The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis

Abstract

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.

Figure 1

Summary of the main functions of cytochrome c. The left side of the figure highlights its involvement in life-sustaining functions, whereas its involvement in cellular death functions is shown on the right side.

Figure 2

Stereo view of the cytochrome c structure in the conventional orientation. Crystallographic data from horse Cytc was used (Sanishvili et al., 1995) and processed with the program Swiss PDB viewer (version 3.7). Residues Thr28, Ser/Thr47, Tyr48, and Tyr97 that can be phosphorylated in mammals are highlighted in purple (Lee et al., 2006; Yu et al., 2008; Zhao et al., 2010). A-side residues Lys72, Lys73, and Lys87 (yellow ribbon) and C-side residue 52 (turquoise ribbon) proposed to be involved in phospho-lipid binding through electrostatic interaction with the negatively charged lipid head group (Kagan et al., 2009) are highlighted. Amino acids Glu69, Asn70, and Lys88 indicated in red ribbon form an ATP binding pocket together with C-side residues Lys72, Lys86, and Lys87 (yellow ribbon) (McIntosh et al., 1996). Key residues required for Apaf-1 binding and caspase activation are highlighted in blue sticks. Phosphorylation sites localize to the right side of the molecule whereas amino acids involved in binding of phospholipids and ATP are on the left side.

Figure 3. Cytochrome c alignment

Human (NM_018947), mouse somatic (CAA25899) and testis (NP_034119) cytochrome c show 81% sequence identity with individual comparisons revealing 91% (human-mouse somatic), 82% (human-mouse testis), and 86% (mouse somatic-mouse testis) conservation. Thr28, Ser47, Tyr48, and Tyr97 which can be phosphorylated in vivo are indicated with an arrow. Note that human Cytc contains an additional fifth tyrosine residue, Tyr46, which is immediately adjacent to the phospho-epitope containing Ser47 and Tyr48. Future work should explore if this residue is targeted for human-specific regulation of Cytc via phosphorylation.

Figure 4. Regulation of mitochondrial respiration, ROS production, and apoptosis through phosphorylation of cytohrome c

Our model proposes changes in the phosphorylation state of Cytc during cellular stress as a switch for the execution of the cell death program: Under healthy unstressed conditions Cytc is phosphorylated leading to partial inhibition of mitochondrial respiration and healthy mitochondrial membrane potentials ΔΨ_m in the range of 80-140 mV. These membrane potentials are ideal and sufficient to drive efficient ATP generation, and importantly, no significant amounts of ROS are produced. In addition, under healthy conditions Cytc functions as a ROS scavenger (not shown). Under stressed conditions, including the presence of apoptotic stimuli and excessive calcium, a phosphatase dephosphorylates Cytc, which has multiple consequences. First, maximal ETC flux is now possible resulting in membrane potentials >140 mV. This hyperpolarization triggers excessive ROS production at complexes I and III due to backup of electrons in the ETC and increased half-lives of the semiquinole free radicals. Second, it is the unphosphorylated form of Cytc which catalyzes the oxidation of cardiolipin, a step which is required to dissociate Cytc from the inner mitochondrial membrane and thus to release it from the mitochondria during apoptosis. Cardiolipin oxidation requires ROS (e.g., peroxide equivalents), which are provided by ΔΨ_m hyperpolarization-mediated ROS production. Third, p66^shc might also contribute to ROS production through the interaction with unphosphorylated Cytc. Finally, Cytc is released from the mitochondria, and it is the unphosphorylated form that is required for apoptosome formation, another safeguard mechanism for regulating this committing step as part of the death program. There is strong evidence that the other components of the OxPhos system are also regulated by phosphorylation, but Cytc appears to be the central player due to its dual active involvement in both respiration and apoptosis.