Mitochondrial
cytochrome c is a highly conserved
protein in eukaryotes. Certain functions of cytochrome c have been tuned during evolution. For instance, the intrinsic peroxidase
activity of human cytochrome c is much lower than
that of the yeast counterpart. Structural studies on K72A yeast iso-1-cytochrome c [McClelland, L. J., et al. (2014) Proc. Natl.
Acad. Sci. USA, 111, 6648–6653] revealed
that residues 81 and 83 in Ω-loop D (residues 70–85)
may be gatekeeper residues for the peroxidase activity linked to intrinsic
apoptosis. Amino acids at both positions evolve to more sterically
demanding amino acids. We hypothesized that residues 81 and 83 evolved
such that steric constraints at these positions tune down the peroxidase
activity of human cytochrome c. To test this hypothesis,
I81A and V83G variants of human cytochrome c were
prepared. Our results show that the I81A substitution significantly
influences the thermodynamics and kinetics of access to alternate
conformers of human cytochrome c, while the V83G
substitution has a more modest effect on these properties. The I81A
variant also shows a significant enhancement in peroxidase activity,
particularly below pH 7, whereas the V83G variant has a similar peroxidase
activity to wild-type human cytochrome c. However,
neither variant increases the peroxidase activity of human cytochrome c to the level of yeast iso-1-cytochrome c, indicating that other substructures of cytochrome c are also involved in tuning the intrinsic peroxidase activity of
mitochondrial cytochrome c.