Search

Overhauser DNP enhancements of toluene were measured at a magnetic field of 0.35 Tesla in a series of chemically functionalized nitroxide radicals. We observe that the enhancements increase systematically with polarizer size and rotational correlation time. Examination of the saturation factor of 14N nitroxides by pulsed ELDOR spectroscopy led to a quantitative interpretation of the enhancements, for which the saturation factor increases up to almost unity due to enhanced nuclear (14N) relaxation in the nitroxide radical. The observation has a direct impact on the choice of optimum DNP polarizers in liquids. [ABSTRACT FROM AUTHOR]

Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to F− cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron‐electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the Mg2+‐bound apo, and the F−‐bound holo form. 19F ENDOR (electron‐nuclear double resonance) was used to investigate the active site structure of the F−‐bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing the periphery of the RNA fold, revealed conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub‐angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques. [ABSTRACT FROM AUTHOR]

Phosphorus‐centered disbiradicals, in which the radical sites exist as individual spin doublets with weak spin‐spin interaction have not been known so far. Starting from monoradicals of the type [⋅P(μ‐NTer)2P−R], we have now succeeded in linking two such monoradical phosphorus centers by appropriate choice of a linker. To this end, biradical [⋅P(μ‐NTer)2P⋅] (1) was treated with 1,6‐dibromohexane, affording the brominated species {Br[P(μ‐NTer)]2}2C6H12 (3). Subsequent reduction with KC8 led to the formation of the disbiradical {⋅[P(μ‐NTer)]2}2C6H12 (4) featuring a large distance between the radical phosphorus sites in the solid state and formally the highest biradical character observed in a P‐centered biradical so far, approaching 100 %. EPR spectroscopy revealed a three‐line signal in solution with a considerably larger exchange interaction than would be expected from the molecular structure of the single crystal. Quantum chemical calculations revealed a highly dynamic conformational space; thus, the two radical sites can approach each other with a much smaller distance in solution. Further reduction of 4 resulted in the formation of a potassium salt featuring the first structurally characterized P‐centered distonic radical anion (5−). Moreover, 4 could be used in small molecule activation. [ABSTRACT FROM AUTHOR]

Phosphor‐zentrierte Disbiradikale, in denen die Radikalzentren als einzelne Spin‐Dubletts mit schwacher Spin‐Spin‐Wechselwirkung vorliegen, waren bisher nicht bekannt. Ausgehend von Monoradikalen des Typs [⋅P(μ‐NTer)2P−R] ist es uns nun gelungen, zwei monoradikalische Phosphorzentren durch geeignete Wahl eines Linkers zu verknüpfen. Zu diesem Zweck wurde das Biradikal [⋅P(μ‐NTer)2P⋅] (1) mit 1,6‐Dibromhexan umgesetzt, wodurch die bromierte Spezies {Br[P(μ‐NTer)]2}2C6H12 (3) entstand. Die anschließende Reduktion mit KC8 führte zur Bildung des Disbiradikals {⋅[P(μ‐NTer)]2}2C6H12 (4), das sich durch einen großen Abstand zwischen den radikalischen Phosphorzentren im Festkörper auszeichnet und mit annähernd 100 % den bisher höchsten Biradikalcharakter in einem P‐zentrierten Biradikal aufweist. Die EPR‐spektroskopische Untersuchung ergab ein Drei‐Linien‐Signal in Lösung mit einer erheblich größeren Austauschwechselwirkung als es aufgrund der Molekülstruktur im Einkristall zu erwarten wäre. Quantenchemische Berechnungen ergaben einen hochdynamischen Konformationsraum; demnach können sich die beiden Radikalstellen in Lösung mit einem viel geringeren Abstand einander nähern. Die weitere Reduktion von 4 führte zur Bildung eines Kaliumsalzes mit dem ersten strukturell charakterisierten P‐zentrierten distonischen Radikalanion (5−). Außerdem konnte 4 für die Aktivierung kleiner Moleküle eingesetzt werden. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5–10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes. [ABSTRACT FROM AUTHOR]

The article focuses on the Iron ENDOR spectroscopy and the use of sulfur cluster in substrate reduction of heterodisulfide reductase. Heterodisulfide reductase (Hdr) from methanogenic archea is an iron-sulfur protein that catalyzes the reversible two-electron reduction of the mixed disulfide CoM-S-S-CoB to the thiol coenzymes. In the final step of methanogenesis, the disulfide functions as the terminal electron acceptor for an energy-conserving electron transport chain, a process called disulfide respiration. It is unusual that this enzyme uses an iron-sulfur cluster to mediate disulfide reduction in two one-electron steps via site-specific cluster chemistry.

