Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47

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 c1 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.Peer reviewe

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iß by cytochrome c

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key re- gulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chap- erone SET/TAF-Iß interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iß to core histones, thereby locking its histone-binding domains and inhibiting its nucle- osome assembly activity. In addition, we have used NMR spectros- copy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iß. Overall, these findings estab- lish a framework for understanding the molecular basis of cyto- chrome c-mediated blocking of SET/TAF-Iß, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iß’s histone chaperone activity

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

A republican command post, a dolmen and a mexican painter in Azutan, Toledo

La excavación arqueológica de una estructura de la guerra civil española no deja indiferente a nadie. Muchos son los datos que nos aporta para conocer mejor aspectos de esta época que están poco estudiados, como, por ejemplo, la vida cotidiana en el frente o el papel que juegan determinadas posiciones militares dentro de amplios territorios a cubrir. Además, resulta que el puesto de mando estudiado en Azután (Toledo) está relacionado con el dolmen ubicado en las proximidades y que fue excavado durante 1981 por la catedrática Primitiva Bueno y el doctor Juan Pereira. A esto hay que sumar que las tropas desplegadas en esta zona durante el conflicto estuvieron comandadas por un exitoso y polémico pintor mexicano apodado “El Coronalazo”. Por otro lado, se ha añadido, de una forma que creemos interesante, una extensión de las nuevas tecnologías a diferentes etapas del proceso de trabajo, de modo que se contó desde con documentación 3D en la excavación hasta reconstrucciones virtuales de apoyo para planteamientos de hipótesis y divulgación.Everyone become impresive because of an Archaeology dig of Spanish Civil War. The information we have due to this are so necessary to study some facts very unkwons, for exemple, daily life or the significance of a military position between longs zones to take care. In addition, the post studied in Azutan (Toledo) is related with the dolmen, localizated near, which was digged out in 1981 by the professor Primitiva Bueno and the doctor Juan Pereira. During the war, troops spread out in this zone were commanded by a successful and polemical mexican painter, called “El Coronalazo”. In the other hand, new technologys have been used during the works, because we think it is more interesant to improve the information we get, since 3D documentation to virtual reconstruction to do stronger our hypothesis and to disseminate the results

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

The cytochrome f–plastocyanin complex as a model to study transient interactions between redox proteins

AbstractTransient complexes, with a lifetime ranging between microseconds and seconds, are essential for biochemical reactions requiring a fast turnover. That is the case of the interactions between proteins engaged in electron transfer reactions, which are involved in relevant physiological processes such as respiration and photosynthesis. In the latter, the copper protein plastocyanin acts as a soluble carrier transferring electrons between the two membrane-embedded complexes cytochrome b6f and photosystem I. Here we review the combination of experimental efforts in the literature to unveil the functional and structural features of the complex between cytochrome f and plastocyanin, which have widely been used as a suitable model for analyzing transient redox interactions

The atypical iron-coordination geometry of cytochrome f remains unchanged upon binding to plastocyanin, as inferred by XAS

The transient complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by X-ray Absorption Spectroscopy in solution, using both proteins in their oxidized and reduced states. Fe K-edge data mainly shows that the atypical metal coordination geometry of cytochrome f, in which the N-terminal amino acid acts as an axial ligand of the heme group, remains unaltered upon binding to its redox partner, plastocyanin. This fact suggests that cytochrome f provides a stable binding site for plastocyanin and minimizes the reorganization energy required in the transient complex formation, which could facilitate the electron transfer between the two redox partners

Antimalarial activity of cupredoxins: the interaction of Plasmodium Merozoite Surface Protein 119 (MSP119) and Rusticyanin

Background: The interaction of MSP119 with the cupredoxin azurin inhibits the growth of Plasmodium falciparum in red blood cells. Results: Rusticyanin forms a well-defined complex with MSP119 upon binding at the same surface area than inhibitory antibodies. Conclusion: Rusticyanin becomes an excellent therapeutic agent for malaria. Significance: Knowing the rusticyanin- MSP119 interface will allow the design of novel anti-malarial drugsJunta de Andalucía P08-CVI-3876, BIO198Ministerio de Economía y Competitividad SAF2011- 26611Fundación Séneca de la Región de Murcia 15354/PI/10Ministerio de Ciencia e Innovación BFU2010-19451Medical Research Council U117574558, U11753206