ENDOR Research Articles

The Soybean Lipoxygenase-Substrate Complex: Correlation between the Properties of Tunneling-Ready States and ENDOR-Detected Structures of Ground States.

Hydrogen tunneling in enzymatic C-H activation requires a dynamical sampling among ground-state enzyme-substrate (E-S) conformations, which transiently generates a tunneling-ready state (TRS). The TRS is characterized by a hydrogen donor-acceptor distance (DAD) of 2.7 Å, ∼0.5 Å shorter than the dominant DAD of optimized ground states. Recently, a high-resolution, 13C electron-nuclear double-resonance (ENDOR) approach was developed to characterize the ground-state structure of the complex of the linoleic acid (LA) substrate with soybean lipoxygenase (SLO). The resulting enzyme-substrate model revealed two ground-state conformers with different distances between the target C11 of LA and the catalytically active cofactor [Fe(III)-OH]: the active conformer "a", with a van der Waals DAD of 3.1 Å between C11 and metal-bound hydroxide, and an inactive conformer "b", with a distance that is almost 1 Å longer. Herein, the structure of the E-S complex is examined for a series of six variants in which subtle structural modifications of SLO have been introduced either at a hydrophobic side chain near the bound substrate or at a remote residue within a protein network whose flexibility influences hydrogen transfer. A remarkable correlation is found between the ENDOR-derived population of the active ground-state conformer a and the kinetically derived differential enthalpic barrier for D versus H transfer, ΔEa, with the latter increasing as the fraction of conformer a decreases. As proposed, ΔEa provides a "ruler" for the DAD within the TRS. ENDOR measurements further corroborate the previous identification of a dynamical network coupling the buried active site of SLO to the surface. This study shows that subtle imperfections within the initial ground-state structures of E-S complexes are accompanied by compromised geometries at the TRS.

Room Temperature Electrically Detected Nuclear Spin Coherence of NV Centres in Diamond

We demonstrate electrical detection of the 14N nuclear spin coherence of NV centres at room temperature. Nuclear spins are candidates for quantum memories in quantum-information devices and quantum sensors, and hence the electrical detection of nuclear spin coherence is essential to develop and integrate such quantum devices. In the present study, we used a pulsed electrically detected electron-nuclear double resonance technique to measure the Rabi oscillations and coherence time (T2) of 14N nuclear spins in NV centres at room temperature. We observed T2 ≈ 0.9 ms at room temperature, however, this result should be taken as a lower limit due to limitations in the longitudinal relaxation time of the NV electron spins. Our results will pave the way for the development of novel electron- and nuclear-spin-based diamond quantum devices.

Comprehensive investigation of the triplet state electronic structure of free-base 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin by a combined advanced EPR and theoretical approach.

The nature of the photoexcited triplet state of free-base 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (H2TPPS4-) has been investigated by advanced Electron Paramagnetic Resonance (EPR) techniques combined with quantum chemical calculations. The zero-field splitting (ZFS) parameters, D and E, the orientation of the transition dipole moment in the ZFS tensor frame, and the proton hyperfine couplings have been determined by magnetophotoselection-EPR and pulse electron-nuclear double resonance spectroscopy. Both time-resolved and pulse experiments exploit the electron spin polarization of the photoexcited triplet state. Comparison of the magnetic observables with computational results, including CASSCF calculations of the ZFS interaction tensor, provides an accurate picture of the triplet-state electronic structure. The theoretical investigation has been integrated with a systematic analysis on the parent free-base porphyrin molecule to assess the effect of the sulfonatophenyl substituents on the magnetic tensors. Additionally, the magnetophotoselection effects are discussed in terms of tautomerization in the excited singlet state of H2TPPS4-.

