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, pH1/2, 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
pH1/2 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.