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The A51V variant of human cytochrome cis linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH1/2for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the kcatvalues for its peroxidase activity increase by 6- to 15-fold in the pH range of 5–8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of Ω-loop C (residues 40–57) in modulating the peroxidase activity of cytochrome cearly in apoptosis.

Mitochondrial cytochrome cis a highly conserved protein in eukaryotes. Certain functions of cytochrome chave been tuned during evolution. For instance, the intrinsic peroxidase activity of human cytochrome cis 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 cwere 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 cto the level of yeast iso-1-cytochrome c, indicating that other substructures of cytochrome care also involved in tuning the intrinsic peroxidase activity of mitochondrial cytochrome c.