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Cytochrome crelease from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome cfrom the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome crelease. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome cfrom beef‐heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial‐mediated ROS generation resulted in a large dissociation of cytochrome cfrom SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome cfrom IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome cfrom the mitochondria to the cytosol.

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome coxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate‐respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome coxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS‐induced loss of cytochrome coxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome coxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS‐induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.

Cardiolipin is a major mitochondrial membrane lipid and plays a pivotal role in mitochondrial function. We have recently suggested a possible involvement of this phospholipid in the age‐linked decline of cytochrome coxidase activity in rat heart mitochondria [G. Paradies et al. (1993) Arch. Gerontol. Geriatr. 16, 263–272]. The aim of this work was to test our earlier proposal. We have investigated whether addition of exogenous cardiolipin to mitochondria is able to reverse, in situ, the age‐linked decrease in the cytochrome oxidase activity. The method of fusion of liposomes with mitochondria developed by Hackenbrock [Hackenbrock and Chazotte (1986) Methods Enzymol. 125, 35–45] was employed in order to enrich the mitochondria cardiolipin content. We demonstrate that the lower cytochrome coxidase activity in heart mitochondria from aged rats can be fully restored to the level of young control rats by exogenously added cardiolipin. No restoration was obtained with other phospholipids or with peroxidized cardiolipin. Our data support a key role for cardiolipin in the age‐linked decline of rat heart mitochondrial cytochrome coxidase activity.