Conformational Heterogeneity of Cytochrome c Probed by Resonance Raman Spectroscopy as a function of pH and Temperature

Abstract

The oxidized state of cytochrome c is a subject of continuous interest due to the multitude of conformations the protein adopts. Despite numerous studies, native and non-native states of ferricytochrome c have not been comprehensively analyzed regarding the influence of solvent conditions on structure, function, and thermodynamic equilibrium. Compared to the oxidized state, the reduced state of cytochrome c is rather stable since it adopts one conformation over a broad pH (2-12) and temperature (∼100°C) range. In the current study, we have analyzed the high frequency (1200-1800cm−1) Soret and Q-band resonance Raman spectra of oxidized and reduced horse heart cytochrome c (hhc) in terms of depolarization ratios and normalized Raman intensities, as a function of increasing pH and temperature. Initial analysis of our data collected for the Soret band resonance indicates that the depolarization ratios of A1g modes v2, v3, and v4 of the native state III and the alkaline state IV are practically identical. They deviate from the respective D4h-value, indicating that B1g (triclinic) or B2g (rhombic) type distortions affect the Raman tensor. For state III, the depolarization ratios of B1g modes v10, v11, and v13 deviate substantially from the expectation value indicating that these modes are affected by a large B1g type distortion. The alkaline III-> IV transition moves the depolarization ratios of these B1g modes closer to their D4h-value of 0.75, indicating a substantial decrease of the rhombic B1g-type deformation. Data from pH 12, which favor the population of the V-state, suggest a substantial increase of rhombic deformations, apparently caused by the replacement of a lysine by a hydroxyl ligand. Currently we are analyzing the depolarization ratios for the Q band resonance and the high temperature data for both resonances.