Cytochrome c is a metalloprotein with primary physiological functions in the respiratory chain and in the regulation of cell death signals. Investigating the mechanisms leading to cytochrome c fibril formation is of primary importance for understanding its misfunctioning and, in a wider perspective, for its technologic applications in the field of bio-nanoscience. In this work, we analyzed the morphology and the spectroscopic properties of cytochrome c aggregates, combining the outcomes from electron microscopy, fluorescence, infrared and Raman spectroscopies and making use of statistical tools for the data analysis. The morphology scenario is quite complex, as it points out the presence of aggregates in the shape of platelets as well as fibers at micrometric scale. By infrared and Raman spectroscopy we analyzed the secondary and tertiary structures of unordered aggregates and fibrils, drawing a pathway for their formation at the timescales from tents to hundreds of minutes. Dependence of the fibrillation route on environmental pH, above and below the isoelectric point, and on protein concentration has also been explored. We found that it is possible to direct the process towards the formation of superstructures with different morphologies and different sizes along with fibrils, after destabilization of the native fold and the formation of β-sheet rich structures. A different mechanism characterizes aggregate/fibril elongation of cyt c in Tris-HCl, in comparison with NaOH environment.
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