Physical and functional effects of substituting coevolved residues from Ω-loop C of yeast Iso-1-cytochrome c into human cytochrome c

Omega loop C (residues 40–57) of cytochrome c (Cytc) is a common location for naturally-occurring variants of human Cytc that cause thrombocytopenia 4 (THC4). These variants are characterized by significant increases in the intrinsic peroxidase activity of Cytc, which appears to be linked to increased dynamics in Ω-loop D (residues 71–85). The mutations in Ω-loop C enhance the dynamics of Ω-loop D by decreasing the acid dissociation constant of the trigger group (pK_H) of the alkaline conformational transition. The intrinsic peroxidase activity of human Cytc is considerably lower than that of yeast iso-1-Cytc. We identified three sites in or near Ω-loop C, which show evidence of coevolution and reasoned that they could play a role in minimizing the intrinsic peroxidase activity of human Cytc so that this activity can be an effective switch in the early stages of apoptosis. We prepared T40S, I57V and T63N variants of human Cytc, which substitute the amino acids found in iso-1-Cytc into these sites. Studies on the global and local (alkaline transition) stabilities and peroxidase activities of these variants show modest effects. These results show that evolution of Ω-loop C has acted to preserve the dynamics of Ω-loops C and D, which surround the heme. Like the THC4 variants, the T40S and T63N variants show a significant decrease in the pK_H of the alkaline transition. However, unlike the THC4 variants this is coupled to changes in pK_C that disfavor the alkaline state, thereby leaving the intrinsic peroxidase activity unaffected.