Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals.

Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation of a wide range of biological processes including some light-induced ones, such as the photolyase-mediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. One of the factors that enable versatile flavin-coenzyme biochemistry and biophysics is the fine-tuning of the cofactor's frontier orbital by interactions with the protein environment. Probing the singly-occupied molecular orbital (SOMO) of the intermediate radical state of flavins is therefore a prerequisite for a thorough understanding of the diverse functions of the flavoprotein family. This may be ultimately achieved by unravelling the hyperfine structure of a flavin by electron paramagnetic resonance. In this contribution we present a rigorous approach to obtaining a hyperfine map of the flavin's chromophoric 7,8-dimethyl isoalloxazine unit at an as yet unprecedented level of resolution and accuracy. We combine powerful high-microwave-frequency/high-magnetic-field electron-nuclear double resonance (ENDOR) with 13C isotopologue editing as well as spectral simulations and density functional theory calculations to measure and analyse 13C hyperfine couplings of the flavin cofactor in DNA photolyase. Our data will provide the basis for electronic structure considerations for a number of flavin radical intermediates occurring in blue-light photoreceptor proteins.

Yersinia pseudotuberculosis cytotoxic necrotizing factor interacts with glycosaminoglycans.

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.

Synthesis and Application of a Perfluorinated Ammoniumyl Radical Cation as a Very Strong Deelectronator.

The perfluorinated dihydrophenazine derivative (perfluoro-5,10-bis(perfluorophenyl)-5,10-dihydrophenazine) ("phenazineF ") can be easily transformed to a stable and weighable radical cation salt by deelectronation (i.e. oxidation) with Ag[Al(ORF )4 ]/ Br2 mixtures (RF =C(CF3 )3 ). As an innocent deelectronator it has a strong and fully reversible half-wave potential versus Fc+ /Fc in the coordinating solvent MeCN (E°'=1.21 V), but also in almost non-coordinating oDFB (=1,2-F2 C6 H4 ; E°'=1.29 V). It allows for the deelectronation of [FeIII Cp*2 ]+ to [FeIV (CO)Cp*2 ]2+ and [FeIV (CN-t Bu)Cp*2 ]2+ in common laboratory solvents and is compatible with good σ-donor ligands, such as L=trispyrazolylmethane, to generate novel [M(L)x ]n+ complex salts from the respective elemental metals.

Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy.

Fluorescence resonance energy transfer in single enzyme molecules (smFRET, single-molecule measurement) allows the measurement of multicomponent distance distributions in complex biomolecules similar to pulsed electron-electron double resonance (PELDOR, ensemble measurement). Both methods use reporter groups: FRET exploits the distance dependence of the electric interaction between electronic transition dipole moments of the attached fluorophores, whereas PELDOR spectroscopy uses the distance dependence of the interaction between the magnetic dipole moments of attached spin labels. Such labels can be incorporated easily to cysteine residues in the protein. Comparison of distance distributions obtained with both methods was carried out with the H+-ATPase from Escherichia coli (EF0F1). The crystal structure of this enzyme is known. It contains endogenous cysteines, and as an internal reference two additional cysteines were introduced (EF0F1-γT106C-εH56C). These positions were chosen to allow application of both methods under optimal conditions. Both methods yield very similar multicomponent distance distributions. The dominating distance distribution (> 50%) is due to the two cysteines introduced by site-directed mutagenesis and the distance is in agreement with the crystal structure. Two additional distance distributions are detected with smFRET and with PELDOR. These can be assigned by comparison with the structure to labels at endogenous cysteines. One additional distribution is detected only with PELDOR. The comparison indicates that under optimal conditions smFRET and PELDOR result in the same distance distributions. PELDOR has the advantage that different distributions can be obtained with ensemble measurements, whereas FRET requires single-molecule techniques.

Guilty and charged: a stable solution of the hexamethylbenzene radical cation as a ligand forming oxidising agent.

While it is often longed for very gentle oxidising agents with ideally close to no interaction with the generated substrate, it may sometimes be beneficial to have an oxidising agent at hand which is able to stabilise a reactive species directly after its generation. Here we present the synthesis of the hexamethylbenzene radical cation as a ligand forming oxidising agent. Its utility was demonstrated by the exemplarily chosen access to the mono-arene complex-cations of univalent gallium and indium [M(C6Me6)]+[Al(ORF)4]- (M = Ga and In).

Global analysis of complex PELDOR time traces.

Pulsed electron-electron double resonance (PELDOR, alternatively called DEER for double electron-electron resonance) pulse sequences allow for the detection of echo decay curves that are modulated by dipole-dipole-coupling frequencies of interacting electron spins. With increasing distance between them, the echo decay needs to be monitored over a progressively extended time period. However, since the echo intensity typically falls off exponentially with increasing time, this might be problematic with respect to the minimum signal-to-noise ratio required for a sound data analysis. In this contribution we present the new PELDOR analysis tool GloPel (Global analysis of PELDOR data), an open-source Python-based application, that allows to extract improved-quality distance distributions from PELDOR data for which no ideal signal-to-noise ratio can be achieved for a very long observation window. By using Tikhonov regularization, GloPel allows for the simultaneous analysis of two time traces acquired for a sample in two different observation time windows, thus taking advantage of both, the typically high signal-to-noise ratio of the time trace acquired at early times of the echo decay, and the best possible background function fitted for the decay at later times, which is in most cases superimposed with considerable noise. In this way, short distances are not overseen in the higher noise of the longer time traces while long distances are not artificially shortened by limiting the observation time window of the experiment. Following our suggested data acquisition procedure, a significant reduction of the measurement time may also be achieved.

Ordering of PCDTBT revealed by time-resolved electron paramagnetic resonance spectroscopy of its triplet excitons.

Time-resolved electron paramagnetic resonance (TREPR) spectroscopy is shown to be a powerful tool to characterize triplet excitons of conjugated polymers. The resulting spectra are highly sensitive to the orientation of the molecule. In thin films cast on PET film, the molecules' orientation with respect to the surface plane can be determined, providing access to sample morphology on a microscopic scale. Surprisingly, the conjugated polymer investigated here, a promising material for organic photovoltaics, exhibits ordering even in bulk samples. Orientation effects may significantly influence the efficiency of solar cells, thus rendering proper control of sample morphology highly important.

[1,2,4]Triazolo[4,3-a]pyridines as bridging ligands--magnetism of azole-bridged dinuclear copper(II) complexes influenced by the trigonal distortion parameter τ.

The dinuclear doubly azole-bridged copper(II) complexes [Cu(II)2(L)2(MeCN)4](ClO4)4·3.73MeCN·0.80H2O and [Cu(II)2(L)6](ClO4)4·solvent (solvent = 2MeCN·H2O; 2MeCN·2H2O; 1.5MeOH·3.5H2O) were prepared [L = 3-(6-methyl-2-pyridyl)-[1,2,4]triazolo[4,3-a]pyridine]. Structural characterizations revealed very different local geometries about the copper(II) ions, being trigonal bipyramidal for the former (τ = 0.76) and square pyramidal for the latter (τ = 0.07, 0.15, 0.07) complex. Magnetic measurements of bulk material [Cu(II)2(L)2(H2O)4](ClO4)4 and [Cu(II)2(L)6](ClO4)4·2H2O revealed antiferromagnetic coupling in both complexes, however, of very different strengths. Electron paramagnetic resonance (EPR) spectroscopy was applied to investigate magnetic properties of the complexes in detail. These experimental findings were supported by broken-symmetry DFT calculations. Systematic magneto-structural correlations are discussed.