Endohedral fullerenes are closed carbon shells encapsulating molecular or ionic species in their inner space. Obtained for the first time in 1985, endohedral metallofullerenes (EMFs) remain in focus of research for many years with a broad variety of metal atoms, endohedral cluster and cage sizes being reported. Electrochemical studies of endohedral metallofullerenes are of particular interest because of the more complex redox behavior in comparison to empty fullerenes. The EMF molecules can be considered as a combinations of positively charged cluster and negatively charged carbon shell “ligand”, and both constituents can be redox active. A cage-based electrochemical activity is more common, in particular, the most abundant nitride clusterfullerenes generally have redox-active cages. Cluster-based electrochemical activity is less common and can be revealed via unexpected redox behavior (e.g., shifted potential when compared to analogous molecules, potential metal dependence) and with the use of spectroscopic methods. Here we report electrochemical and EPR studies of three EMF families: (i) M2@C82-C3v and M2@C82-Cs dimetallofullerenes with a covalent bonding between two metal atoms, (ii) M2@C80(CH2Ph) dimetallofullerene derivatives with single-occupied metal-bonding orbital, and (iii) M2TiC@C80 EMFs with endohedral Ti(IV) (M is either Sc or Y or a lanthanide). For the first two families, the metal-metal bonding orbital has been found to be redox active: in M2@C82, the double-occupied M-M bonding orbital is involved in the first oxidation process, while in M2@C80(CH2Ph) the unoccupied component of single-occupied metal-bonding orbital acts as the LUMO, accepting one electron during the first reduction step. Thus, single electron transfer reactions in both cases lead to the changes in the magnetic properties of EMFs, which is especially well revealed by EPR spectroscopy. For the series of M2TiC@C80 EMFs, the first reduction predominantly occurs on internal Ti atom and can be described as TiIV/TiIII redox process. Due to the variation of the size of the Ti ion in different oxidation states, reduction changes the inner strain of the cluster, leading to a large variability of the TiIV/TiIII reduction potential in dependence on the size of the formally inert lanthanide metal in M2TiC@C80.

L’ACC Oxydase est une enzyme à Fe(II) non-hémique impliquée dans la biosynthèse de l’éthylène chez les plantes. Notre compréhension du mécanisme ainsi le rôle des différents cofacteurs nécessite l’obtention des données structurales. Une structure cristallographique a été publiée montrant la partie C-terminale (C-term) éloignée du site actif. Ce n’est pas la conformation active car la partie C-term est essentielle à l’activité. Un modèle structural a été construit dans lequel la partie C-term est tournée vers le site actif. Différentes conformations semblent donc possibles. Le marquage de spin couplé à la spectroscopie RPE est une technique puissante pour sonder la dynamique structurale des protéines. Elle implique la liaison de nitroxydes sur des cystéines. Il est possible d’analyser la mobilité des sondes pour obtenir des informations sur leur environnement local. Par l’utilisation de techniques de RPE avancées, des mesures de distances entre deux sondes sont possibles. Des mutants portant une ou deux cystéines ont été conçus. La dynamique des mutants marqués a été étudiée in vitro par RPE. Par RPE impulsionnelle, des distances ont été mesurées pour l’ACCO en présence de différentes combinaisons de cofacteurs. Les distances expérimentales ont été comparées à celles prédites à partir des structures cristallographiques et du modèle structural et aussi à celles obtenues par des calculs de dynamique moléculaire. Pour cibler d’autres positions sur l’ACCO, l’introduction d’un acide aminé non naturel a été réalisée avec succès permettant d’obtenir de premières données structurales. Des données structurales préliminaires par RPE in cell sont également présentées / ACC Oxidase is a nonheme iron(II) containing enzyme involved in the biosynthesis of ethylene in plants. ACCO reaction mechanism and the role of the various cofactors are not well understood and structural and dynamic data are still required. A crystallographic structure has been reported showing the C-terminal part (C-term) away from the active site. This is not the active conformation as it has been shown that the C-term is essential. Later, a structural model has been proposed in which the C-term is folded towards the active site. Different conformations can be hypothesized. A technique well suited to monitor protein dynamics is site-directed spin labeling followed by EPR spectroscopy. It relies on the insertion of a nitroxide derivative on cysteines. Using this approach, it is possible to analyze the mobility of the label in order to obtain information on its local environment. Moreover using advanced EPR techniques, it is possible to acquire interspin distances between two incorporated probes. Mutants bearing one or two cysteines at desirable positions were designed. The dynamics of labeled mutants were studied in vitro using continuous wave EPR. By pulsed EPR, distances were recorded for ACCO in presence of different combinations of cofactors. The experimental distances were compared to the predicted ones obtained from the crystallographic and model structures, and also to the calculated ones obtained by molecular dynamic simulations. A successful introduction of an unnatural amino acid onto the sequence of ACCO was performed, allowing to obtain earliest results. The achievement of preliminary structural data by in cell EPR are also presented

