Redox biointeractome of cytochromes in respiration and photosynthesis

Protein complex formation is at least a two-step process in which the formation of a final, well-defined complex entails the initial formation of a dynamic encounter complex. Highly transient complexes, with lifetimes in the order of microseconds-milliseconds, exhibit moderate or low binding affinities, with dissociation constants in the micromolar-millimolar range. Electron transfer reactions mediated by soluble redox proteins exchanging electrons between large membrane complexes in respiration and photosynthesis are excellent examples of transient interactions. Here, experimental approaches based on diamagnetic and paramagnetic Nuclear Magnetic Resonance (NMR) spectroscopy and/or Isothermal Titration Calorimetry, combined with computational methods, have been used to study the molecular recognition processes of particular redox complexes involved in respiration and photosynthesis. The studies presented in this PhD thesis go into detail about the structural and biophysical basis of the following redox complexes: cytochrome c¿cytochrome c1, cytochrome c-cytochrome c oxidase and cytochrome c-galactonolactone dehydrogenase interactions in respiration and the cytochrome c6¿cytochrome f adduct in photosynthesis. All these ET ensembles exhibit proper coupling between the redox centers although they differ in their dynamic behavior, which can be ascribed to its distinct functionality depending on the organism and its biological context. Moreover, post-translational regulation can alter the usual mechanisms of such proteins. In this work, the structure determination of a phosphomimetic mutant of cytochrome c has also been addressed by means of solution NMR. Needless to say, such a multidisciplinary methodology, combining experimental and computational methods, opens new perspectives in our understanding of the dynamic, transient adducts formed between proteins beyond the model systems herein analyzed. The current document has been prepared by following the guidelines required by the University of Seville to submit the PhD thesis as a collection of journal papers and book chapters. It consists of the following sections: II. The list of Abbreviations. III. The list of Publications and merits of the PhD candidate. IV. A brief Summary of the PhD thesis. V. An Introduction and the state-of-the-art. VI. A section covering the Objectives of the PhD thesis. VII. A brief description of the Results and Discussion, including main achievements and outcomes. VIII. The global Conclusions. IX. The list of References which have been cited in previous sections. X. The Appendix I, containing the published journal papers and an accepted book chapter, on which the PhD thesis is based. All papers were published in scientific journals indexed in Journal Citation Reports (JCR) database. XI. The Appendix II, containing an additional manuscript in preparation focused on the structure and dynamics of a phosphomimetic variant of cytochrome c. XII. The Appendix III, including three datasets deposited in the Biological Magnetic Resonance Data Bank (BMRB) and the Protein Data Bank (PDB).Premio Extraordinario de Doctorado U

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

Assessing Urban Accessibility in Monterrey, Mexico: A Transferable Approach to Evaluate Access to Main Destinations at the Metropolitan and Local Levels

Cities demand urgent transformations in order to become more affordable, livable, sustainable, walkable and comfortable spaces. Hence, important changes have to be made in the way cities are understood, diagnosed and planned. The current paper puts urban accessibility into the centre of the public policy and planning agenda, as a transferable approach to transform cities into better living environments. To do so, a practical example of the City of Monterrey, Mexico, is presented at two planning scales: the metropolitan and local level. Both scales of analysis measure accessibility to main destinations using walking and cycling as the main transport modes. The results demonstrate that the levels of accessibility at the metropolitan level are divergent, depending on the desired destination, as well as on the planning processes (both formal and informal) from different areas of the city. At the local level, the Distrito Tec Area is diagnosed in terms of accessibility to assess to what extent it can be considered a part of a 15 minutes city. The results show that Distrito Tec lacks the desired parameters of accessibility to all destinations for being a 15 minutes city. Nevertheless, there is a considerable increase in accessibility levels when cycling is used as the main travelling mode. The current research project serves as an initial approach to understand the accessibility challenges of the city at different planning levels, by proving useful and disaggregated data. Finally, it concludes providing general recommendations to be considered in planning processes aimed to improve accessibility and sustainability

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

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

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. 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 c6 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 c6 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 c6 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. © 2014 Elsevier B.V.Peer Reviewe