Ultraviolet resonance Raman spectra of cytochrome c conformational states

Abstract

Ultraviolet resonance Raman (UV RR) spectra are reported for ferricytochrome c from tuna and horse heart at pH 1.6, 7, 10, and 13, representing distinct conformational states of the protein (states II, III, IV, and V, respectively). The spectra were obtained with pulsed laser excitation at 200 and 218 nm, via H2 Raman shifting the fourth harmonic output of a pulsed YAG laser. At these deep UV wavelengths, strong enhancement is observed for vibrational modes associated with tryptophan, tyrosine, and phenylalanine side chains and with the amide groups of the polypeptide backbone. The amide I peak frequency is consistent with a dominant contribution from .alpha.-helical regions, although a broad high-frequency tail reflects a variety of unordered conformations. The peak frequency is 12 cm-1 higher for cytochrome c from tuna than from horse, suggesting a less tightly wound structure, which is consistent with the lower denaturation temperature previously reported for the tuna protein. The amide I peak broadens when native protein (state III) is converted to the low- or high-pH forms (states II and IV), reflecting some disordering of the polypeptide chain, but the peak frequencies are unshifted, establishing that the .alpha.-helical segments are not completely unfolded in these states. Raising the pH to 13 (state V), however, does produce a frequency upshift, reflecting helix unfolding. The amide II and III frequencies are likewise consistent with a dominant .alpha.-helix contribution in the native proteins; they gain intensity, and amide III is shifted to a lower frequency, in states II and IV, consistent with partial disordering. Dissolution of protein in D2O leads to the disappearance of the amide III band and to a very strong amide II'' band, as recently observed for sample peptides, consistent with stretching of the amide C-N bond in the resonant excited state, and a concentration of the C-N stretching coordinate in the amide II'' mode upon D/H exchange of the amide proton. The tyrosine band intensities change significantly between state III and state II, becoming similar to those displayed by tyrosine itself in water; these changes are attributed to disruption of internal H bonds for two of the four tyrosines (five for tuna) in the protein. Similar, but less marked, changes in the intensity pattern are seen for state IV. In state V, several spectral changes are observed which are consistent with the expected tyrosine ionization at pH 13. Tryptophan modes also show changes in intensity, and in band frequency, suggesting disruption of the internal H bond for the one tryptophan of the horse protein (and one of the two residues in the tuna protein) in states II and V.