Tissue-specific regulation of cytochrome c by post-translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis

Abstract

Cytochrome c (Cyt c) plays a vital role in the mitochondrial electron transport chain (ETC). In addition, it is a key regulator of apoptosis. Cyt c has multiple other functions including ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import. Cyt c is tightly regulated by allosteric mechanisms, tissue-specific isoforms, and post-translational modifications (PTMs). Distinct residues of Cyt c are modified by PTMs, primarily phosphorylations, in a highly tissue-specific manner. These modifications downregulate mitochondrial ETC flux and adjust the mitochondrial membrane potential (ΔΨ_m), to minimize reactive oxygen species (ROS) production under normal conditions. In pathologic and acute stress conditions, such as ischemia-reperfusion, phosphorylations are lost, leading to maximum ETC flux, ΔΨ_m hyperpolarization, excessive ROS generation, and the release of Cyt c. It is also the dephosphorylated form of the protein that leads to maximum caspase activation. We discuss the complex regulation of Cyt c and propose that it is a central regulatory step of the mammalian ETC that can be rate limiting in normal conditions. This regulation is important because it maintains optimal intermediate ΔΨ_m, limiting ROS generation. We examine the role of Cyt c PTMs, including phosphorylation, acetylation, methylation, nitration, nitrosylation, and sulfoxidation and consider their potential biological significance by evaluating their stoichiometry.-Kalpage, H. A., Bazylianska, V., Recanati, M. A., Fite, A., Liu, J., Wan, J., Mantena, N., Malek, M. H., Podgorski, I., Heath, E. I., Vaishnav, A., Edwards, B. F., Grossman, L. I., Sanderson, T. H., Lee, I., Hüttemann, M. Tissue-specific regulation of cytochrome c by post-translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis.

Keywords: electron transport chain; ischemia–reperfusion; phosphorylation; reactive oxygen species; signal transduction.

Figure 1

Pro-life (green arrows) and pro-death functions (red arrows) of Cytc.

Figure 2

Tissue-specific phosphorylation sites are located on the right side of Cytc, whereas cardiolipin and ATP binding sites are located on the left side. Crystallographic data from oxidized rat Cytc were used (76) and analyzed with the molecular-graphics, -modeling and -simulation program YASARA (http://www.yasara.org/). Cytc is shown in the conventional view. The heme group is shown in red, and amino acids known to be phosphorylated in a tissue-specific manner are highlighted in yellow, together with the organs in which they have been identified under baseline condition. Amino acids implicated in ATP binding are Lys88, Arg91, and Glu62 (150) and are shown in green. Residues involved in cardiolipin binding are Lys87, Lys86, Lys73, Lys72 (A site), and Asn52 (C site) (5) and are shown in magenta. A third site for electrostatic interaction of Cytc with phospholipids consists of Lys22, Lys25, Lys27, His26, and His33 (151).

Figure 3

Sequence alignment of Cytc with highlighted post-translational modifications identified in mammals in vivo. Top, somatic Cytc from selected mammalian species with matching sequences of the corresponding testes-specific isoforms (T) where available (bottom). Note that humans express only a single ubiquitous Cytc (middle), which shares sequence homology with the somatic and testis isoforms. Ac, acetylation; P, phosphorylation. Met80 of the heme group shown in red is likely involved in sulfoxidation and nitrosylation as a result of nitro-oxidative stress. UniProt (https://www.uniprot.org/) Protein sequence accession numbers were F7D4V9 (horse); P62894 (bull); P62895 (pig); A0A0C4Y1X3 (goat); P00011 (dog); P62897 (mouse); P62898 (rat); S7QBN9 (bat); I6VH08 (dolphin); P99999 (human); Q3SZT9 (bull testes); P00015 (mouse testes); and P10715 (rat testes).

Figure 4

A model of the connection of the phosphorylation state of Cytc in the regulation of electron flux in the ETC, ΔΨ_m, and ROS production. In normal conditions (middle) Cytc is phosphorylated, which lowers electron flux in the ETC, maintaining healthy intermediate ΔΨ_m levels, which are sufficient for effective ATP generation, but limit the production of ROS that are generated exponentially at ΔΨ_m levels exceeding 140 mV. During stress conditions, such as ischemia (left), the tissue becomes energy depleted because of the lack of oxygen, leading to dephosphorylation of Cytc and other mitochondrial proteins. This occurrence renders the ETC primed for hyperactivation. When the tissue is reperfused and oxygen re-enters the cells, the ETC generates high ΔΨ_m levels, leading to ROS production and initialization of cell death cascades, adding to the damage already generated during the ischemic phase.