Modulation of the Peroxidase Activity of Human Cytochrome C by Ω Loops C and D

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.