Structural studies of yeast iso-1 cytochrome c mutants by resonance Raman spectroscopy.
The Ser82 and Phe82 variants of yeast iso-1 cytochrome c were studied by resonance Raman spectroscopy. In both oxidation states, distinct spectral changes were observed for some of those bands in the low-frequency region, which sensitively respond to conformational perturbations of the protein environment of the heme. These bands can be assigned to modes which include strong contributions of vibrations largely localized in the propionate-carrying pyrrole rings A and D. This indicates structural differences in the deeper part of the heme crevice, remote from the mutation site. This conclusion is in line with previous results from X-ray crystallography and NMR spectroscopy. No differences in the resonance-Raman spectra were observed which can be directly correlated with conformational changes of the heme pocket in the vicinity of the mutation site. Temperature-dependent resonance Raman experiments of the oxidized mutants revealed spectral changes which are closely related to those observed for cytochrome c upon adsorption to charged silver surfaces by surface-enhanced resonance Raman spectroscopy. These spectral changes can be attributed to an opening of the heme crevice accompanied by a weakening of the iron-methionine ligand bond. The temperature-dependent conformational transition occurs at approximately 30 degrees C for the Ser82 variant and at about 45 degrees C for the Phe82 variant, implying that the Phe----Ser substitution significantly lowers the thermal stability of the heme pocket. The reduced forms of both mutants are stable up to 65 degrees C.
Hildebrandt
P
P
Max-Planck-Institut für Strahlenchemie, Mülheim, Federal Republic of Germany.
Pielak
G J
GJ
Williams
R J
RJ
D003460:Crystallization; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004355:Drug Stability; D004563:Electrochemistry; D006418:Heme / Q000737:chemistry; D006358:Hot Temperature; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D013059:Spectrum Analysis, Raman
D003574:Cytochrome c Group; D006418:Heme