pH-Induced Changes in the Secondary Structure of Cytochrome c: an Infrared Spectroscopic Study

Abstract The infrared spectra of 3mM solutions of horse heart cytochrome c were recorded as a function of pD(corresponding to acid high spin form, acidic low spin form, native form, lysine form, the so-termed “strained lysine form” as well as the so-termed “A state” of cytochrome c). An analysis of the pH-induced changes in the secondary structure was performed based on changes in the conformation-sensitive amide I bands of this protein. In lysine and strained lysine forms, the contents of random structure increase at the expense of α-helix. In acidic low. spin form (pH4–2.5), cytochrome c was almost unfolded. The proportion of α-helix had a substantial decrease while the contents of both random structure and 310-helices got a rise. In acidic high spin form (when pH is below 2.5), a further decrease of pD gave no rise to a continued unfolding in cytochrome c but made it refold to the “A state” with properties similar to those of a molten globular state. Adding enough KC1 to the cytochrome c solution(pD2.2 with 0.5MKC1 and pD3.0 with 1.5 MKCl) in which cytochrome c was nearly fully unfolded also led to the formation of A state of cytochrome c. The compositions of all types of secondary structures in A state were evaluated. The behavior of 310-helices in acidic high/low spin form indicated they might be intermediates between α-helices and random structure in acidic solution as proposed by Miick et al.