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It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation. Electron transfer between mitochondrial cytochrome c and subunit of cytochrome bc1 can proceed at long distance. Here the authors investigate further the mechanism and show phosphorylation regulation of the interactions between the protein partners in the electron transport chain. [ABSTRACT FROM AUTHOR]

Despite mitochondria being key for the control of cell homeostasis and fate, their role in DNA damage response is usually just regarded as an apoptotic trigger. However, growing evidence points to mitochondrial factors modulating nuclear functions. Remarkably, after DNA damage, cytochrome c (Cc) interacts in the cell nucleus with a variety of well‐known histone chaperones, whose activity is competitively inhibited by the haem protein. As nuclear Cc inhibits the nucleosome assembly/disassembly activity of histone chaperones, it might indeed affect chromatin dynamics and histone deposition on DNA. Several histone chaperones actually interact with Cc Lys residues through their acidic regions, which are also involved in heterotypic interactions leading to liquid–liquid phase transitions responsible for the assembly of nuclear condensates, including heterochromatin. This relies on dynamic histone–DNA interactions that can be modulated by acetylation of specific histone Lys residues. Thus, Cc may have a major regulatory role in DNA repair by fine‐tuning nucleosome assembly activity and likely nuclear condensate formation. [ABSTRACT FROM AUTHOR]

SUMMARY: Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra‐mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14‐3‐3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase‐like activity. This finding establishes the 14‐3‐3ι protein as a relevant factor at the onset of plant H2O2‐induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14‐3‐3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14‐3‐3ι and human 14‐3‐3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14‐3‐3ι so as to inhibit caspase‐like proteases, which in turn promote cell death upon H2O2 treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death. Significance Statement: Common features of the cytochrome c‐dependent pathways leading to programmed cell death in plants and humans are herein revealed. In response to oxidative stress, cytochrome c is released from mitochondria to the cytoplasm to hamper the iota isoform of the 14‐3‐3 protein family, thereby decreasing the inhibition of caspase‐like activity and likely contributing to promote cell death in plants. [ABSTRACT FROM AUTHOR]

Cytochrome c (Cc) is a protein that functions as an electron carrier in the mitochondrial respiratory chain. However, Cc has moonlighting roles outside mitochondria driving the transition of apoptotic cells from life to death. When living cells are damaged, Cc escapes its natural mitochondrial environment and, once in the cytosol, it binds other proteins to form a complex named the apoptosome—a platform that triggers caspase activation and further leads to controlled cell dismantlement. Early released Cc also binds to inositol 1,4,5‐triphosphate receptors on the ER membrane, which stimulates further massive Cc release from mitochondria. Besides the well‐characterized binding proteins contributing to the proapoptotic functions of Cc, many novel protein targets have been recently described. Among them, histone chaperones were identified as key partners of Cc following DNA breaks, indicating that Cc might modulate chromatin dynamics through competitive binding to histone chaperones. In this article, we review the ample set of recently discovered antiapoptotic proteins—involved in DNA damage, transcription, and energetic metabolism—reported to interact with Cc in the cytoplasm and even the nucleus upon DNA breaks. [ABSTRACT FROM AUTHOR]

Over the past decade, evidence has emerged suggesting a broader role for cytochrome c (Cyt c) in programmed cell death. Recently, we demonstrated the ability of Cyt c to inhibit the nucleosome assembly activity of histone chaperones SET/template‐activating factor Iβ and NAP1‐related protein during DNA damage in humans and plants respectively. Here, we hypothesise a dual concentration‐dependent function for nuclear Cyt c in response to DNA damage. We propose that low levels of highly cytotoxic DNA lesions – such as double‐strand breaks – induce nuclear translocation of Cyt c, leading to the attenuation of nucleosome assembly and, thereby, increasing the time available for DNA repair. If DNA damage persists or is exacerbated, the nuclear Cyt c concentration would exceed a given threshold, causing the haem protein to block DNA remodelling altogether. [ABSTRACT FROM AUTHOR]

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl- L-phenylalanine ( pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48 pCMF mutation significantly destabilizes the FeMet bond in the ferric form of cytochrome c, thereby lowering the p Ka value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation. [ABSTRACT FROM AUTHOR]