Oxygen-vacancy donor-electron center in Y3Al5O12 garnet crystals: Electron paramagnetic resonance and dielectric spectroscopy study

F+ center, an electron trapped at oxygen vacancy (VO), was investigated in the oxygen deficient Y3Al5O12 (YAG) crystals by EPR. The measurements were performed at temperatures 5-450 K and frequencies 9.4-350 GHz with using both the continue wave and pulse EPR technique. The pulse electron-nuclear double resonance was applied to resolve the hyperfine interaction of the trapped electron with surrounding nuclei. The measurements show that at low temperatures, T < 50 K, EPR spectrum of the F+ center is anisotropic with g factors in the range 1.999-1.988 and originates from three magnetically inequivalent positions of the center in garnet lattice according to different directions of the Al(IV)-VO-Al(VI) chains, where Al(IV) and Al(VI) are the tetrahedral and octahedral Al sites, respectively. As the temperature increases, the EPR spectrum becomes isotropic suggesting a motional averaging of the anisotropy due to motion of electron between neighboring oxygen vacancies. With further increase of the temperature to T > 200 K, we observed delocalization of the electron into the conduction band with the activation energy about 0.4-0.5 eV that resulted in substantial narrowing of the EPR spectral line with simultaneous change of its shape from the Gaussian to Lorentzian due to diminish up to zero of the Fermi contact hyperfine field at 27Al and 89Y nuclei. Such temperature behavior of the F+-center electron in YAG is completely similar to behavior of a donor electron in a semiconductor. Our findings is further supported by measurements of the conductivity and dielectric properties. In particular, these data show that the conduction electrons are not homogeneously distributed in the crystal: there are high-conductive regions separated by poorly-conductive dielectric layers. This leads to the so-called Maxwell-Wagner dielectric relaxation with huge apparent dielectric constant at low frequencies.

Multiple drug binding modes in Mycobacterium tuberculosis CYP51B1

The Mycobacterium tuberculosis (Mtb) genome encodes 20 different cytochrome P450 enzymes (CYPs), many of which serve essential biosynthetic roles. CYP51B1, the Mtb version of eukaryotic sterol demethylase, remains a potential therapeutic target. The binding of three drug fragments containing nitrogen heterocycles to CYP51B1 is studied here by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques to determine how each drug fragment binds to the heme active-site. All three drug fragments form a mixture of complexes, some of which retain the axial water ligand from the resting state. Hyperfine sublevel correlation spectroscopy (HYSCORE) and electron-nuclear double resonance spectroscopy (ENDOR) observe protons of the axial water and on the drug fragments that reveal drug binding modes. Binding in CYP51B1 is complicated by the presence of multiple binding modes that coexist in the same solution. These results aid our understanding of CYP-inhibitor interactions and will help guide future inhibitor design.

Structure and dynamics of catalytically competent but labile paramagnetic metal-hydrides: the Ti(iii)-H in homogeneous olefin polymerization.

Metal hydride complexes find widespread application in catalysis and their properties are often understood on the basis of the available crystal structures. However, some catalytically relevant metal hydrides are only spontaneously formed in situ, cannot be isolated in large quantities or crystallised and their structure is therefore ill defined. One such example is the paramagnetic Ti(iii)-hydride involved in homogeneous Ziegler–Natta catalysis, formed upon activation of CpTi(iv)Cl3 with modified methylalumoxane (MMAO). In this contribution, through a combined use of electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies we identify the nature of the ligands, their bonding interaction and the extent of the spin distribution. From the data, an atomistic and electronic model is proposed, which supports the presence of a self-assembled ion pair between a cationic terminal Ti-hydride and an aluminate anion, with a hydrodynamic radius of ca. 16 Å.

Methyl groups matter: Photo-CIDNP characterizations of the semiquinone radicals of FMN and demethylated FMN analogs.

In this contribution, the relative hyperfine couplings are determined for the 1H nuclei of the flavin mononucleotide (FMN) radical in an aqueous environment. In addition, three structural analogs with different methylation patterns are characterized and the influence of the substituents at the isoalloxazine moiety on the electronic structure of the radicals is explored. By exploiting nuclear hyperpolarization generated via the photo-CIDNP (chemically induced dynamic nuclear polarization) effect, it is possible to study the short-lived radical species generated by in situ light excitation. Experimental data are extracted by least-squares fitting and supported by quantum chemical calculations and published values from electron paramagnetic resonance and electron-nuclear double resonance. Furthermore, mechanistic details of the photoreaction of the investigated flavin analogs with l-tryptophan are derived from the photo-CIDNP spectra recorded at different pH values. Thereby, the neutral and anionic radicals of FMN and three structural analogs are, for the first time, characterized in terms of their electronic structure in an aqueous environment.

