Oxidative stress is tightly regulated by cytochrome c phosphorylation and respirasome factors in mitochondria

Respiratory cytochrome c has been found to be phosphorylated at tyrosine 97 in the postischemic brain upon neuroprotective insulin treatment, but how such posttranslational modification affects mitochondrial metabolism is unclear. Here, we report the structural features and functional behavior of a phosphomimetic cytochrome c mutant, which was generated by site-specific incorporation at position 97 of p-carboxymethyl-l-phenylalanine using the evolved tRNA synthetase method. We found that the point mutation does not alter the overall folding and heme environment of cytochrome c, but significantly affects the entire oxidative phosphorylation process. In fact, the electron donation rate of the mutant heme protein to cytochrome c oxidase, or complex IV, within respiratory supercomplexes was higher than that of the wild-type species, in agreement with the observed decrease in reactive oxygen species production. Direct contact of cytochrome c with the respiratory supercomplex factor HIGD1A (hypoxia-inducible domain family member 1A) is reported here, with the mutant heme protein exhibiting a lower affinity than the wild-type species. Interestingly, phosphomimetic cytochrome c also exhibited a lower caspase-3 activation activity. Altogether, these findings yield a better understanding of the molecular basis for mitochondrial metabolism in acute diseases, such as brain ischemia, and thus could allow the use of phosphomimetic cytochrome c as a neuroprotector with therapeutic applications.España, Junta de Andalucía BIO-198España MINECO BFU2015-71017/BM

Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c

Higher-order plants and mammals use similar mechanisms to repair and tolerate oxidative DNA damage. Most studies on the DNA repair process have focused on yeast and mammals, in which histone chaperone-mediated nucleosome disassembly/reassembly is essential for DNA to be accessible to repair machinery. However, little is known about the specific role and modulation of histone chaperones in the context of DNA damage in plants. Here, the histone chaperone NRP1, which is closely related to human SET/TAF-Iβ, was found to exhibit nucleosome assembly activity in vitro and to accumulate in the chromatin of Arabidopsis thaliana after DNA breaks. In addition, this work establishes that NRP1 binds to cytochrome c, thereby preventing the former from binding to histones. Since NRP1 interacts with cytochrome c at its earmuff domain, that is, its histone-binding domain, cytochrome c thus competes with core histones and hampers the activity of NRP1 as a histone chaperone. Altogether, the results obtained indicate that the underlying molecular mechanisms in nucleosome disassembly/reassembly are highly conserved throughout evolution, as inferred from the similar inhibition of plant NRP1 and human SET/TAF-Iβ by cytochrome c during DNA damage response.Junta de Andalucía BIO198Ministerio de Economía y Competitividad BFU2015-71017 / BMC y BFU2013-4706

Respiratory complexes III and IV can each bind two molecules of cytochrome c at low ionic strength

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 c1 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.España, MINECO Grant Nos. BFU2010-19451/BMC and BFU2012-31670/BMCJunta de Andalucía Grant PAI, BIO198España Ministerio de Educación, y European Social Fund-ERDF AP2009-409

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methylL-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.España, MINECO BFU2015-71017-P/BMC and BFU2015- 19451/BMCUnión Europea, Bio-NMR-00130 and CALIPSO-312284España, Ministerio de Educación AP2009-409

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-L-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.Financial support was provided by the Spanish Ministry of Economy and Competitiveness (Grants BFU2015-71017-P/BMC and BFU2015-19451/BMC, cofounded by FEDER EU), European Union (Bio-MR-00130 and CALIPSO-312284), Ramon Areces Foundation, and Andalusian Government (BIO198). B.M.-B. was awarded a PhD fellowship from the Spanish Ministry of Education (AP2009-4092) and a short-term traveling fellowship from the European Bio-NMR Project. A.G.-C. was awarded a PhD fellowship from the CSIC (JaePre-2011-01248).Peer reviewe

Mitochondrial Factors in the Cell Nucleus

The origin of eukaryotic organisms involved the integration of mitochondria into the ancestor cell, with a massive gene transfer from the original proteobacterium to the host nucleus. Thus, mitochondrial performance relies on a mosaic of nuclear gene products from a variety of genomes. The concerted regulation of their synthesis is necessary for metabolic housekeeping and stress response. This governance involves crosstalk between mitochondrial, cytoplasmic, and nuclear factors. While anterograde and retrograde regulation preserve mitochondrial homeostasis, the mitochondria can modulate a wide set of nuclear genes in response to an extensive variety of conditions, whose response mechanisms often merge. In this review, we summarise how mitochondrial metabolites and proteins—encoded either in the nucleus or in the organelle—target the cell nucleus and exert different actions modulating gene expression and the chromatin state, or even causing DNA fragmentation in response to common stress conditions, such as hypoxia, oxidative stress, unfolded protein stress, and DNA damage.Junta de Andalucía P18-FR-348

A common signalosome for programmed cell death in humans and plants

Peer reviewe

Cytochrome c1 exhibits two binding sites for cytochrome c in plants

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 c1, 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 c1 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. © 2014 Elsevier B.V.Peer Reviewe

Crystal structure of human nucleophosmin-core in complex with cytochrome c

Dataset generated during the current study is available from the PDB accession code 5EHD.Peer reviewe

New Arabidopsis thaliana cytochrome c partners: A look into the elusive role of cytochrome c in programmed cell death in plants

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets upon programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280Peer reviewe