The transient interactions of respiratory cytochrome c with complexes III and IV is herein investigated by using heterologous proteins, namely human cytochrome c , the soluble domain of plant cytochrome c 1 and bovine cytochrome c oxidase. The binding molecular mechanisms of the resulting cross-complexes have been analyzed by Nuclear Magnetic Resonance and Isothermal Titration Calorimetry. Our data reveal that the two cytochrome c -involving adducts possess a 2:1 stoichiometry – that is, two cytochrome c molecules per adduct – at low ionic strength. We conclude that such extra binding sites at the surfaces of complexes III and IV can facilitate the turnover and sliding of cytochrome c molecules and, therefore, the electron transfer within respiratory supercomplexes. [ABSTRACT FROM AUTHOR]

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c 1 , namely a non-productive (or distal ) site with a long heme-to-heme distance and a functional (or proximal ) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c 1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a “floating boat bridge” of cytochrome c molecules (between complexes III and IV) in plant respirasome. [ABSTRACT FROM AUTHOR]

Abstract: Under nitroxidative stress, a minor fraction of cytochrome c can be modified by tyrosine nitration. Here we analyze the specific effect of nitration of tyrosines 46 and 48 on the dual role of cytochrome c in cell survival and cell death. Our findings reveal that nitration of these two solvent-exposed residues has a negligible effect on the rate of electron transfer from cytochrome c to cytochrome c oxidase, but impairs the ability of the heme protein to activate caspase-9 by assembling a non-functional apoptosome. It seems that cytochrome c nitration under cellular stress counteracts apoptosis in light of the small amount of modified protein. We conclude that other changes such as increased peroxidase activity prevail and allow the execution of apoptosis. [Copyright &y& Elsevier]

Cytochrome c (C c) is a key protein in cell life (respiration) and cell death (apoptosis). On the one hand, it serves as a mitochondrial redox carrier, transferring electrons between the membrane-embedded complexes III and IV. On the other hand, it acts as a cytoplasmic apoptosis-triggering agent, forming the apoptosome with apoptosis protease-activating factor-1 (Apaf-1) and activating the caspase cascade. The two functions of cytochrome c are finely tuned by the phosphorylation of tyrosines and, in particular, those located at positions 48 and 97. However, the specific cytochrome c-phosphorylating kinase is still unknown. To study the structural and functional changes induced by tyrosine phosphorylation in cytochrome c, we studied the two phosphomimetic mutants Y48E and Y97E, in which each tyrosine residue is replaced by glutamate. Such substitutions alter both the physicochemical features and the function of each mutant compared with the native protein. Y97E is significantly less stable than the WT species, whereas Y48E not only exhibits lower values for the alkaline transition p K and the midpoint redox potential, but it also impairs Apaf-1-mediated caspase activation. Altogether, these findings suggest that the specific phosphorylation of Tyr48 makes cytochrome c act as an anti-apoptotic switch. [ABSTRACT FROM AUTHOR]

• Respiratory chain organization is highly dynamic and adapts to cellular needs. • Supercomplex formation facilitates cytochrome c 2D sliding from complex III to IV. • Physical contact contributes to redox potential gap and electron transfer. • Long-distance electron transfer allows a faster turnover of cytochrome c. The supramolecular arrangement of respiratory complexes into supercomplexes is widely accepted. The common feature observed in the supercomplex architecture from organisms of diverse phylogenetic origin is the reduction of the distance between cytochrome bc 1 (complex III) and cytochrome c oxidase (complex IV). Such an arrangement reduces the dimensionality (from 3D to 2D) of the diffusional search of cytochrome c as the electron carrier that connects both complexes. In this scenario, our recent finding of additional binding sites for cytochrome c reinforces the concept of a "restrained 2D sliding pathway" onto the supercomplex surface. Herein, we analyze novel mechanistic insights into electron transfer towards cytochrome c , including modulation of the redox potential by physical contact as well as gated, long-range electron transfer through an aqueous solution. These data establish a new horizon for the understanding of electron transfer mechanisms beyond unique and well-orientated protein complexes. Multiple and dynamic long-distance conformational ensembles compatible with electron transfer could indeed contribute to the rapid adjustment of electron flow in response to changing cellular conditions. [ABSTRACT FROM AUTHOR]

Mitochondria are the powerhouses of the cell, whilst their malfunction is related to several human pathologies, including neurodegenerative diseases, cardiovascular diseases, and various types of cancer. In mitochondrial metabolism, cytochrome c is a small soluble heme protein that acts as an essential redox carrier in the respiratory electron transport chain. However, cytochrome c is likewise an essential protein in the cytoplasm acting as an activator of programmed cell death. Such a dual role of cytochrome c in cell life and death is indeed fine-regulated by a wide variety of protein post-translational modifications. In this work, we show how these modifications can alter cytochrome c structure and functionality, thus emerging as a control mechanism of cell metabolism but also as a key element in development and prevention of pathologies. [ABSTRACT FROM AUTHOR]

