Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN′ by NMR

We have determined by ¹⁵N, ¹H, and ¹³C NMR, the chemical behavior of the six histidines in subtilisin BPN′ and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every ¹⁵N, ¹H, C^ε1 and C^δ2 resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK_a = 7.30 ± 0.03 at 25°C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK_a value of 7.9 ± 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C^ε1-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C^ε1-H^⋯O = C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N^δ1-H tautomer, exhibiting ¹³C^δ1 chemical shifts ∼9 ppm higher than those for N^ε2-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by ¹⁵N-¹H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK_a value of 7.47 ± 0.02.