High affinity binding of Bcl-xL to cytochrome c: Possible relevance for interception of translocated cytochrome c in apoptosis

The release of cytochrome c from mitochondria and apoptosis relies on several preferential and selective interactions involving the Bcl-2 family of proteins. There is, however, no direct evidence for the interaction of cytochrome c with these proteins at any stage of apoptosis. To investigate if any pro-survival protein from the Bcl-2 family could intercept cytochrome c after its translocation from mitochondria, the interaction of cytochrome c with bacterially expressed human Bcl-xL was studied at pH 7. In size-exclusion chromatography, purified full-length His6-tagged Bcl-xL migrated as both dimer and monomer, of which the monomeric fractions were used for experiments. Coimmunoprecipitation studies show that cytochrome c interacts with Bcl-xL. The extent of caspase activity in cell lysate elicited by externally added cytochrome c is reduced when a preincubated mixture of Bcl-xL and cytochrome c is used instead. Equilibrium binding monitored by optical absorption of cytochrome c as a function of titrating concentrations of Bcl-xL yields the association constant, Kass = 8.4(± 4) ×106 M− 1 (binding affinity, Kdiss = 1/Kass ≈ 120 nM) which decreases at high ionic strength. The rates for binding of Bcl-xL to cytochrome c, studied by stopped-flow kinetics at pH 7, show that the bimolecular rate constant for binding, kbi = 0.24 × 106 M− 1 s− 1. Values of the thermodynamic and kinetic parameters for Bcl-xL–cytochrome c interaction are very similar to those known for regulatory protein–protein interactions in apoptosis.