Abstract
Abstract The circular dichroic (CD) behavior of ferri- and ferrocytochrome c in the presence of methanol, ethanol, and 1-propanol was 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 1-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 α 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 ferro- and ferricytochrome c under these conditions is a reflection of interactions between the distorted heme coordination sphere and the altered heme environment.
Highlights
The circular dichroic (CD) behavior of ferri- and 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
In earlier papers we reported on the effect of alcohols and ethers on the Soret absorbance of ferricytochrome c [15, 16] and on the alcohol-induced autoxidation of ferrocytochrome c [17]
The extrema in the Soret region at 417( -)nm and 402( +)nm rapidly altered with increasing alcohol concentration above a certain level, which was dependent on the alcohol used
Summary
Alcoholic solutions of ferricytochrome c were prepared as described in an earlier paper [17]. The buffer-alcohol mixture was bubbled with oxygen-free, water-saturated argon in a Thiinberg cuvette. Ferrocytochrome c and enough solid dithionite to reduce any portion of the cytochrome c, which may have become oxidized, was placed in the side arm and was mixed with the solvent after 30 min of argon bubbling. Absorbance spectra of the solutions were determined in the 550.nm region with a Cary 14 spectrophotometer before and after determination of the circular dichroism spectra. An experiment was repeated if any change in absorbance at 550 nm was noted. Concentrations of, cytochrome c are reported in the figure legends and were determined with molar extinction coefficients as reported by
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