When the thymine base is oxidized, the resulting cation may deprotonate reversibly at N3, or irreversibly at >C5-CH3. In all thymine derivatives studied so far in the solid state, there is always a significant concentration of a radical formed by net H-abstraction from the >C5-CH 3. DFT calculations on this allyl-like radical are in good agreement with the experimental results for both the isotropic and anisotropic hyperfine couplings. There is a tendency for the thymine cation to deprotonate at N3 in solution. Calculations on the N3 deprotonated thymine cation yield two structures, one planar radical with an unusually large N1-C2 bond length, and one nonplanar radical with the N3 more than 25° out of the molecular plane. Calculations show that the structure with the lowest energy is the allyl-like radical.

This review contains the results of EPR/ENDOR experiments on DNA constituents in the solid-state. Most of the results presented involve single crystals of the DNA bases, nucleosides and nucleotides. The emphasis is on low-temperature ENDOR results. Typical experiments involve irradiations at or near helium temperatures in attempts to determine the primary radiation induced oxidation and reduction products. The use of the ENDOR technique allows one to determine the protonation state of the initial products. Subsequent warming of the sample facilitates a study of the reactions that the primary products undergo. A summary of the results is provided to show the relevance the study of model compounds has in understanding the radiation chemistry of DNA.

Detailed EPR and ENDOR experiments on the cocrystalline complex of 1-methyluracil:9-Ethyladenine (MUEA) have revealed that the major radiation-induced products observed at 10 K on MU are: MUEA1, a radical formed by net hydrogen abstraction from the N1-CH3 methyl group, MUEA2, the MU radical anion, and MUEA3, the C5 H-addition radical. The following four products were observed on the adenine moiety at 10 K, MUEA4, the N3 protonated adenine anion, MUEA5, the native adenine cation, MUEA6, the amino deprotonated adenine cation, and MUEA7, the C8 H-addition radical formed by net H-addition to C8 of the adenine base. The geometries, energetics, and hyperfine properties of all possible radicals of MU and EA, the native anions and cations, as well as radicals formed via net hydrogen atom abstraction (deprotonated cations) or addition (protonated anions) were investigated theoretically. All systems were optimized using the hybrid Hartree-Fock-density functional theory functional B3LYP, in conjunction with the 6-31G(d,p) basis set of Pople and co-workers. Calculations of the anisotropic hyperfine couplings for all the radicals observed in MUEA are presented and are shown to compare favorably with the experimentally measured hyperfine couplings. The calculated ionizations potentials indicate that EA would be the preferred oxidation site. In MUEA, both the adenine cation and its N4-deprotonated derivative were observed. The calculated electron affinities indicate that MU would be the preferred reduction site. In MUEA radical, MUEA2 is a uracil reduction product, however the protonation state of this radical could not be determined experimentally. Calculations suggest that MUEA2 is actually the C4=O protonated anion.

In this work, we studied three proteins from three different organisms by EPR spectroscopy: NpHtrII is part of the phototaxis system found in halophilic archaea, ChR2 is a cation-selective channel isolated from a green alga, and HsaCCA is an enzyme involved in the protein biosynthesis of humans. The goal was to identify characteristic conformations and dynamics in each of the studied proteins that were linked to their specific functions. The scientific disciplines employed in this work include biochemistry (site-directed spin-labeling), bioinformatics (data analysis, molecular modeling), informatics (device control, software development), molecular biology (mutagenesis, transformation, heterologous protein expression, protein purification, protein characterization), and physics (EPR spectroscopy, optical spectroscopy, experimental assembly). The experimental results show a strong interdependence between protein structure, conformers, dynamics, and function. Hydrogen bonds, although being a transient electrostatic attraction between polar molecules, are the key molecular interactions required for the conservation of protein functionality: Hydrogen bond networks in NpSRII and ChR2 stabilize the helix bundles, and hydrogen bond networks in HsaCCA mediate interdomain flexibility. However, the resulting structural alterations observed in our proteins manifest on a much larger scale: We have detected changes in the protein backbone mobility of the HAMP2 domain in NpHtrII after signaling. We have discovered the TMH B movement in ChR2 accompanying channel opening. And we have documented a substrate-dependent motion of the head domain in HsaCCA during catalysis.

