Cytochrome c (Cytc) has both life-sustaining and cellular death-related functions, depending on subcellular localization. Within mitochondria, Cytc acts as a single electron carrier as part of the electron transport chain (ETC). When released into the cytosol after cellular insult, Cytc triggers the assembly of the apoptosome, committing the cell to intrinsic apoptosis. Due to these dual natures, Cytc requires strong regulation by the cell, including post-translational modifications, such as phosphorylation and acetylation. Six phosphorylation sites and three acetylation sites have been detected on Cytc in vivo. Phosphorylations at T28, S47, Y48, T49, T58, and Y97 tend to be present under basal conditions in a tissue-specific manner. In contrast, the acetylations at K8, K39, and K53 tend to be present in specific pathophysiological conditions. All of the phosphorylation sites and two of the three acetylation sites partially inhibit respiration, which we propose serves to maintain an optimal, intermediate mitochondrial membrane potential (ΔΨ_m) to minimize reactive oxygen species (ROS) production. Cytc phosphorylations are lost during ischemia, which drives ETC hyperactivity and ΔΨ_m hyperpolarization, resulting in exponential ROS production thus causing reperfusion injury following ischemia. One of the acetylation sites, K39, shows a unique behavior in that it is gained during ischemia, stimulating respiration while blocking apoptosis, demonstrating that skeletal muscle, which is particularly resilient to ischemia-reperfusion injury compared to other organs, possesses a different metabolic strategy to handle ischemic stress. The regulation of Cytc by these post-translational modifications underscores the importance of Cytc for the ETC, ΔΨ_m, ROS production, apoptosis, and the cell as a whole.

Mitochondria are essential to cellular function as they generate the majority of cellular ATP, mediated through oxidative phosphorylation, which couples proton pumping of the electron transport chain (ETC) to ATP production. The ETC generates an electrochemical gradient, known as the proton motive force, consisting of the mitochondrial membrane potential (ΔΨ_m, the major component in mammals) and ΔpH across the inner mitochondrial membrane. Both ATP production and reactive oxygen species (ROS) are linked to ΔΨ_m, and it has been shown that an imbalance in ΔΨ_m beyond the physiological optimal intermediate range results in excessive ROS production. The reaction of cytochrome c oxidase (COX) of the ETC with its small electron donor cytochrome c (Cytc) is the proposed rate-limiting step in mammals under physiological conditions. The rate at which this redox reaction occurs controls ΔΨ_m and thus ATP and ROS production. Multiple mechanisms are in place that regulate this reaction to meet the cell's energy demand and respond to acute stress. COX and Cytc have been shown to be regulated by all three main mechanisms, which we discuss in detail: allosteric regulation, tissue-specific isoforms, and post-translational modifications for which we provide a comprehensive catalog and discussion of their functional role with 55 and 50 identified phosphorylation and acetylation sites on COX, respectively. Disruption of these regulatory mechanisms has been found in several common human diseases, including stroke and myocardial infarction, inflammation including sepsis, and diabetes, where changes in COX or Cytc phosphorylation lead to mitochondrial dysfunction contributing to disease pathophysiology. Identification and subsequent targeting of the underlying signaling pathways holds clear promise for future interventions to improve human health. An example intervention is the recently discovered noninvasive COX-inhibitory infrared light therapy that holds promise to transform the current standard of clinical care in disease conditions where COX regulation has gone awry.

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the functional effects of K53 modification in vitro, K53 was mutated to acetylmimetic glutamine (K53Q), and to arginine (K53R) and isoleucine (K53I) as controls. Cytochrome c oxidase (COX) activity analyzed with purified Cytc variants showed reduced oxygen consumption with acetylmimetic Cytc compared to the non-acetylated Cytc (WT), supporting the Warburg effect. In contrast to WT, K53Q Cytc had significantly lower caspase-3 activity, suggesting that modification of Cytc K53 helps cancer cells evade apoptosis. Cardiolipin peroxidase activity, which is another proapoptotic function of the protein, was lower in acetylmimetic Cytc. Acetylmimetic Cytc also had a higher capacity to scavenge reactive oxygen species (ROS), another pro-survival feature. We discuss our experimental results in light of structural features of K53Q Cytc, which we crystallized at a resolution of 1.31 Å, together with molecular dynamics simulations. In conclusion, we propose that K53 acetylation of Cytc affects two hallmarks of cancer by regulating respiration and apoptosis in prostate cancer xenografts.