Apparatus for electrically detected electron nuclear double resonance in solid state electronic devices.

We have developed a sensitive electron nuclear double resonance spectrometer in which the detection takes place through electrically detected magnetic resonance. We demonstrate that the spectrometer can provide reasonably high signal to noise spectra of 14N interactions with deep level centers in a fully processed bipolar junction transistor at room temperature.

Adsorption of Olefins at Cupric Ions in Metal-Organic Framework HKUST-1 Explored by Hyperfine Spectroscopy.

Metal-organic frameworks (MOFs) represent a promising platform for gas storage and separation. In this work, adsorption of olefins in Zn-doped HKUST-1 metal-organic framework is explored with hyperfine spectroscopy. By means of electron-nuclear double resonance and hyperfine sublevel correlation spectroscopy, we detect the interaction between the electron spins of the Cu2+ sites of the MOF and the 1H nuclear spins of adsorbed C2H4 and C4H8. Further analysis of the measured spectra allows us to precisely locate the protons in the vicinity of the Cu2+ ions and thereby establish ensemble-averaged structural models of the olefin molecules adsorbed at the open metal sites of HKUST-1.

Measurement of Angstrom to Nanometer Molecular Distances with 19 F Nuclear Spins by EPR/ENDOR Spectroscopy.

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using 19F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.

Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy

AbstractSpectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using 19F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

Effect of the Diamagnetic Single-Crystalline Host on the Angular-Resolved Electron Nuclear Double Resonance Experiments: Case of Paramagnetic [nBu4N]2[Cu(opba)] Embedded in Diamagnetic [nBu4N]2[Ni(opba)

The electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) spectroscopies are frequently used to determine hyperfine (A) and quadrupole (Q) tensors for the paramagnetic transition-metal complexes, which knowledge enables estimation of the spin density distribution in the complex and the assessment of magnetic exchange pathways in polynuclear molecular networks. The most accurate results can be obtained if paramagnetic complexes are isolated in a single-crystalline diamagnetic host. In this work we were able to detect angle-resolved ESR/ENDOR spectra of the paramagnetic [nBu4N]2[Cu(opba)] (opba = o-phenylenebis(oxamato) complex hosted by the single crystal of diamagnetic [nBu4N]2[Ni(opba)], which allow recovery of A, Q, and g-tensors, all at once. Remarkably, with the aid of theoretical modeling, our data have unveiled the effect of multipositional hosting of the paramagnetic complex in the diamagnetic matrix. This effect can have a profound influence on the data handling and must be carefully considered while interpreting similar experiments.

Influence of Al on the Structure and in Vitro Behavior of Hydroxyapatite Nanopowders.

Nanopowders of aluminum-substituted (0-20 mol %) hydroxyapatite (HA) with the average size of 40-60 nm were synthesized by the precipitation method from nitrate solutions. A series of samples were studied by various analytical tools to elucidate the peculiarities of Al introduction. Electron paramagnetic resonance and pulsed electron-nuclear double resonance data demonstrate that incorporation of Al resulted in a decrease in the concentration of impurity carbonate anions and lead to an increase in the number of protons in the distant environment of the impurity nitrogen species. Density functional theory calculations show that the Al3+ incorporation is accompanied by the local positional rearrangement and the distortion of anion channel geometry. An in vitro test conducted on MG-63 cells demonstrates the cytocompatibility and magnification of the surface matrix characteristics with Al doping.

Electrically detected electron nuclear double resonance in 4H-SiC bipolar junction transistors

We demonstrate high signal-to-noise electrically detected electron-nuclear double resonance measurements on fully processed bipolar junction transistors at room temperature. This work indicates that the unparalleled analytical power of electron-nuclear double resonance in the identification of paramagnetic point defects can be exploited in the study of defects within fully functional solid-state electronic devices.

