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.

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.

No abstract

Cytochrome c (Cytc) is a globular protein within the inner mitochondrial membrane that shuttles electrons from Complex III to Complex IV in the electron transport chain. Upon release from the mitochondria, Cytc participates in initiation of the caspase cascade leading to apoptosis. Cytc binds to cardiolipin in the inner mitochondrial membrane leading to loss of Met80-heme ligation, enabling Cytc to function as a peroxidase capable of oxidizing cardiolipin. The affinity of Cytc for oxidized cardiolipin is decreased and therefore Cytc can dissociate from the membrane facilitating release from mitochondria and propagation of apoptosis. In order for the heme coordination site to open for peroxidase activity, the heme crevice loop must undergo structural rearrangement similar to the alkaline transition. Unlike most residues in the heme crevice loop, positions 81 and 83 evolve to more sterically demanding residues in higher eukaryotes. Previous results suggest that although residues 81 and 83 are key residues for regulating the peroxidase activity of Cytc, there is evidence that substitutions in U-loop C can affect the stability of the heme crevice loop. We hypothesize that U loops C and D have evolved interactively to optimize the peroxidase activity of Cytc. To perform this test, we prepared a series of human-to-yeast substitutions in U-loop C of human Cytc. Using circular dichroism and UV-visible spectroscopies, we have investigated global stability of the protein and local stability of the heme crevice loop. Using stoppedflow methods, we investigated the peroxidase activity for both I57V and N54K human Cytc. We observe that the N54K substitution strongly enhances the peroxidase activity of human Cytc, whereas the I57V substitution has little effect.

No abstract