We previously reported that serine-47 (S47) phosphorylation of cytochrome c (Cytc) in the brain results in lower cytochrome c oxidase (COX) activity and caspase-3 activity in vitro. We here analyze the effect of S47 modification in fibroblast cell lines stably expressing S47E phosphomimetic Cytc, unphosphorylated WT, or S47A Cytc. Our results show that S47E Cytc results in partial inhibition of mitochondrial respiration corresponding with lower mitochondrial membrane potentials (ΔΨ_m) and reduced reactive oxygen species (ROS) production. When exposed to an oxygen-glucose deprivation/reoxygenation (OGD/R) model simulating ischemia/reperfusion injury, the Cytc S47E phosphomimetic cell line showed minimal ROS generation compared to the unphosphorylated WT Cytc cell line that generated high levels of ROS upon reoxygenation. Consequently, the S47E Cytc cell line also resulted in significantly lower cell death upon exposure to OGD/R, confirming the cytoprotective role of S47 phosphorylation of Cytc. S47E Cytc also resulted in lower cell death upon H₂O₂ treatment. Finally, we propose that pro-survival kinase Akt (protein kinase B) is a likely mediator of the S47 phosphorylation of Cytc in the brain. Akt inhibitor wortmannin abolished S47 phosphorylation of Cytc, while the Akt activator SC79 maintained S47 phosphorylation of Cytc. Overall, our results suggest that loss of S47 phosphorylation of Cytc during brain ischemia drives reperfusion injury through maximal electron transport chain flux, ΔΨ_m hyperpolarization, and ROS-triggered cell death.

Arsenic is a well-known human carcinogen associated with a number of cancers, including lung cancers. We have previously shown that long-term exposure to an environmentally relevant concentration of inorganic arsenic (As³⁺) leads to the malignant transformation of the BEAS2B cells, and some of the transformed cells show cancer stem-like features (CSCs) with a significant upregulation of glycolysis and downregulation of mitochondrial oxidative phosphorylation. In the present report, we investigate the short-term effect of As³⁺ on the endoplasmic reticulum (ER) stress response—the “unfolded protein response (UPR)” and metabolism in human bronchial epithelial cell line BEAS-2B cells. Treatment of the cells with inorganic As³⁺ upregulated both glycolysis and mitochondrial respiration. Analysis of ER UPR signaling pathway using a real-time human UPR array revealed that As³⁺ induced a significant up-regulation of some UPR genes, including ATF6, CEBPB, MAPK10, Hsp70, and UBE2G2. Additional tests confirmed that the induction of ATF6, ATF6B and UBE2G2 mRNAs and/or proteins by As³⁺ is dose dependent. Chromosome immunoprecipitation and global sequencing indicated a critical role of Nrf2 in mediating As³⁺-induced expression of these UPR genes. In summary, our data suggest that As³⁺ is able to regulate the ER stress response, possibly through activating the ATF6 signaling.

Cytochrome c (Cytc) is important for both mitochondrial respiration and apoptosis, both of which are altered in cancer cells that switch to Warburg metabolism and manage to evade apoptosis. We earlier reported that lysine 53 (K53) of Cytc is acetylated in prostate cancer. K53 is conserved in mammals that is known to be essential for binding to cytochrome c oxidase and apoptosis protease activating factor-1 (Apaf-1). Here we report the effects of this acetylation on the main functions of cytochrome c by expressing acetylmimetic K53Q in cytochrome c double knockout cells. Other cytochrome c variants analyzed were wild-type, K53R as a control that maintains the positive charge, and K53I, which is present in some non-mammalian species. Intact cells expressing K53Q cytochrome c showed 49% decreased mitochondrial respiration and a concomitant increase in glycolytic activity (Warburg effect). Furthermore, mitochondrial membrane potential was decreased, correlating with notably reduced basal mitochondrial superoxide levels and decreased cell death upon challenge with H₂O₂ or staurosporine. To test for markers of cancer aggressiveness and invasiveness, cells were grown in 3D spheroid culture. K53Q cytochrome c-expressing cells showed profoundly increased protrusions compared to WT, suggesting increased invasiveness. We propose that K53 acetylation of cytochrome c is an adaptive response that mediates prostate cancer metabolic reprogramming and evasion of apoptosis, which are two hallmarks of cancer, to better promote tumor survival and metastasis.