IMPORTANCE OF THE REDOX STATE OF CYTOCHROME-C DURING CASPASE ACTIVATION IN CYTOSOLIC EXTRACTS

The export of cytochrome c from mitochondria to the cytoplasm has been detected during apoptosis. Addition of cytochrome c to cytosolic extr acts can activate the caspases, suggesting that this export could be a n important intracellular signal for initiating the apoptotic programm e. We have investigated the mechanism of caspase activation by cytochr ome c. Mitochondrial cytochrome c normally shuttles electrons between complexes :(II and IV of the electron transport chain. Interaction wit h these complexes is dependent on electrostatic interactions via a pol ylysine binding pocket. Cytosolic caspase activation was only observed with intact holocytochrome c, and increasing the ionic composition of the extracts prevented activation, suggesting that stringent alloster ic interactions between cytochrome c and other cytoplasmic factors are necessary. Cytochrome c was fully reduced within 5 min of addition to the cytosolic extracts. Potassium ferricyanide could maintain cytochr ome c in an oxidized state, but care was taken to use ferricyanide at concentrations where its polyanion effect did not cause interference. The oxidized form of cytochrome c was able to activate the caspases. W e conclude that reduced cytochrome c will function in the cytoplasm; h owever, its reduction is not a critical step, and electron transfer fr om cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis.