We test the hypothesis that Lys72 suppresses the intrinsic peroxidase activity of human cytochrome c, as observed previously for yeast iso-1-cytochrome c [McClelland et al. PNAS 111, 6648–6653 (2014)]. A 1.25 Å X-ray structure of K72A human cytochrome c shows that the mutation minimally affects structure. Guanidine hydrochloride denaturation demonstrates that the K72A mutation increases global stability by 0.5 kcal/mol. The K72A mutation also increases the apparent pKa of the alkaline transition, a measure of the stability of the heme crevice, by 0.5 units. Consistent with the increase in the apparent pKa, the rate of formation of the dominant alkaline conformer decreases and this conformer is no longer stabilized by proline isomerization. Peroxidase activity measurements show that the K72A mutation increases kcat by 1.6– to 4–fold at pH values from 7 to 10, a larger effect than for the yeast protein. X-ray structures of wild type and K72A human cytochrome c indicate that direct interactions of Lys72 with the far side of Ω-loop D, which are seen in X-ray structures of horse and yeast cytochrome c and could suppress peroxidase activity, are lacking. Instead, we propose that the larger effect of the K72A mutation on peroxidase activity of human versus yeast cytochrome c results from relief of steric interactions between the side chains at positions 72 and 81 (Ile in human versus Ala in yeast), which suppress the dynamics of Ω-loop D necessary for the intrinsic peroxidase activity of cytochrome c.

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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.

The A51V variant of human cytochrome c is 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 pH1/2 for 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 k cat values 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 c early in apoptosis.

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