Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation

Abstract

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.

Fig. 1

Mapped phosphorylation sites on cytochrome c and cytochrome c oxidase. Crystal structure data of horse heart cytochrome c [119] and cow heart COX [12] were used and processed with the program Swiss-PDBViewer 3.7. Identified phosphorylated amino acids in mammals are shown in sticks. See Table 1 for a detailed description of the sites including phospho-epitopes and references. Note that Thr52 in rabbit corresponds to Ser52 in cow COX subunit IV.

Fig. 2

Nano-LC-ESI-MS/MS analysis of Blue Native-PAGE-separated, BrCN and trypsin cleaved COX subunit II isolated from cow heart. Prior to analysis by tandem mass spectrometry (MS/MS) the phosphopeptides were enriched with titanium dioxide as described [54, 120]. The MS/MS spectrum unambiguously identifies the peptide IPTpSELKPGELR. The localization of Ser126 as the site of phosphorylation was possible after fragmentation of the doubly charged precursor ion (710.5 Th) with collision-induced dissociation (CID) and the triply charged ion (474.3 Th) with electron transfer dissociation (ETD). A, the CID-MS/MS spectrum contains a CID-typical neutral loss of phosphoric acid from the phosphoserine of the precursor ion. The y₁₀ and b₃ fragment ions as well as the neutral loss ions y₉ and b₅ (both -H₃PO₄) indicate phosphoserine. B, the ETD fragmentation resulted in a spectrum specifying the peptide amino acid sequence including the phosphosite-specific C-terminal z₈ and z₉ ions and the corresponding N-terminal c₃ and c₄ ions. Furthermore, on the basis of the fragmentation pattern phosphorylation of Thr125 was excluded by the presence of ions c₂, z₉ and z₁₀.

Fig. 3

Proposed model linking COX activity with the mitochondrial membrane potential, ATP and ROS production under healthy conditions and in two distinct pathological states. Under healthy conditions (middle) COX and Cytc are phosphorylated leading to a partial inhibition of respiration (Cytc is not shown). This generates healthy mitochondrial membrane potentials ΔΨ_m of about 120 mV, sufficient for efficient ATP generation but too low for ROS production. Two distinct pathological conditions are shown, both of which can lead to cell death: 1) acute inflammatory signaling via TNFα causes COX subunit I Tyr304 phosphorylation (pY304), leading to strong COX inhibition, decreased ΔΨ_m levels, and eventually energy depletion. In patients with sepsis this model would explain organ failure and death through energy failure; 2) the opposite mode of action takes place during ischemia/reperfusion. During ischemia, nutrients and oxygen become depleted. This stress causes excessive calcium release leading to changes in the phosphorylation pattern and/or dephosphorylation of Cytc and COX. The electron transport chain is now ‘primed’ for hyperactivity. During reperfusion, in the presence of oxygen and metabolites, OxPhos resumes to rebuild ΔΨ_m and ATP. Since the ETC complexes are in a hyperactive state, flux is maximal, which leads to a hyperpolarization of ΔΨ_m, reaching levels at which excessive ROS are generated. ROS cause extensive damage to the cell and trigger apoptosis.