Protein influences on porphyrin structure in cytochrome c: evidence from Raman difference spectroscopy.

To probe the details of protein heme interactions, we have developed a Raman difference spectroscopic technique, which allows reliable detection of very small, approximately equal to 0.01 cm-1, frequency differences. When this technique is applied to heme proteins, structural differences in the protein which perturb the porphyrin macrocycle may be examined by obtaining Raman difference data on the porphyrin vibrational modes which are strongly enhanced in the Raman spectrum produced with visible laser excitation. We report here Raman difference spectroscopic data on cytochromes c from 24 species. The differences in the Raman spectrum of the porphyrin between the cytochromes c of any two species are small, confirming that all of the cytochromes we have examined have the same "cytochrome fold". However, many small (0.02-2 cm-1) but systematic differences were detected which indicate structural differences among these proteins. These differences could be classified into three different groups and interpreted in terms of different types of structural variations resulting from specific differences in the amino acid sequences. First, direct interactions between near-heme residues and the porphyrin influence the electron density in the pi orbitals of the porphyrin macrocycle. Second, variation in the residue at position 92, far removed from the heme, affects the frequency of the core-size marker line at 1584 cm-1. Third, the conformation near cysteine 14 affects the shape of the Raman mode which is sensitive to the pyrrole ring substituents (approximately 1313 cm-1). From these data we conclude that there are several ways in which the protein amino acid sequence may regulate the oxidation-reduction potential and several ways in which the sequence can modify the binding site between cytochrome c and its redox partners.