Proton occupancies in histidine side chains of carbonic anhydrase II by neutron crystallography and NMR : differences, similarities and opportunities

Histidine is a key amino-acid residues in proteins that can exist in three different protonation states: two different neutral tautomeric forms and a protonated, positively charged one. It can act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Human Carbonic Anhydrase II (HCA II) is a pivotal enzyme catalyzing the reversible hydration of carbon dioxide. It contains 12 histidine residues: six are surface exposed, two buried, three are active site zinc ion ligands, and one is a proton shuttle. Comparing results from NMR spectroscopy with previously determined neutron protein crystal structures enabled a side-by-side investigation of the proton occupancies and preferred tautomeric states of the histidine residues in HCA II. Buried and zinc coordinating histidines remain in one neutral tautomeric state across the entire pH range studied, as validated by both methods. In contrast, solvent-exposed histidines display high variability in proton occupancies. While the data were overall remarkably consistent between methods, some discrepancies were observed, shedding light on the limitations of each technique. Therefore, combining these methods with full awareness of the advantages and drawbacks of each, provides insights into the dynamic protonation landscape of HCA II histidines, crucial for elucidating enzyme catalytic mechanisms.