Methanol-Induced Unfolding and Refolding of Cytochrome b5 and Its P40V Mutant Monitored by UV-Visible, CD, and Fluorescence Spectra

In order to illustrate the structural importance of proline-40 of cytochrome b₅ (Cyt b₅), the P40V mutant gene was constructed. Unfolding and refolding of Cyt b₅ induced by methanol was investigated by means of the UV-visible spectrum, circular dichroism, and the fluorescence spectrum. Methanol denaturation of Cyt b₅ is a cooperative process, that is, the heme group dissociates from the heme pocket accompanied by unfolding of the polypeptide chain both in the secondary and tertiary structures. Substitution of proline by valine reduces the stability of the mutant under methanol denaturation. The unfolding process is almost reversible by dilution. During refolding, the denatured polypeptide must be folded to a more ordered structure prior to the heme capture. Pro40 plays an important role in modulating the protein's stability. The role of tyrosine in the unfolding and refolding of Cyt b₅ is evaluated for the first time. A mechanism of methanol denaturation is also proposed.