Colicin A is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of target bacteria. In the present thesis, we aimed at studying the closed channel state, the membrane insertion mechanism, the acidic pH induced molten globule state and the interaction of colicin A in living E. coli cells. For that, we used Electron Paramagnetic Resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) method to explore the structural details of colicin A. The EPR studies of the membrane-bound colicin A (reconstituted into proteoliposomes) suggest the transmembrane orientation of the hydrophobic hairpin in the closed channel state. The pH dependent membrane insertion studies indicate that the membrane binding efficiency is significantly enhanced at pH < 3. Moreover, in the presence of a membrane potential, the pH induced membrane-bound state is able to open channels in the liposomes. The membrane-bound conformation (induced by acidic pH) is similar to the conformation of reconstituted colicin A which support the umbrella model for the closed channel state of colicin A. The studies on pH dependent conformational changes suggest that colicin A forms a molten globule at pH 2. The molecular details of pH induced conformational changes were analyzed by molecular dynamic simulations. The results of the MD simulations agree with the EPR results. Conformational changes of colicin A upon interaction with living E. coli cells could also be followed. Comparison between colicin A in wild type (WT) cells and tolB knock-out mutants suggest that the observed conformational changes originate from colicin A which has been already translocated to the inner membrane.

In der vorliegenden Arbeit wurde mit Hilfe von Embedded-Cluster-Simulationen die Struktur einiger typischer Defektzentren des technologisch vielversprechenden Perowskitkristalls BaTiO3 untersucht. Embedded-Cluster-Simulationen bestehen aus einer quantenmechanischen ab-initio Beschreibung eines begrenzten Cluster-Bereiches aus ca. 20-30 Ionen entweder in Hartree-Fock-Theorie oder in Dichtefunktionaltheorie. Zur Simulation der Kristallumgebung wird der Cluster in ein mit klassischen Potentialmethoden beschriebenes Punktladungsfeld eingebettet. Zur Erfassung elektronischer Polarisierungsanteile bietet sich hierfür das Schalenmodell an. Zunächst wurde das Verfahren auf das experimentell gut bekannte Ti3+-Jahn-Teller-Polaron angewendet. Es ergab sich eine gute Übereinstimmung mit den Resultaten aus vorherigen Elektronenspinresonanz-Untersuchungen. Der Energiegewinn durch die Ankopplung an die totalsymmetrische Atmungsmode des Gitters und durch den zusätzlichen symmetriereduzierenden Jahn-Teller-Effekt sowie der Vergleich mit dem Energiegewinn durch Bandbildung ergab eine geringfügige Begünstigung des polaronischen Zustands von ca. 0.1-0.2 eV. Ebenso konnte gezeigt werden, daß die dem Jahn-Teller-Polaron zuzuordnende Ladungsdichte auf mehrere benachbarte Titan-Ionen delokalisiert ist (intermediäres Polaron). Nach einer Reduktionsbehandlung werden in der ESR weitere axiale Zentren mit Ti3+-Charakter beobachtet, die bisher durch den Elektroneneinfang an einem Ti4+-Ion in der Nachbarschaft von Sauerstoff-Leerstellen (Ti3+-VO) interpretiert wurden. Aus den beobachteten g-Werten wird weiterhin ein Orbital mit t2-Symmetrie gefolgert. In Embedded-Cluster-Simulationen wird dagegen für das Ti3+-VO-Zentrum ein elektronischer Grundzustand mit e-Symmetrie gefunden. Auch eine aus der ESR vermutete Anlagerung von Akzeptoren führte nicht zu einem t2-artigen Grundzustand. Als Modell, welches mit der ESR kompatibel ist, wurde dagegen ein Elektroneneinfang in der Nachbarschaft von Akzeptoren ohne die Annahme einer VO gefunden (Ti3+-O2--A+). Im Rahmen von Schalenmodellrechnungen wurde die tendenzielle Möglichkeit der Stabilität solch einer Konfiguration gezeigt und in Embedded-Cluster-Simulationen die Jahn-Teller-Aufspaltung berechnet, die in guter Übereinstimmung mit den in der ESR gefundenen liegt. Der Grund, weshalb der theoretisch erwartete Elektroneneinfang in e-Symmetrie in der Nachbarschaft einer VO in der ESR nicht beobachtet wird, wird in einem Einfang von zwei Elektronen an der VO vermutet. In der Simulation dieses sogenannten Bipolarons ergab sich durch die Ankopplung an das äußere Gitter eine deutliche Reduktion der effektiven Elektron-Elektron Wechselwirkung auf einige Zehntel eV, so daß in weitergehenden Rechnungen mit einem größeren Cluster und einem besseren Basissatz eine Bipolaronen-Bildung möglich erscheint. / In this thesis several typical defect centres in the technologically important perovskite BaTiO3 have been analysed using the embedded-cluster approach. The embedded-cluster-approach consists of a quantum mechanical simulation of an inner cluster region of about 20-30 atoms or ions either with the Hartree-Fock theory or with the Density-Functional theory. The simulation of the crystal environment is realized by embedding the cluster in a point-charge field which is treated by means of classical potential methods. In order to take electronic polarization effects into account the shell-model is used. At first we simulated the experimentally well-known Ti3+ - Jahn-Teller polaron. A good agreement with previous EPR measurements has been found. The energy gained by coupling to the breathing-mode of the surrounding lattice and by the additional symmetry-reducing Jahn-Teller effect in comparison with the energy gain related to delocalization showed a small preference of the polaronic state of 0.1-0.2 eV. In addition a slight delocalization of the polaronic charge over several neighbouring Ti-ions was found (intermediate polaron). After a reduction treatment further axial centres of Ti3+-type were found with EPR, which have been interpreted as an electron capture at a Ti-ion near an oxygen vacancy (Ti3+-VO). Because of the measured g-values the polaronic orbital was expected to have t2-like symmetry. In contrast, the embedded cluster simulations revealed a ground-state with e-character. Even an electron trapping near a repulsive acceptor (Ti3+-VO - A+) did not have the proposed t2 ground state symmetry. In agreement with the experimental expectations, we suggest a model consisting of an electron with t2 symmetry captured near an acceptor without taking into account an oxygen vacancy (Ti3+-O2--A+). Using shell-model calculations the stability of such a complex could be shown. In embedded cluster simulations the Jahn-Teller splittings were found to be in good agreement with the experimentally observed ones. A possible explanation for the non-observability of the theoretically proposed Ti-centres with e-symmetry near the oxygen vacancy could be the existence of neutral vacancies, i.e. capture of two electrons near the vacancy. In simulations of this so called bipolaron we showed that the effective electron-electron interaction is effectively reduced by coupling to the crystal lattice to some tenths of eV, so that a stable bipolaronic state seems to be possible. Further calculations with a larger cluster and a larger basis set need to be done in the future.

