Cytochrome c is a relatively small (~12 kDa) polycationic mitochondrial protein. It was first identified as an electron shuttle in the electron transport chain. In healthy cells, the protein is found in the mitochondrial intermembrane space; it is associated with the inner mitochondrial membrane cardiolipin (CL), a lipid which comprises approximately 9-13 mol% (18-25% by weight) of that membrane. In 1996, Liu et al. found that the release of cytochrome c from the mitochondrial intermembrane space into the cytosol can induce apoptosis and that addition of exogenous cytochrome c to the cytosol causes a similar response.
The mitochondrial release of cytochrome c proceeds by mechanisms not yet entirely determined. It requires both a loosening of the tight cytochrome c-CL bonding which anchors the protein to the inner mitochondrial membrane and an exit via mitochondrial outer membrane permeabilization (MOMP).
There is some discussion about the potential role of post-translational modifications of cytochrome c as a switch between its electron transport function and its apoptotic function. Acetylation or deacetylation, lysine trimethylation, heme nitrosylation, tyrosine phosphorylation, and tyrosine nitration have been proposed, but the role of these modifications in vivo is unclear.
The first step of mitochondrial cytochrome c may be related to peroxynitrite decomposition and peroxidase activities catalyzed by the cytochrome c-CL complex. The peroxidized lipids formed via the peroxidase pathway are found to bind less tightly to cytochrome c than unreacted lipids, releasing the protein more readily from the inner mitochondrial membrane during apoptosis.
Alternatively, when cytochrome c remains lipid-bound, it can interact further with the lipid hydroperoxides, via a radical intermediate pathway, to form downstream products including lipid epoxides and aldehydes are observed to covalently modify cytochrome c at multiple positions, which causes partial protein unfolding including displacement of the heme Met80. Finally, cytochrome c crosslinking can occur, either as a result of oxidized lipids binding to multiple protein molecules or due to the combination of cytochrome c tyrosine radicals to form dityrosine linkages. It will be interesting to see how these complicated feedback events affect the apoptotic signaling of cytochrome c.
For the second step, the MOMP, a number of mechanisms are proposed, some of which may act cooperatively. The most popular explanation links permeabilization to the binding of Bcl-2-homology proteins BAX and BAK to the outer membrane, followed by oligomerization and the formation of a protein or lipid pore. Other Bcl-2-type proteins are involved in regulating this process: Bcl-2 and Bcl-XL are anti-apoptotic members of the protein family, while tBid is a pro-apoptotic protein with several proposed roles. tBid is thought to preferentially bind to CL at contact sites between the inner and outer membrane, assist in the targeting of BAK and BAX to the mitochondria and their subsequent oligomerization, and oligomerize itself to form pores. Lipidic pores formed due to membrane curvature effects have also been observed when membranes are treated with tBid and BAX, or with BAX alone.