Effects of Humanlike Mutations on Yeast Iso-1-Cytochrome C

Abstract

Apoptosis is a key process in various human diseases. It is initiated in the mitochondria, where changes in the local environment cause cytochrome c (cytc) to become competent for peroxidase activity. Human cytc has 20- to 30-fold lower peroxidase activity than yeast cytc, which we hypothesize allows for tighter control of apoptosis in higher eukaryotes. Many residues that have evolved between yeast and human are found in Ω-loops C and D, which are some of the most dynamic regions of cytc. The Ω-loops are thought to control the dynamics of opening the heme crevice, and thus peroxidase activity. Residues present in human are larger and more hydrophobic than in yeast cytochrome c, so mutations have been made in yeast to reflect that. By mutating residues S40, N63, V57, and A81I, which are larger and more hydrophobic in human cytc, both individually and pairwise in yeast iso-1-cytc, we expect to see corresponding changes in peroxidase activity with minimal changes in overall structure. Circular dichroism was used to monitor guanidine unfolding and assess changes in global stability between each of the variants. The alkaline transition was monitored to estimate the effect of these mutations on Ω-loop D stability. Peroxidase activity measurements were conducted using stopped-flow with guaiacol as a substrate. Our results indicate that single mutations usually have small effects, however in some instances double mutations have more pronounced effects than either single mutation alone. These results seem to indicate that the combined effects of these residues form a network that modulates the peroxidase activity in human cytochrome c, as opposed to one single residue being mainly responsible for these differences.