The importance of vibronic perturbations in ferrocytochrome c spectra: A reevaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations.

We have measured and analyzed the low-temperature (T=10 K) absorption spectrum of reduced horse heart and yeast cytochrome c. Both spectra show split and asymmetric Q₀ and Q_v bands. The spectra were first decomposed into the individual split vibronic sidebands assignable to B_1g (ν₁₅) and A_2g (ν₁₉, ν₂₁, and ν₂₂) Herzberg-Teller active modes due to their strong intensity in resonance Raman spectra acquired with Q₀ and Q_v excitations. The measured band splittings and asymmetries cannot be rationalized solely in terms of electronic perturbations of the heme macrocycle. On the contrary, they clearly point to the importance of considering not only electronic perturbations but vibronic perturbations as well. The former are most likely due to the heterogeneity of the electric field produced by charged side chains in the protein environment, whereas the latter reflect a perturbation potential due to multiple heme-protein interactions, which deform the heme structure in the ground and excited states. Additional information about vibronic perturbations and the associated ground-state deformations are inferred from the depolarization ratios of resonance Raman bands. The results of our analysis indicate that the heme group in yeast cytochrome c is more nonplanar and more distorted along a B_2g coordinate than in horse heart cytochrome c. This conclusion is supported by normal structural decomposition calculations performed on the heme extracted from molecular-dynamic simulations of the two investigated proteins. Interestingly, the latter are somewhat different from the respective deformations obtained from the x-ray structures. [ABSTRACT FROM AUTHOR]