Molecular Mechanisms of Acid Denaturation: The Role of Histidine Residues in the Partial Unfolding of Apomyoglobin

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Apomyoglobin adopts a partly folded intermediate conformation (I), sometimes referred to as a molten globule intermediate, near pH 4. To determine which histidine residues trigger this partial unfolding reaction, we made mutants in which nine of the twelve histidine residues in the protein are substituted individually. We then measured acid and urea-induced unfolding curves for these substituted proteins. Two acid unfolding transitions are observed: native (N) to intermediate (I), and I to unfolded (U). These data were fitted using a simple three-state model which has been shown to give an adequate description of acid and urea-induced unfolding of wild-type apomyoglobin. The aim is to quantify changes in the apparent standard Gibbs energy differences between N, I and U, as well as the unfolding mechanism, that result from these substitutions, and to test how well the model fits data for substituted proteins. In most cases, the model fits the data reasonably well, and significant changes in fitted unfolding parameters of various mutants are also clearly visible in the primary data. The following conclusions are drawn. (1) Histidines 24 and 119 synergistically stabilize native apomyoglobin (N) at pH 8, but together destabilize N as pH is decreased below seven. (2) Histidine 36 stabilizes N when it is protonated. (3) Histidine substitutions in the heme-binding pocket (residues 64, 93 and 97) have little effect on the stability of N, suggesting that the heme-binding pocket is open. (4) Histidine substitutions affect the N to I transition but have little effect on the I to U transition. (5) The simple model we use to describe the unfolding of apomyoglobin cannot account for all the data, particularly the effects of the H36Q mutation. The effect of protonated histidine 36 on stabilizing N is not included in the model. We suggest that breaking the hydrogen bond between histidines 24 and 119 by protonation when the pH is decreased from 6 to 4 is an important part of triggering the partial unfolding of N to I, and likewise that formation of the hydrogen bond between histidines 24 and 119 may be a rate-determining step in the kinetic process of forming N from I during refolding.