchange rate in terms of log­(<i>k</i>₂^max/<i>k</i>₂) representing the deviation of <i>k</i>₂ from <i>k</i>₂^max. In the catalytic site of bovine ribonuclease A, His12 showed a change in log­(<i>k</i>₂^max/<i>k</i>₂) much larger than that of His119 upon binding with cytidine 3′-monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, we need to consider the hydrogen bonds of the imidazole group with nondissociable groups to interpret an extremely slow H/D exchange rate of His48 in a partially solvent-exposed situation