The pK(a) of His-24 in the folding transition state of apomyoglobin

In native apomyoglobin, His-24 cannot be protonated, although at pH 4 the n ative protein forms a molten globule folding intermediate in which the hist idine residues are readily protonated. The inability to protonate His-24 in the native protein dramatically affects the unfolding/refolding kinetics, as demonstrated by simulations for a simple model. Kinetic data for wild ty pe and for a mutant lacking His-24 are analyzed. The pK(a) values of histid ine residues in native apomyoglobin are known from earlier studies, and the average histidine pK(a) in the molten globule is determined from the pH de pendence of the equilibrium between the native and molten globule forms. An alysis of the pH-dependent unfolding/refolding kinetics reveals that the av erage pK(a) of the histidine residues, including His-24. is closely similar in the folding transition state to the value found in the molten globule i ntermediate. Consequently, protonation of His-24 is not a barrier to refold ing of the molten globule to the native protein. Instead, the normal pK(a) of His-24 in the transition state, coupled with its inaccessibility in the native state, promotes fast unfolding at low pH. The analysis of the wild-t ype results is confirmed and extended by using the wild-type parameters to fit the unfolding kinetics of a mutant lacking His-24.