Phosphorylation of cytochrome C: functional and structural features

Post-translational modifications often modulate protein function. Actually, phosphorylation of cytochrome c occurs in vivo at threonine 28, serine 47 and tyrosines 48 and 97. Phosphorylation of the two latters, in particular, is related to a wide range of human diseases as cytochrome c plays a pleiotropic role serving as an electron carrier in the respiratory electron transfer and acting as a cell death signal at the onset of apoptosis. The effect that phosphorylation of threonine 28 and serine 47 bears on the physiological functions of this protein remains concealed. The low yield of phosphorylated cytochrome c purification from cell extracts makes its analysis challenging. Also the specific kinases acting on the protein remain unknown. Hence, it has resorted to mutations to mimic targeted phosphorylation. Here, we have replaced threonine 28 and serine 47 by aspartate. And the analysis of tyrosine 48 and 97 phosphorylation has been performed by using the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF), which is a close phosphorylation mimic of tyrosine. Noteworthy, the Y48pCMF mutation significantly lowers the value for the alkaline transition pKa of oxidized cytochrome c. The negative charges at positions 28 and 48 cause a decrease in the midpoint redox potential value of 30 mV and 60 mV, respectively, and lower the affinity towards the distal site of cytochrome c1 in complex III. However, the phosphomimic variants at position 28, 47 and 48 are more efficient as electron donors to cytochrome c oxidase than the wild-type species. Concerning the role of cytochrome c in programmed cell death, negative charges at positions 48 and 97 hinder its ability to triger caspase-3 activation. In addition, any modification of residue 47 affects the pro-apoptotic function of cytochrome c. In summary, phosphorylation of cytochrome c modulates its distinct functions depending on the targeted residue, and can thus be the basis to understand an ample set of molecular diseases.Premio Extraordinario de Doctorado U

Cytochrome c: Surfing Off of the Mitochondrial Membrane on the Tops of Complexes III and IV

The proper arrangement of protein components within the respiratory electron transport chain is nowadays a matter of intense debate, since altering it leads to cell aging and other related pathologies. Here, we discuss three current views-the so-called solid, fluid and plasticity models-which describe the organization of the main membrane-embedded mitochondrial protein complexes and the key elements that regulate and/or facilitate supercomplex assembly. The soluble electron carrier cytochrome c has recently emerged as an essential factor in the assembly and function of respiratory supercomplexes. In fact, a 'restricted diffusion pathway' mechanism for electron transfer between complexes III and IV has been proposed based on the secondary, distal binding sites for cytochrome c at its two membrane partners recently discovered. This channeling pathway facilitates the surfing of cytochrome c on both respiratory complexes, thereby tuning the efficiency of oxidative phosphorylation and diminishing the production of reactive oxygen species. The well-documented post-translational modifications of cytochrome c could further contribute to the rapid adjustment of electron flow in response to changing cellular conditions.Spanish Ministry of Economy and Competitiveness (BFU2015-71017/BMC MINECO/FEDER and PGC2018-096049-B-I00 BIO/BMC MICINN/FEDER, EU

Long distance electron transfer through the aqueous solution between redox partner proteins

Despite the importance of electron transfer between redox proteins in photosynthesis and respiration, the inter-protein electron transfer rate between redox partner proteins has never been measured as a function of their separation in aqueous solution. Here, we use electrochemical tunneling spectroscopy to show that the current between two protein partners decays along more than 10 nm in the solution. Molecular dynamics simulations reveal a reduced ionic density and extended electric field in the volume confined between the proteins. The distance-decay factor and the calculated local barrier for electron transfer are regulated by the electrochemical potential applied to the proteins. Redox partners could use electrochemically gated, long distance electron transfer through the solution in order to conciliate high specificity with weak binding, thus keeping high turnover rates in the crowded environment of cells.España, MINECO BFU2015-71017-P/España, Gobierno de Andalucía (BIO198)Comisión de Universidades e Investigación del Departamento de Innovación, Universidades y Empresa de la Generalitat de Catalunya.(2017 SGR 1442, 2014 SGR 1442, and 2017 SGR 1189