The hydration structure of nitroxide radicals in aqueous solutions is elucidated by advanced 17O hyperfine (hf) spectroscopy with support of quantum chemical calculations and MD simulations. A piperidine and a pyrrolidine‐based nitroxide radical are compared and show clear differences in the preferred directionality of H‐bond formation. We demonstrate that these scenarios are best represented in 17O hf spectra, where in‐plane coordination over σ ${\sigma }$ ‐type H‐bonding leads to little spin density transfer on the water oxygen and small hf couplings, whereas π ${{\rm \pi }}$ ‐type perpendicular coordination generates much larger hf couplings. Quantitative analysis of the spectra based on MD simulations and DFT predicted hf parameters is consistent with a distribution of close solvating water molecules, in which directionality is influenced by subtle steric effects of the ring and the methyl group substituents. [ABSTRACT FROM AUTHOR]

The hydration structure of nitroxide radicals in aqueous solutions is elucidated by advanced 17O hyperfine (hf) spectroscopy with support of quantum chemical calculations and MD simulations. A piperidine and a pyrrolidine‐based nitroxide radical are compared and show clear differences in the preferred directionality of H‐bond formation. We demonstrate that these scenarios are best represented in 17O hf spectra, where in‐plane coordination over σ ${\sigma }$ ‐type H‐bonding leads to little spin density transfer on the water oxygen and small hf couplings, whereas π ${{\rm \pi }}$ ‐type perpendicular coordination generates much larger hf couplings. Quantitative analysis of the spectra based on MD simulations and DFT predicted hf parameters is consistent with a distribution of close solvating water molecules, in which directionality is influenced by subtle steric effects of the ring and the methyl group substituents. [ABSTRACT FROM AUTHOR]

The article titled "Cover Picture: Rational Design of a Phosphorus-Centered Disbiradical" in the journal Angewandte Chemie International Edition features research on arranging diatoms (tiny algae) under a microscope using a single hair. The diatoms represent the formula of a disbiradical described in the article and its characteristic three-line EPR signal. The background image of the Milky Way symbolizes the interaction through space that explains the unique EPR signal. The article also includes research on photoswitch azobenzene-spiropyran dyads, lignin O-demethylation, and the design of a large polyoxometalate cluster with specific properties. [Extracted from the article]

The article titled "Titelbild: Design eines Phosphor-zentrierten Disbiradikals" in the journal Angewandte Chemie features research on arranging diatoms, small algae, under a microscope using a single hair. The diatoms represent the formula of a disbiradical described in the article and its characteristic three-line EPR signal. The background image of the Milky Way symbolizes the interaction through space that explains the unique EPR signal. The article also includes research on photoswitch azobenzene-spiropyran dyads, lignin O-demethylation, and the design of a large polyoxometalate cluster with specific properties. [Extracted from the article]

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics. [ABSTRACT FROM AUTHOR]

• We demonstrate high-field 1H ENDOR spectroscopy on a ∼ 15 pmol amino acid radical. • The 263 GHz ENDOR setup and a new 263 GHz/400 MHz ENDOR probe head are described. • High orientational selectivity at 9 T allows disentangling new 1H resonances of a tyrosyl radical. • Enhanced resolution and sensitivity should expand the application range of ENDOR spectroscopy. We present and discuss the performance of 1H electron-nuclear double resonance (ENDOR) at 263 GHz/9.4 T by employing a prototype, commercial quasi optical spectrometer. Basic instrumental features of the setup are described alongside a comprehensive characterization of the new ENDOR probe head design. The performance of three different ENDOR pulse sequences (Davies, Mims and CP-ENDOR) is evaluated using the 1H BDPA radical. A key feature of 263 GHz spectroscopy – the increase in orientation selectivity in comparison with 94 GHz experiments – is discussed in detail. For this purpose, the resolution of 1H ENDOR spectra at 263 GHz is verified using a representative protein sample containing approximately 15 picomoles of a tyrosyl radical. Davies ENDOR spectra recorded at 5 K reveal previously obscured spectral features, which are interpreted by spectral simulations aided by DFT calculations. Our analysis shows that seven internal proton couplings are detectable for this specific radical if sufficient orientation selectivity is achieved. The results prove the fidelity of 263 GHz experiments in reporting orientation-selected 1H ENDOR spectra and demonstrate that new significant information can be uncovered in complex molecular systems, owing to the enhanced resolution combined with high absolute sensitivity and no compromise in acquisition time. [ABSTRACT FROM AUTHOR]