In this work 3 different proteins are subjected to investigations on their structural, dynamic and functional properties by SDSL EPR spectroscopy, combined with in silico structure prediction and modeling: the pore-forming bacterial toxin colicin A in its membrane-bound form and its corresponding immunity protein Cai, and the Na+/proline symporter PutP. Colicin A (ColA) is a plasmid-encoded water-soluble pore-forming toxin produced by certain E. coli strains that kills unprotected cells of related strains by inserting a pore-forming subdomain into the cytoplasmic membrane to form voltage-dependent ion channels. Detailed structural data for the membrane-bound channel, in the closed as well as in the open state, is still missing, thus in the present study, the in vitro investigation by site-directed spin labeling and EPR spectroscopy has been substantially extended. The results indicate that a larger fraction of the protein than previously suggested penetrates into the hydrophobic core of the membrane, and distance measurements by pulse and cw EPR spectroscopy provide evidence that ColA in lipid bilayer membranes forms an oligomeric structure. Pulse EPR distance measurements under in vivo conditions reveal clear indications for an oligomeric ColA structure also in vivo. The results of all EPR measurements were combined to construct a dimer model for the colicin A closed channel state conformation. The immunity protein Cai, an integral inner membrane protein, protects the producing E. coli cell from the cytotoxic activity of its corresponding toxin (colicin A), by preventing channel opening by a yet unknown mechanism. ESR measurements for single spin label probes attached to ColA in the presence and absence of the immunity protein Cai reveal a clear influence on the ColA helices of the pore-forming domain in the presence of Cai as previously postulated. The data suggest that Cai induces a conformational change in/for the voltage sensor helix H6 of ColA, forming a “locked” inactive channel conformation that is not capable of voltage sensing and channel opening. Initial experiments with spin labeled wt-Cai in the presence and absence of unlabeled ColA suggest a more compact structure in the presence of ColA. PutP is an integral membrane protein located in the cytoplasmic membrane of E. coli, being responsible for the coupled transport of Na+ and proline in a 1:1 stoichiometry. It belongs to the family of sodium solute symporters (SSSF). Three dimensional structural data for PutP are at the moment not available, but a homology model has been developed based on the crystal structure of another member of this protein family, the Na+/galactose symporter vSGLT of Vibrio parahaemolyticus. The observed periodicity in spin label mobility and polarity measurements suggest a secondary structure of the extracellular Loop eL4 of PutP of two α-helical segments eL4a and eL4b, and imply the idea of eL4 functioning as an external gate to the SSSF. The ligand-induced changes observed in mobility, polarity and accessibility upon substrate binding support this notion, thus providing further insights into the mechanistic basis of sodium solute symport.

No description available.