Cytochrome c (Cc) binding to apoptosis protease activation factor-1 (Apaf-1) is a critical activation step in the execution phase of apoptosis. Here we report studies that help define the Cc:Apaf-1 binding surface. It is shown that a large number of Cc residues, including residues 7, 25, 39, 62-65, and 72, are involved in the Cc:Apaf-1 interaction. Mutation of residue 72 eliminated Cc activity whereas mutations of residues 7, 25, 39, and 62-65 showed reduced activity in an additive fashion. The implications of this binding model for both recognition and modulation of protein-protein interactions are briefly discussed.

In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. Here, we show through fluorescence polarization experiments that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c⅐Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c⅐Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. We also report here that the cytochrome c⅐Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins.