Tautomeric stabilities of 4-fluorohistidine shed new light on mechanistic experiments with labeled ribonuclease A

Highlights: • The π-tautomer of 4-fluorohistidine is found to be predominant via MP2 and DFT calculations. • Proton transfer in 4-fluorohistidine labeled RNase A via the π-tautomer of His119 is indicated. • Tautomerization in wild-type RNase A should be considered in future mechanistic work. Ribonuclease A is the oldest model for studying enzymatic mechanisms, yet questions remain about proton transfer within the active site. Seminal work by Jackson et al. (1994) labeled Ribonuclease A with 4-fluorohistidine, concluding that active-site histidines act as general acids and bases. Calculations of 4-fluorohistidine indicate that the π-tautomer is predominant in all simulated environments (by ∼17 kJ/mol), strongly suggesting that fluoro-labeled ribonuclease A functions with His119 in π-tautomer. The tautomeric form of His119 during proton transfer and tautomerism as a putative mechanistic step in wild-type RNase A remain open questions and should be considered in future mechanistic studies.