Ultrafast, laser induced pH jump with time resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (apoMb) under mild denaturing conditions (0.2 M GuHCl). At pre-pulse pH = 7, when apoMb is in its native state (N), two protonation processes can be clearly distinguished, that can be attibuted to the binding of a proton to the imidazole ring of a His and to the carboxy residues of either either a Glu or an Asp. Reaction with imidazole leads to an unusually large contraction of (-35± 6) ml/mol and an apparent bimolecular rate constant of (1.2± 0.1) 10¹⁰ M^-1 s^-1. Our experiments evidence a rate limiting step for this process at high apoMb concentrations, characterized by a value k₃^p = (0.6± 0.1) 10⁶ s^-1. Neutralization of the carboxylic groups is accompanied by an expansion of (3.9± 0.2) ml/mol, which is a bit lower than expected for the protonation of carboxyls in proteins, and occurs with an apparent bimolecular rate constant of (1.1± 0.1) 10¹¹ M^-1 s^-1. The reaction enthalpy for both processes is very small. The activation energies for the protonation of imidazole and carboxyls are (6.7± 1.0) kcal/mol and (16± 3) kcal/mol, respectively.

When the prepulse pH is lowered at 4.5 apoMb is in state I, the contraction due to the reaction with imidazole disappears, whereas the volume change and the rate constant for protonation of carboxyls become (3.3±0.2) ml/mol and (4.1± 0.1) 10¹⁰ M^-1 s^-1, respectively. The activation energy (6.5± 0.9 kcal/mol) for the protonation of the carboxyls is identical to the values measured at neutrality.

The unusually large contraction and the high energetic barrier observed for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate limiting step following protonation of the imidazole ring, leading to a substantial solvation of the structure with very little enthalpy change.