H2 Activation and Direct Access to Terminal Nitride and cyclo-P3 Complexes by an Acceptor-Free Rhenium(II) β-Diketiminate

The rhenium(II) complex Re(η5-Cp)(BDI) (B; BDI = N,N'-bis(2,6-diisopropylphenyl)-3,5-dimethyl-β-diketiminate; Cp = cyclopentadienyl) was subjected to a number of reactivity studies that highlight its unique combination of oxidation state and acceptor-free coordination environment. In the reactions under study we found that this low-valent paramagnetic species yields diamagnetic rhenium(III) or rhenium(V) products, indicating a combination of reducing power and radical character are the primary variables that control its reactivity. Namely, B has been found to promote the cleavage of dihydrogen to yield the rhenium(III) hydride Re(H)(η5-Cp)(BDI) (C), as well as the three-electron reduction of organic azides to yield a rhenium(V) terminal nitride complex NRe(η3-Cp)(BDI) (3), which features a low-hapticity Cp ligand. Additionally, we found that B readily activates white phosphorus (P4) to form the rhenium(V) cyclo-P3 complex (cyclo-P3)Re(η5-Cp)(BDI) (4). One-electron oxidation of 4 gave the paramagnetic rhenium(VI) salt [(cyclo-P3)Re(η5-Cp)(BDI)][OTf] (5-OTf), which was characterized by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy spectroscopies. Density functional theory calculations and X-ray crystallography also provided a useful basis for understanding the reactivity and electronic structure of B and its derivatives.

Influence of the Chemical Modification of the Nanodiamond Surface on Electron Paramagnetic Resonance/Electron-Nuclear Double Resonance Spectra of Intrinsic Nitrogen Defects

A high-frequency/high-magnetic field (94 GHz/3.3 T) electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) study of monocrystalline nanodiamonds (ND) is carried out. Influence of the hydrogen and fluorine modification of the ND surface on EPR and ENDOR signals from the superficial and bulk paramagnetic centers is observed. The effect shows that the ND bulk paramagnetic centers could serve as the intrinsic probe of ND surface modification despite deep localization, shielding from the environment and two-layered surface of the diamond nanoparticle.

Flavodoxin with an air-stable flavin semiquinone in a green sulfur bacterium.

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.

The Elusive 5'-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies.

The 5'-deoxyadenosyl radical (5'-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5'-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5'-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe-4S]+ cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S-C5' bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5' (2pπ) radical (∼70% spin occupancy); the C5'(H)2 plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5'-C4'-(C4'-H) plane, placing a C5'-H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4'-H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.

Metal Bonding with 3d and 6d Orbitals: An EPR and ENDOR Spectroscopic Investigation of Ti3+-Al and Th3+-Al Heterobimetallic Complexes.

Accessing covalent bonding interactions between actinides and ligating atoms remains a central problem in the field. Our current understanding of actinide bonding is limited because of a paucity of diverse classes of compounds and the lack of established models. We recently synthesized a thorium (Th)–aluminum (Al) heterobimetallic molecule that represents a new class of low-valent Th-containing compounds. To gain further insight into this system and actinide–metal bonding more generally, it is useful to study their underlying electronic structures. Here, we report characterization by electron paramagnetic resonance (EPR) and electron–nuclear double resonance (ENDOR) spectroscopy of two heterobimetallic compounds: (i) a Cptt2ThH3AlCTMS3 [TMS = Si(CH3)3; Cptt = 1,3-di-tert-butylcyclopentadienyl] complex with bridging hydrides and (ii) an actinide-free Cp2TiH3AlCTMS3 (Cp = cyclopentadienyl) analogue. Analyses of the hyperfine interactions between the paramagnetic trivalent metal centers and the surrounding magnetic nuclei, 1H and 27Al, yield spin distributions over both complexes. These results show that while the bridging hydrides in the two complexes have similar hyperfine couplings (aiso = −9.7 and −10.7 MHz, respectively), the spin density on the Al ion in the Th3+ complex is ∼5-fold larger than that in the titanium(3+) (Ti3+) analogue. This suggests a direct orbital overlap between Th and Al, leading to a covalent interaction between Th and Al. Our quantitative investigation by a pulse EPR technique deepens our understanding of actinide bonding to main-group elements.