The Human Cytochrome Domain-Swapped Dimer Facilitates Tight Regulation of Intrinsic Apoptosis.

Oxidation of cardiolipin (CL) by cytochrome (cyt) has been proposed to initiate the intrinsic pathway of apoptosis. Domain-swapped dimer (DSD) conformations of cyt have been reported both by our laboratory and by others. The DSD is an alternate conformer of cyt that could oxygenate CL early in apoptosis. We demonstrate here that the cyt DSD has a set of properties that would provide tighter regulation of the intrinsic pathway. We show that the human DSD is kinetically more stable than horse and yeast DSDs. Circular dichroism data indicate that the DSD has a less asymmetric heme environment, similar to that seen when the monomeric protein binds to CL vesicles at high lipid:protein ratios. The dimer undergoes the alkaline conformational transition near pH 7.0, 2.5 pH units lower than that of the monomer. Data from fluorescence correlation spectroscopy and fluorescence anisotropy suggest that the alkaline transition of the DSD may act as a switch from a high affinity for CL nanodiscs at pH 7.4 to a much lower affinity at pH 8.0. Additionally, the peroxidase activity of the human DSD increases 7-fold compared to that of the monomer at pH 7 and 8, but by 14-fold at pH 6 when mixed Met80/HO ligation replaces the lysine ligation of the alkaline state. We also present data that indicate that cyt binding shows a cooperative effect as the concentration of cyt is increased. The DSD appears to have evolved into a pH-inducible switch that provides a means to control activation of apoptosis near pH 7.0.