Unraveling the Mystery of Ferricytochrome C: An Investigation into Induced Non-Native Conformational Changes

Abstract

Cytochrome_c, in its oxidized state, adopts a multitude of conformations depending on solution conditions. Some of these conformations are relevant for the protein's functions in the electron transport process and in apoptosis. As frequently as cytochrome_c has been investigated limited experiments have been carried out under low ionic conditions, which is of significant biological importance since it's required for Apaf-1 complex formation and anionic lipids. This investigation explores the energy landscape of cytochrome_c under well-defined thermodynamic conditions. A comparison of CD and absorption of the Soret band region of both the native and non-native states of ferricytochrome c adopted between pH1-13, and temperatures between 278-353K at ionic strengths below 0.1mM was performed. Avoiding the binding of anions to positive patches on the proteins surface. State-I shows the protein unfolded with the iron in a high spin state, as the protein environment was acidified a Cotton band emerges in the CD spectra, the intensity of the bands decreased, starting around pH4. Approaching state-III, the iron enters a low spin state, a stronger couplet emerges reflecting band-splitting, predominantly caused by a combination of electronic and vibronic perturbations, maintained below 343K. Suggesting a conformational transition from the native state, into a thermally activated intermediate state, affecting the internal electric field causing moderate rearrangements of the heme, until it enters a thermally unfolded state. This state of the protein consistently becomes populated at higher temperatures across the pH range. This couplet remains into pH9 possible reflecting an intermediate transition of state III-IV, moving more alkaline this couplet disappears. Using Kuhn anisotropy, Δɛ/e vs temperature, the population of intermediates is indicated as temperature increased. Characterization of ferricytochrome_c transitions at low ionic strength showed significant heterogeneity of the protein throughout the pH range.