Abstract: Double electron–electron resonance (DEER) at W-band (95GHz) was applied to measure the distance between a pair of nitroxide and Gd3+ chelate spin labels, about 6nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700MHz) between the narrow, intense signal of the Gd3+ central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12μs in a protein. This opens the possibility of accessing distances of 8nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd3+–nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels. [Copyright &y& Elsevier]

The Eschericliia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: a and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4'-radical of a 3'-keto-2'-deoxycytidine 5'-diphosphate. To identify the structure of the nucleotide radical [1'-2H], [2'-2H], [4'-2H], [5'-2H], [U-13C, 15N], [U-15N], and [5,6 -2H] CDP and [β-2H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35GHz ENDOR spectroscopy. Substitution of 2H at C4' and C1' altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-lll/B3LYP/ TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate. The relationship of this radical to the disulfide radical anion is discussed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Resolution of 19F chemical shift anisotropies (CSA) in ENDOR spectra at 9.4 T. • CSA reveals the orientation of 19F moieties and rotation of CF 3 groups. • CSA determined from analysis of ENDOR spectra. • Code of simulation routine is made available. Pulsed 19F ENDOR spectroscopy provides a selective method for measuring angstrom to nanometer distances in structural biology. Here, the performance of 19F ENDOR at fields of 3.4 T and 9.4 T is compared using model compounds containing one to three 19F atoms. CF 3 groups are included in two compounds, for which the possible occurrence of uniaxial rotation might affect the distance distribution. At 9.4 T, pronounced asymmetric features are observed in many of the presented 19F ENDOR spectra. Data analysis by spectral simulations shows that these features arise from the chemical shift anisotropy (CSA) of the 19F nuclei. This asymmetry is also observed at 3.4 T, albeit to a much smaller extent, confirming the physical origin of the effect. The CSA parameters are well consistent with DFT predicted values and can be extracted from simulation of the experimental data in favourable cases, thereby providing additional information about the geometrical and electronic structure of the spin system. The feasibility of resolving the CSA at 9.4 T provides important information for the interpretation of line broadening in ENDOR spectra also at lower fields, which is relevant for developing methods to extract distance distributions from 19F ENDOR spectra. [ABSTRACT FROM AUTHOR]

The article presents a study that demonstrates 1hydrogen (H) and 13carbon (C) dynamic nuclear polarization (DNP) enhancements on water solutions with a two-field shuttle DNP spectrometer. It notes that the results define and optimize the principle for the feasibility of a shuttle DNP experiment. The findings show that DNP in aqueous solution mixed with high resolution nuclear magnetic resonance (NMR) detection is possible in a shuttle DNP spectrometer.

The article examines the distance between residues involved in the radical propagation pathway of Escherichia (E.) coli ribonucleotide reductase (RNR). The researchers consider the docking model which is generated from the individual structures of α2 and ß2 based on shape and charge complementarities in analyzing the distances. Based on the results, the study shows the position of the critical Tyr pathway residues in the α2ß2 complex.

Photolysis of a platinum(II) azide complex in the presence of styrenes enables C=C double bond cleavage upon dissociative olefin imination to aldimido (Pt II -N=CHPh) and formimido (Pt II -N=CH 2 ) complexes as the main products. Spectroscopic and quantum chemical examinations support a mechanism that commences with the decay of the metallonitrene photoproduct (Pt II -N) via bimolecular coupling and nitrogen loss as N 2 . The resulting platinum(I) complex initiates a radical chain mechanism via a dinuclear radical-bridged species (Pt II -CH 2 CHPhN • -Pt II ) as a direct precursor to C-C scission. The preference for the Pt I mediated route over styrene aziridination is attributed to the distinct nucleophilicity of the triplet metallonitrene., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Nuclear magnetic resonance (NMR) is fundamental in the natural sciences, from chemical analysis and structural biology, to medicine and physics. Despite its enormous achievements, one of its most severe limitations is the low sensitivity, which arises from the small population difference of nuclear spin states. Methods such as dissolution dynamic nuclear polarization and parahydrogen induced hyperpolarization can enhance the NMR signal by several orders of magnitude, however, their intrinsic limitations render multidimensional hyperpolarized liquid-state NMR a challenge. Here, we report an instrumental design for 9.4 Tesla liquid-state dynamic nuclear polarization that enabled enhanced high-resolution NMR spectra in one and two-dimensions for small molecules, including drugs and metabolites. Achieved enhancements of up to two orders of magnitude translate to signal acquisition gains up to a factor of 10,000. We show that hyperpolarization can be transferred between nuclei, allowing DNP-enhanced two-dimensional 13 C- 13 C correlation experiments at 13 C natural abundance. The enhanced sensitivity opens up perspectives for structural determination of natural products or characterization of drugs, available in small quantities. The results provide a starting point for a broader implementation of DNP in liquid-state NMR., (© 2024. The Author(s).)

The hydration structure of nitroxide radicals in aqueous solutions is elucidated by advanced 17 O hyperfine (hf) spectroscopy with support of quantum chemical calculations and MD simulations. A piperidine and a pyrrolidine-based nitroxide radical are compared and show clear differences in the preferred directionality of H-bond formation. We demonstrate that these scenarios are best represented in 17 O hf spectra, where in-plane coordination over σ ${\sigma }$ -type H-bonding leads to little spin density transfer on the water oxygen and small hf couplings, whereas π ${{\rm \pi }}$ -type perpendicular coordination generates much larger hf couplings. Quantitative analysis of the spectra based on MD simulations and DFT predicted hf parameters is consistent with a distribution of close solvating water molecules, in which directionality is influenced by subtle steric effects of the ring and the methyl group substituents., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Dynamic nuclear polarization (DNP) is a method to enhance the low sensitivity of nuclear magnetic resonance (NMR) via spin polarization transfer from electron spins to nuclear spins. In the liquid state, this process is mediated by fast modulations of the electron-nuclear hyperfine coupling and its efficiency depends strongly on the applied magnetic field. A peculiar case study is triphenylphosphine (PPh 3 ) dissolved in benzene and doped with BDPA radical because it gives 31 P-NMR signal enhancements of two orders of magnitude up to a magnetic field of 14.1 T. Here we show that the large 31 P enhancements of BDPA/PPh 3 in benzene at 1.2 T (i) decrease when the moieties are dissolved in other organic solvents, (ii) are strongly reduced when using a nitroxide radical, and (iii) vanish with pentavalent 31 P triphenylphosphine oxide. Those experimental observations are rationalized with numerical calculations based on density functional theory that show the tendency of BDPA and PPh 3 to form a weak complex via non-covalent interaction that leads to large hyperfine couplings to 31 P (Δ A iso ≥ 13 MHz). This mechanism is hampered in other investigated systems. The case study of 31 P-DNP in PPh 3 is an important example that extends the current understanding of DNP in the liquids state: non-covalent interactions between radical and target can be particularly effective to obtain large NMR signal enhancements.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α 2 β 2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β 2 , nucleotide reduction in α 2 . Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y 356 (β) and Y 731 (α) across the α/β interface. Using 2,3,5-F 3 Y 122 -β 2 with 3,5-F 2 Y 731 -α 2 , GDP (substrate) and TTP (allosteric effector), a Y 356 • intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19 F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y 356 • and the 19 F nuclei of 3,5-F 2 Y 731 in this RNR mutant. Similar experiments with the double mutant E 52 Q/F 3 Y 122 -β 2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F 2 Y 731 . Remarkably, one conformation is consistent with 3,5-F 2 Y 731 within the H-bond distance to Y 356 • , whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y 356 and Y 731 . The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1 H/ 2 H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)