Mitochondrial regulation of apoptosis by the redox state of cytochrome

Abstract

Cytochrome c release from mitochondria induces caspase activation in the cytosol, however, the question whether the redox state of cytosolic cytochrome c is important in caspase activation is still under debate. Using cytosols isolated from rat hearts and other mammalian cells we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added TMPD (tetramethyl-phenylenediamine), ascorbate, dithiothreitol or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Treatment with TMPD of intact cells or perfused hearts strongly inhibited activation of caspases and cell death induced by ischaemia/reperfusion or other proapoptotic stimuli. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase were inhibited. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain cytochrome c oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.