Cytochrome c (Cyt c) has two main functions in mitochondria, caspase activation and electron transport. In healthy cells, cytochrome c (Cyt c) is located in the mitochondrial intermembrane space/tetrascapular space, acts as an electron shuttle in the respiratory chain, and interacts with cardiolipin (CL). Several pro-apoptotic stimuli induce outer membrane permeabilization, promote communication between the intermembrane space and the tetrascapular space, and promote the mobilization of Cyt c from the CL, resulting in the release of Cyt c from mitochondria into the cytoplasm. In the cytoplasm, Cyt c mediates the ectopic activation of caspase-activating factor 1, which is required for the proteolytic maturation of caspase-9 and caspase-3. Activated caspases ultimately lead to apoptotic cell disintegration.

Cyt c in mitochondria

Cyt c is a peripheral protein of the mitochondrial inner membrane (IM) that acts as an electron shuttle between complex III and complex IV of the respiratory chain, and its activity is essential for life. In mice, disruption of the unique somatic Cyt c gene causes embryonic lethality. Cyt c is synthesized in the cytoplasm as a carrier protein that binds to a heme repair group after transport to the mitochondria. Therefore, functional Cyt c, or holocytochrome c, consists of a single polypeptide chain of 104 amino acid residues covalently bound to a heme group. Based on crystallographic data, Cyt c is roughly spherical with a diameter of 3.4 nanometers. At physiological pH, Cyt c is mostly protonated, which means that most of Cyt c is bound to acidic phospholipids through electrostatic bonds, which are abundant in mitochondria. At least 15% of Cyt c in mitochondria is tightly bound through electrostatic and hydrophobic interactions. The rest of Cyt c is loosely attached to the IM through weak electrostatic interactions and can be readily mobile. The loosely and tightly bound Cyt c pools have different functions. The former participates in electron transport, inhibits the formation of reactive oxygen species (ROS), and prevents oxidative stress. The latter may bind to cardiolipin (CL), an unusual lipid that is mainly confined to the intracellular membrane. In fact, CL seems to be essential for the intercalation of myelin in cardiomyocytes.

Mitochondria release pro-apoptotic molecules

The mitochondrial intermembrane space (IMS) contains a heterogeneous class of proteins whose release promotes cell death. Soluble molecules from the IMS include Cyt c, which, once in the cytoplasm, can interact with Apaf-1 to generate the apoptosome, a molecular platform for the activation of caspase-9. Catalytic maturation of caspase-9 activates the caspase cascade, ultimately favoring the formation of an apoptotic morphology. These events are controlled by several HSPs at different levels of the apoptotic cascade and by X-linked inhibitor of apoptosis protein (XIAP), which negatively regulates caspase-3 and -9. AIF and Endo G translocate to the nucleus and trigger DNA fragmentation and chromatin condensation in a caspase-independent manner.

It is important to note that the two functions of Cyt c are located in different domains of the protein. Replacing the iron atom in the heme prosthetic group abolishes Cyt c-mediated electron transfer but does not affect its potential to mediate caspase activation. This means that the redox and pro-apoptotic activities of Cyt c follow different biochemical principles. Thus, Cyt c from Saccharomyces cerevisiae is able to replace its mammalian counterpart in the respiratory chain but does not induce the formation of the apoptotic body in mammalian cells.

In addition, Cyt c has been implicated in the differentiation of spermatids in Drosophila. The cyt-c-d gene (which is expressed at lower levels than the replacement gene cyt-c-p for the Drosophila Cyt c gene) does not appear to have an important role in mitochondrial respiration but is essential for sperm differentiation that depends on dendritic enzymes. Cyt-c-d mutant males are viable but sterile.

Cyt c-induced caspase activation may also play a role in B-cell proliferation. During B-cell cycle progression, caspase-3 is activated. Mice lacking caspase-3 have increased numbers of splenic B cells, normal apoptosis, but enhanced proliferation following mitogenic stimulation in vivo and in vitro. Thus, Cyt c release induces caspase activation, which in turn promotes important processes such as cell death or differentiation and proliferation.

References:

[1] VLADIMIR GOGVADZE; Boris Z; Sten Orrenius. Multiple pathways of cytochrome c release from mitochondria in apoptosis[J]. Biochimica et Biophysica Acta-Bioenergetics, 2006, 1757 5: Pages 639-647. DOI:10.1016/j.bbabio.2006.03.016.