Tautomeric States of the Histidine Residues

Natural abundance *‘C NMR (at 67.9 MHz) is used to study the nonprotonated aromatic carbons of bovine pancreatic ribonuclease A. The CY resonances of the 4 histidines are identified and assigned to specific residues. The effect of pH on the chemical shifts of these resonances yields information about the tautomeric state of the imidazole form of each histidine residue. Only His-119 exists predominantly or entirely in the “common” N”-H (N-H) tautomeric state. His-12 and His-48 exist mainly or entirely in the N”-H (N”-H) tautomeric state. His-105 exhibits about 50 to 90% of the N”-H tautomer. These results, when taken together with known crystallographic coordinates for ribonuclease A, strongly suggest that H” of His-105 is hydrogen bonded to the COOH-terminal carboxylate group, that H” of His-48 is hydrogen bonded to the carboxylate group of Asp-14 or (less likely) to the hydroxyl oxygen of Thr-17, and that HR’ of His-12 is probably hydrogen bonded to the hydroxyl oxygen of Thr-45.