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 extracts can activate the caspases, suggesting that this export could be an important intracellular signal for initiating the apoptotic programme. We have investigated the mechanism of caspase activation by cytochrome c. Mitochondrial cytochrome c normally shuttles electrons between complexes III and IV of the electron transport chain. Interaction with these complexes is dependent on electrostatic interactions via a polylysine 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 allosteric 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 cytochrome 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. We conclude that reduced cytochrome c will function in the cytoplasm; however, its reduction is not a critical step, and electron transfer from cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis.
Hampton
M B
MB
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden.
Zhivotovsky
B
B
Slater
A F
AF
Burgess
D H
DH
Orrenius
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004579:Electron Transport; D004789:Enzyme Activation; D005292:Ferricyanides / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D012697:Serine Endopeptidases / Q000378:metabolism*
D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D005292:Ferricyanides; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C026600:2'-deoxyadenosine triphosphate; D004229:Dithiothreitol; C028033:potassium ferricyanide