Circular dichroism studies of the perturbations of cytochrome c by alcohols.

The circular dichroic (CD) behavior of ferriand ferrocytochrome c in the presence of methanol, ethanol, and 1-propanol u-as investigated in an attempt to elucidate some structure-function relationships of the protein. The circular dichroic spectrum of ferricytochrome c in the Soret region exhibited extrema at 417 and 402 nm, which were susceptible to the presence of alcohols in solution. In concentrations of I-propanol exceeding 6 mole % the protein became modified to a form that showed a positive CD band at 406 nm with a large increase in ellipticity and the disappearance of the 417~nm trough. Similar changes were also observed in solvents containing greater than 13 mole % of ethanol and greater than 21 mole % of methanol. These changes in the Soret region occurred at the same alcohol concentration as those in the aromatic region, thus possibly implicating the aromatic residues in heme crevice conformation changes. Changes in the intrinsic region of ferricytochrome c indicated the enhancement of o( helix content at elevated alcohol concentrations. In contrast, ferrocytochrome c under anaerobic conditions showed no changes in either absorption or CD spectra even in 50 volume per cent of 1-propanol. These spectra were, however, converted immediately by higher alcohol concentrations to that resembling the modified ferricytochrome c. The heme of this apparently “autoxidized” cytochrome c was reactive to carbon monoxide and thus the iron was still in the reduced state. These results indicate that alcohols perturb effectively the interactions between the heme and the protein moiety forming the heme crevice. At increasing alcohol concentrations, ferrocytochrome c which is less susceptible to modification than ferricytochrome c is gradually deformed. Consequently, the heme becomes exposed and reactive toward CO. The similarity in the CD spectra of ferroand ferricytochrome c under these conditions is a reflection of interactions between the distorted heme coordination sphere and the altered heme environment.