Abstract: The Reactive Nitrogen and Oxygen Species (the so-called RNOS), which are well-known radicals formed in the mitochondria under nitro-oxidative cell stress, are responsible for nitration of tyrosines in a wide variety of proteins and, in particular, in cytochrome c (Cc). Only three out of the five tyrosine residues of human Cc, namely those at positions 67, 74 and 97, have been detected in vivo as nitrotyrosines. However, nitration of the two other tyrosines, namely those at positions 46 and 48, has never been detected in vivo despite they are both well-exposed to solvent. Here we investigate the changes in heme coordination and alkaline transition, along with the peroxidase activity and in cell degradation of Cc mutants in which all their tyrosine residues – with the only exception of that at position 46 or 48 – are replaced by phenylalanines. In Jurkat cell extracts devoid of proteases inhibitors, only the high-spin iron nitrated forms of these monotyrosine mutants are degraded. Altogether the resulting data suggest that nitration of tyrosines 46 and 48 makes Cc easily degradable upon turning the heme iron state to high-spin. [Copyright &y& Elsevier]

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c , in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c – namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death – is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c 1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death. [ABSTRACT FROM AUTHOR]

Abstract: The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b 6 f with the electron transfer proteins plastocyanin and cytochrome c 6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c 6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c 6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c 6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process. [Copyright &y& Elsevier]

Abstract: Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Human cytochrome c, with five tyrosine residues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mitochondrial respiratory electron transport chain, and on the other hand, it serves as a cytoplasmic apoptosis-triggering agent. In a previous paper, we reported the effect of nitration on physicochemical and kinetic features of monotyrosine cytochrome c mutants. Here, we analyse the nitration-induced changes in secondary structure, thermal stability, haem environment, alkaline transition and molecular dynamics of three of such monotyrosine mutants – the so-called h-Y67, h-Y74 and h-Y97 – which have four tyrosines replaced by phenylalanines and just keep the tyrosine residue giving its number to the mutant. The resulting data, along with the functional analyses of the three mutants, indicate that it is the specific nitration of solvent-exposed Tyr74 which enhances the peroxidase activity and blocks the ability of Cc to activate caspase-9, thereby preventing the apoptosis signaling pathway. [Copyright &y& Elsevier]

In oxidative phosphorylation, the transfer of electrons from reduced cofactors to molecular oxygen via the electron transport chain (ETC) sustains the electrochemical transmembrane potential needed for ATP synthesis. A key component of the ETC is complex III (CIII, cytochrome bc 1), which transfers electrons from reduced ubiquinone to soluble cytochrome c (C c) coupled to proton translocation into the mitochondrial intermembrane space. One electron from every two donated by hydroquinone at site P is transferred to C c via the Rieske-cytochrome c 1 (C c 1) pathway. According to recent structural analyses of CIII and its transitory complex with C c, the interaction between the Rieske subunit and C c 1 switches intermittently during CIII activity. However, the electrochemical properties of C c 1 and their function as a wire between Rieske and C c are rather unexplored. Here, temperature variable cyclic voltammetry provides novel data on the thermodynamics and kinetics of interfacial electron transfer of immobilized C c 1. Findings reveal that C c 1 displays two channels for electron exchange, with a remarkably fast heterogeneous electron transfer rate. Furthermore, the electrochemical properties are strongly modulated by the binding mode of the protein. Additionally, we show that electron transfer from C c 1 to C c is thermodynamically favored in the immobilized C c 1 -C c complex. Nuclear Magnetic Resonance, HADDOCK, and Surface Plasmon Resonance experiments provide further structural and functional data of the C c 1 -C c complex. Our data supports the Rieske-C c 1 -C c pathway acting as a unilateral switch thyristor in which redox potential modulation through protein-protein contacts are complemented with the relay-like Rieske behavior. • C c 1 redox potential is modulated by binding to its physiological protein partners. • Directional electron transfer from C c 1 to C c is thermodynamically favored within the C c 1 -C c complex. • The Rieske-C c 1 -C c electron transfer pathway functions as a unilateral switch thyristor. [ABSTRACT FROM AUTHOR]