Colombia: estación biogeográfica en el origen de su diversidad biótica

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Incursionando en el mundo de la investigación: Orientaciones básicas

Este libro pluridisciplinario coliga la teoría con la metodología de investigación. El contenido examina de modo recíproco en dónde se investiga, la hipótesis, los conceptos, el principio de búsqueda y los enfoques cuantitativo y cualitativo. El texto busca ofrecer de manera didáctica conocimientos de investigación que presenta pedagógicamente, partiendo de la situación problemática y siguiendo con el planteamiento del problema, los objetivos, las consultas de pesquisa y la justificación del estudio. Así mismo, se presenta la configuración del marco teórico, la revisión de la literatura y la construcción de una perspectiva teórica. Un libro indispensable para docentes y estudiantes interesados en la investigación y generación de conocimientos sobre la realidad social. Este libro es parte de ese esfuerzo que como investigadores sentimos la necesidad de ofrecer. En él presentamos las herramientas y los saberes suficientes que, gracias a la investigación y el conocimiento científicos, se pueden generar cotidianamente, con los cuales es posible tratar de construir una mejor calidad de vida y darle solución a muchos de los problemas que enfrenta la humanidad

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

Assessment of Exercise Stroke Volume and Its Prediction From Oxygen Pulse in Paralympic Athletes With Locomotor Impairments: Cardiac Long-Term Adaptations Are Possible

The determinants of cardiac output (CO) during exercise, i.e., stroke volume (SV) and heart rate (HR), could differ in Paralympic athletes (PAthl) with spinal cord injury (SCI) with respect to PAthl with locomotor impairments caused by different health conditions (HCs). The purposes of the present study were the comparisons of two groups of PAthl, one with SCI and the other with either amputation (AMP) or post poliomyelitis syndrome (PM), assessing the (1) peak cardiorespiratory responses and determinants (SV and HR) of CO during maximal and submaximal arm cranking exercise (ACE), respectively; (2) correlations between peak oxygen uptake (VO2peak) and the highest SV obtained during submaximal exercise; and (3) correlations between oxygen pulse (O-2 pulse, ratio between VO2 and HR) and both SV and O-2 arterio-venous difference [(a-v)O(2)diff]. Each athlete (19 PAthl with SCI, 9 with AMP, and 5 with PM) completed a continuous incremental cardiopulmonary ACE test to volitional fatigue to assess peak responses. In a different session, CO was indirectly measured through carbon dioxide (CO2) rebreathing method at sub-maximal exercise intensities approximating 30, 50, and 70% of the VO2peak. There were no significant differences between the PAthl groups in age, anthropometry, and VO2peak. However, peak HR was significantly lower, and peak O-2 pulse was significantly higher in PAthl with AMP/PM compared to those with SCI. During sub-maximal exercise, PAthl with AMP/PM displayed significantly higher SV values (154.8 +/- 17.60 ml) than PAthl with SCI (117.1 +/- 24.66 ml). SV correlated significantly with VO2peak in both PAthl with SCI (R-2 = 0.796) and AMP/PM (R-2 = 0.824). O-2 pulse correlated significantly with SV in both PAthl with SCI (R-2 = 0.888) and AMP/PM (R-2 = 0.932) and in the overall sample (R-2 = 0.896). No significant correlations were observed between O-2 pulse and (a-v)O(2)diff. It was concluded that in PAthl with different HCs: (1) significant differences, as a consequence of the different HC, exist in the determinants of CO at maximal and submaximal ACE; (2) SV is a significant determinant of VO2peak, suggesting cardiac adaptations possible also in PAthl with SCI; and (3) SV can be predicted from O-2 pulse measurements during submaximal exercise in both groups of PAthl

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

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