Modulation of cytochrome c’s peroxidase activity by Ω-loops C and D in monomers and dimers

Mitochondrial cytochrome c is a heme containing protein with flexible heme ligation. Cytochrome c in its native state with Met80 bound to the heme is an electron shuttle in the electron transport chain. Under apoptotic stimuli, cytochrome c oxidizes the mitochondrial lipid cardiolipin, leading to the release of pro-apoptotic factors into the cytoplasm. To oxidize cardiolipin, Met80 in Ω-loop D must be dissociated from the heme iron, which allows cytochrome c to obtain access to alternative conformers. Two segments of cytochrome c, namely Ω-loops C and D, have been suggested to be important in regulating the dissociation of Met80. Unlike most residues in Ω-loop D, positions 81 and 83 have evolve to more sterically demanding residues in higher eukaryotes, leading to a 30-fold decrease in peroxidase activity. We study the effects of evolutionary substitutions at positions 81 and 83 in both human and yeast iso-1 cytochrome c. The side chain volumes at positions 81 and 83 do not fully account for the difference in the peroxidase activity between human and yeast iso-1 cytochrome c, indicating that other segments of cytochrome c contribute significantly to the low intrinsic peroxidase activity of human cytochrome c. All naturally occurring disease-related mutations, namely G41S, Y48H, and A51V, of human cytochrome c, which contain abnormally high peroxidase activity, are located in Ω-loop C, suggesting that Ω-loop C is an important modulator of the peroxidase activity. Investigating the disease mutation, A51V, in human cytochrome c significantly destabilizes Ω-loop D, resulting in enhancement of peroxidase activity. Dimeric cytochrome c with an open heme coordination site has been suggested to mediate the peroxidase activity in the early stage of apoptosis. We have recently solved a structure of human dimeric cytochrome c, demonstrating that the heme ligation in dimeric cytochrome c is pH dependent, providing a pH-inducible switch that controls the peroxidase activity of the human dimer near physiological pH.