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

Area of Science:

• Biochemistry
• Chemical Biology
• Protein Science

Background:

• Histidine's imidazole side chain has unique acid-base properties near physiological pH.
• Histidine's three states (two neutral, one protonated) enable diverse roles in protein function, including catalysis.
• Understanding histidine tautomer exchange is vital for elucidating enzymatic mechanisms where proton transfer rates can be limiting.

Purpose of the Study:

• To determine the exchange kinetics of histidine residues across a physiological pKa range (5-9).
• To investigate the pH-dependent mechanisms of proton exchange between histidine and solvent.
• To provide a comprehensive kinetic model for histidine protonation and tautomerization.

Main Methods:

• Utilized pH-dependent 15N, 13C, and 1H nuclear magnetic resonance (NMR) relaxation rate measurements.
• Measured relaxation rates for five nuclei within the imidazole ring to probe dynamics.
• Analyzed data to determine rate constants and lifetimes for proton exchange and tautomer interconversion.

Main Results:

• Proton exchange is hydronium-mediated at acidic/neutral pH and hydroxide-mediated at basic pH.
• Proton transfer rates approach the diffusion limit near neutral pH.
• Identified a direct pathway for tautomer interconversion, potentially involving water or hydroxide ions.
• Quantified all rate constants for histidine with pKa 7 across the entire pH range.

Conclusions:

• The study provides a detailed kinetic framework for histidine proton exchange and tautomerization.
• These findings offer critical insights into the mechanisms of histidine-involved enzymatic acid-base catalysis.
• The methodology can be applied to study histidine dynamics in various protein systems.