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

The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH_1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH_1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the k_cat values for its peroxidase activity increase by 6-to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis.