Effect of V83G and I81A Substitutions to Human Cytochrome c on Acid Unfolding and Peroxidase Activity below a Neutral pH

Abstract

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.

Naturally Occurring A51V Variant of Human Cytochrome c Destabilizes the Native State and Enhances Peroxidase Activity

“…However, we have recently reported kinetic data for the V83G and I81A variants of Hu Cytc, which we fit assuming two slow phases. 64 Values of pK H for both the k obs,1 and k obs,2 phases were within the range of 10.7−11.2 for kinetic data fit with two slow phases for these variants (Table 6). 64 As for the G41S and Y48H variants, the increased access to alternate conformers for the A51V variant appears to result from a decrease in the pK H of the triggering deprotonation.…”

“…The I81A variant of Hu Cytc also shows enhanced kinetic accessibility of only one of the two kinetically distinguishable alkaline conformers in 0.1 M NaCl. 64 In this case, pK H for k obs,1 is unchanged, whereas pK C is more favorable because k f is larger and k b is smaller (Table 6). Notably, k f and k b derived from k obs,1 for the V83G variant are similar to those of the A51V variant, yielding a similar less favorable pK C for these two variants (Table 6).…”

“…Figure 7 shows plots of the corrected extinction coefficients at 695 nm (Met80 ligation), ε 695corr , and 622 nm (high-spin heme), ε 622corr , versus pH for the acid unfolding of A51V compared to previously reported data for WT Hu Cytc. 64 The plots show that the A51V variant is significantly less stable to acid unfolding than WT Hu Cytc. For both proteins, the data monitored at 622 nm yield a broader transition than the data at 695 nm.…”

Naturally Occurring I81N Mutation in Human Cytochrome c Regulates Both Inherent Peroxidase Activity and Interactions with Neuroglobin

“…Previous studies have shown that mutations in the Ω-loop D (70–85) such as K72A, P76C, K79G/M80X, I81A, F82K, and V83G may alter the heme crevice dynamics and ligand-binding properties. 19 , 32 , 40 − 42 …”

“…The strong correlation between the peroxidase activity and acidic pH indicates that Ile81 is a peroxidase trigger in h Cyt c . It is worth mentioning that the peroxidase activity of the I81A variant ( k cat = 0.96 ± 0.01 s –1 ) 32 is 6-fold higher than that of the I81N ( k cat = 0.16 ± 0.01 s –1 ) and 8.7-fold higher than that of the WT ( k cat = 0.11 ± 0.01 s –1 ) at neutral pH, and thus the I81A mutation may not be allowed naturally.…”