Vibrational Coherence Spectroscopy Investigation of Cytochrome c equilibrium Unfolding

Using vibrational coherence spectroscopy (VCS) we studied guanidinium hydrochloride (GdHCl) induced equilibrium unfolding of ferric cytochrome c excited at 412 nm. Upon unfolding, we observed that the strong 50cm$^{-1}$ mode, which dominates the VCS spectrum of native cyt c, loses its intensity relative to the 80cm$^{-1}$ mode. The 224 cm$^{-1}$ mode ($\gamma _{24})$ also shifts to 205 cm$^{-1}$, reflecting the heme configuration change associated with unfolding. We also compared the amplitude of these unfolding sensitive modes at different GdHCl concentrations using a cyt c-imidazole complex as a model system. The peak of the Soret band does not shift when the cyt c- imidazole complex is unfolded. Because the resonance conditions are invariant, the relative intensities are a direct probe of the heme structural changes. Our results show that the 50 cm$^{-1}$ mode dramatically loses amplitude, while the 80 cm$^{-1}$ mode stays nearly the same. When compared to other Raman studies, which suggest that the heme adopts a more planer structure when cyt c is unfolded, the 50cm$^{-1}$ mode may reflect a similar structural change as the 569 cm$^{-1}$ ($\gamma _{21})$ mode. We suggest that these modes are diagnostic of a protein-induced ruffling distortion.