ELIMINATION OF THE NEGATIVE SORET COTTON EFFECT OF CYTOCHROME-C BY REPLACEMENT OF THE INVARIANT PHENYLALANINE USING SITE-DIRECTED MUTAGENESIS

The circular dichroism (CD) was determined for variants of Saccharomyces cerevisiae iso-1-cytochrome c, in which phenylalanine-87, an invariant residue in eukaryotic and many prokaryotic cytochromes c, is replaced with a serine, tyrosine, of glycine. The major differences between the CD spectra of the wild-type protein and the variants occur in the Soret (385-445 nm) region of the spectrum. Specifically, a negative Cotton effect that occurs between 415 and 418 nm, with an intensity of (5.3-5.7) .times. 104.degree. cm2/dmol in the spectrum of the oxidized state of the wild-type protein, is absent from the spectra of the oxidized variants. Difference spectroscopy of the variants suggest that this same negative Cotton effect may also be present in the spectrum of the reduced wild-type protein. Two mechanisms by which phenylalanine-87 could contribute to the Cotton effect are considered. A direct mechanism involves the interaction of the .pi. to .pi.* transition of the aromatic side chain of phenylalanine-87 with the delocalized .pi.-electron system of the heme prosthetic group. In an indirect mechanism, phenylalanine-87 could contribute to this Cotton effect by defining a unique conformation for the polypeptide surrounding the heme. A review of published data suggests that the occurrence of this negative Cotton effect in different cytochromes c correlates with the presence and orientation, with respect to the plane of the heme, of a phenylalanine residue in a position equivalent to that of phenylalanine-87 in the S. cerevisiae protein.