Electrochemical probing of the solution pH-induced structural alterations around the heme group in myoglobin.

Protein folding-unfolding has received considerable attention because of its fundamental importance in the manifestation of biological functions, in molecular diagnostics, in drug development, and its close relationship to human health. Although many attempts have been made to study and monitor the protein unfolding, a facile and effective method for probing the structural changes of proteins is still highly desired. This work reports electrochemical voltammetry for probing the structural alterations around the heme group in myoglobin (Mb) induced by solution pH. This approach is based on the changes of the electron transfer (ET) signals of Mb upon the structural alteration because the heme group is deeply buried in the hydrophobic pocket in neutral conditions (in the native state), resulting in poor ET signals, while it dissociates from the hydrophobic pocket and becomes exposed to the solution in acidic and basic conditions (in the unfolding state) due to the breakage of the Fe-His93 bond, causing an obvious enhancement in ET signals. The good consistency of the results obtained with our electrochemical method with those of spectroscopic measurements (such as UV-vis, CD, and Raman spectroscopy) and molecular dynamics (MD) simulations substantially validates that our proposed method is a facile and effective way for probing structural changes around the heme group in Mb. Compared with the existing methods used for the study of structural changes of protein, our method demonstrated here could be advantageous in terms of its sensitivity, ease of operation, cost-effective feature, simple use of bare electrodes, and availability of abundant dynamic information. This study essentially offers a facile and effective electrochemical route to probe the structural changes around the heme group and the conformation alterations of heme-containing proteins.