It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82—conserved amino acid and a key actor in cytochrome c intermolecular interactions—with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by 1H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization.
The Human Cytochrome c Domain-Swapped Dimer Facilitates Tight Regulation of Intrinsic Apoptosis
