Using Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.

Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes

New approaches by Site-Directed Spin Labeling combined with Electronic Paramagnetic Resonance spectroscopy : application to the study of structural transitions in proteins

Cette thèse porte sur le développement de nouvelles approches par marquage de spin suivi par spectroscopie RPE. Cette technique est bien adaptée pour suivre la dynamique structurale des protéines. Son principe repose sur l'insertion d'un radical nitroxyde, en un (ou plusieurs) site(s) choisi(s) d'une protéine et permet de sonder localement la structure de la protéine étudiée grâce aux différentes techniques de RPE (en onde continue et impulsionnelle).Dans une première partie, cette technique a été appliquée à la caractérisation de la dynamique structurale de l'IF1 de levure, un peptide inhibiteur de l'ATP-synthase. L'utilisation des spectroscopies de RPE et de dichroïsme circulaire a permis de montrer qu'IF1 de levure dimérise par sa partie médiane et que la partie C-terminale est désordonnée.La seconde partie est plus méthodologique et a pour but d'étudier et de caractériser un marqueur nouvellement synthétisé afin d'élargir les potentialités du marquage de spin. En effet, cette technique est notamment limitée par la faible diversité spectrale offerte par les sondes disponibles (trois raies). Le nouveau marqueur donne un spectre RPE à six raies grâce à la présence d'un noyau magnétique dans l'environnement du radical. Greffé sur une protéine modèle, nous avons montré que ce nouveau marqueur est tout autant capable de rendre compte de variations structurales qu'un marqueur classique. La superposition des signatures spectrales (trois raies + six raies) montre qu'il est possible de différencier les deux signatures spectrales et de sonder simultanément deux sites d'une protéine et de son partenaire.This thesis focuses on the development of new approaches for site-directed spin labeling followed by EPR spectroscopy. This technique is well suited to monitor the structural dynamics of proteins. The insertion of a nitroxide radical, in one (or several) selected site(s) of a protein, allows probing the structure of the protein using different EPR spectroscopy approaches (continuous wave and pulsed).In a first part, this technique has been applied to characterize the structural dynamics of the yeast IF1, an inhibitory peptide of the ATP-synthase. Using EPR and circular dichroïsm spectroscopies we showed that yeast IF1 dimerizes by its central part and that the C-terminal part remains disordered.The second part is more methodological and the aim is to study and characterize a newly synthesized spin label in order to expand the potential of site-directed spin labeling. In particular, the technique is limited by the poor spectral diversity offered by the available labels (three lines). The new label gives a six lines EPR spectrum thanks to the presence of a magnetic nucleus in the environment of the radical. Grafted on a model protein, we demonstrated that this new label is as able as classical ones to report on structural variations. The superposition of the spectral signatures (three lines + six lines) showed that it is possible to differentiate the two spectral signatures and to probe two sites of a protein and its partner simultaneously

The Effect of the Linking Unit on the Electronic and Magnetic Interactions in Copper(II) Porphyrin Dimers Linked by Metal Ions

The syntheses of a series of copper(II) porphyrins and their dimers linked by palladium(II) or platinum(II) are reported. Their electronic properties and their magnetic properties were studied. In particular, the effect of the linking unit on these properties was evaluated. It was discovered that three factors influence the electronic and magnetic interactions between the two metalloporphyrins: the nature of the linking metal ion, the nature of the external coordination site of the porphyrin, and also the nature of the metal ion present in the central core of the aromatic macrocycle

Nitroxide spin labels : fabulous spy spins for biostructural EPR applications

International audienc

Exploring intrinsically disordered proteins using site-directed spin labeling electron paramagnetic resonance spectroscopy

International audienceProteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site-Directed Spin Labeling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events. SDSL-EPR is based on selective grafting of a paramagnetic label on the protein under study and is limited neither by the size nor by the complexity of the system. The objective of this mini-review is to describe the basic strategy of SDSL-EPR and to illustrate how it can be successfully applied to characterize the structural behavior of IDPs. Recent developments aimed at enlarging the panoply of SDSL-EPR approaches are presented in particular newly synthesized spin labels that allow the limitations of the classical ones to be overcome. The potentialities of these new spin labels will be demonstrated on different examples of IDP

Interdigitated conducting tetrathiafulvalene-based coordination networks

Assembly of a novel ethylenedithio-tetrathiafulvalene (EDT-TTF) derivative bearing two adjacent 4-thiopyridyl groups with M(NCS)2 nodes (M = Fe, Co) leads to two isostructural 1D coordination polymers showing an enhancement of their electronic conductivity by six orders of magnitude (10[superscript -6] vs. 10[superscript -12] S cm[superscript -1), upon surface oxidation by iodine and subsequent generation of EDT-TTF-based radicals.National Science Foundation (Award 1122374

Assessing the Extent of Potential Inversion by Cyclic Voltammetry: Theory, Pitfalls, and Application to a Nickel Complex with Redox-Active Iminosemiquinone Ligands

International audiencePotential inversion refers to the situation where a protein cofactor or a synthetic molecule can be oxidized or reduced twice in a cooperative manner, that is the second electron transfer (ET) is easier than the first. This property is very important regarding the catalytic mechanism of enzymes that bifurcate electrons and the properties of bidirectional redox molecular catalysts that function in either direction of the reaction with no overpotential. Cyclic voltammetry is the most common technique for characterizing the thermodynamics and kinetics of ET to or from these molecules. However, a gap in the literature is the absence of analytical predictions to help interpret the values of the voltammetric peak potentials when potential inversion occurs ; the cyclic voltammograms are therefore often analyzed by simulating the data, with no discussion of the possibility of overfitting and often no estimation of the error on the determined parameters. Here we formulate the theory for the voltammetry of freely-diffusing or surface-confined two-electron redox species in the experimentally relevant irreversible limit where the peak separation depends on scan rate. We explain why the model is intrinsically underdetermined, and we illustrate this conclusion by the analysis of the voltammetry of a Ni complex with redox-active iminosemiquinone ligands. Being able to characterize the thermodynamics of two-electron transfer reactions will be crucial for designing more efficient catalysts

Characterization of a novel picornavirus isolated from moribund gilthead seabream ( Sparus aurata ) larvae

International audienceGilthead seabream represents a species of importance in Mediterranean aquaculture. The larval stage is particularly sensitive and frequently impacted in suboptimal environmental or sanitary conditions. In the present study, investigations were carried out in a seabream hatchery following an unusual mortality reaching 70% among 50-day post-hatching. Anorexia, loss of appetite and abnormal swimming behaviour were observed in absence of parasites or pathogenic bacteria. Proliferation of rod-shaped bacteria in the gut lumen was associated with focal degeneration in the intestinal mucosa. Cytopathic effects on an EK-1 cell line after 21 days of culture at 14°C and 20°C in contact with homogenized affected larvae revealed the presence of a viral agent. Molecular characterization by high-throughput sequencing showed a typical picornavirus genome organization with a polyprotein precursor of 2276 amino acids sharing 46.3% identity with that of the Eel Picornavirus-1. A specific real-time PCR confirmed the presence of the viral genome in affected larval homogenate and corresponding cell culture supernatant. We propose the name Potamipivirus daurada for this novel species within the genus Potamipivirus. The etiological role of this virus remains uncertain at this time, and future studies will be necessary to investigate its prevalence in natural and aquaculture-reared populations as well as its ability to cause diseases in gilthead seabream