Electron Nuclear Double Resonance Studies Research Articles

Terminal Hydride Complex of High-Spin Mn.

The iron-molybdenum cofactor of nitrogenase (FeMoco) catalyzes fixation of N2 via Fe hydride intermediates. Our understanding of these species has relied heavily on the characterization of well-defined 3d metal hydride complexes, which serve as putative spectroscopic models. Although the Fe ions in FeMoco, a weak-field cluster, are expected to adopt locally high-spin Fe2+/3+ configurations, synthetically accessible hydride complexes featuring d5 or d6 electron counts are almost exclusively low-spin. We report herein the isolation of a terminal hydride complex of four-coordinate, high-spin (d5; S = 5/2) Mn2+. Electron paramagnetic resonance and electron-nuclear double resonance studies reveal an unusually large degree of spin density on the hydrido ligand. In light of the isoelectronic relationship between Mn2+ and Fe3+, our results are expected to inform our understanding of the valence electronic structures of reactive hydride intermediates derived from FeMoco.

The Active-Site [4Fe-4S] Cluster in the Isoprenoid Biosynthesis Enzyme IspH Adopts Unexpected Redox States during Ligand Binding and Catalysis.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase, or IspH (formerly known as LytB), catalyzes the terminal step of the bacterial methylerythritol phosphate (MEP) pathway for isoprene synthesis. This step converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into one of two possible isomeric products, either isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This reaction involves the removal of the C4 hydroxyl group of HMBPP and addition of two electrons. IspH contains a [4Fe-4S] cluster in its active site, and multiple cluster-based paramagnetic species of uncertain redox and ligation states can be detected after incubation with reductant, addition of a ligand, or during catalysis. To characterize the clusters in these species, 57Fe-labeled samples of IspH were prepared and studied by electron paramagnetic resonance (EPR), 57Fe electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies. Notably, this ENDOR study provides a rarely reported, complete determination of the 57Fe hyperfine tensors for all four Fe ions in a [4Fe-4S] cluster. The resting state of the enzyme (Ox) has a diamagnetic [4Fe-4S]2+ cluster. Reduction generates [4Fe-4S]+ (Red) with both S = 1/2 and S = 3/2 spin ground states. When the reduced enzyme is incubated with substrate, a transient paramagnetic reaction intermediate is detected (Int) which is thought to contain a cluster-bound substrate-derived species. The EPR properties of Int are indicative of a 3+ iron-sulfur cluster oxidation state, and the Mössbauer spectra presented here confirm this. Incubation of reduced enzyme with the product IPP induced yet another paramagnetic [4Fe-4S]+ species (Red+P) with S = 1/2. However, the g-tensor of this state is commonly associated with a 3+ oxidation state, while Mössbauer parameters show features typical for 2+ clusters. Implications of these complicated results are discussed.

High-Field (3.4 T) Electron Paramagnetic Resonance, 1H Electron-Nuclear Double Resonance, ESEEM, HYSCORE, and Relaxation Studies of Asphaltene Solubility Fractions of Bitumen for Structural Characterization of Intrinsic Carbon-Centered Radicals

Petroleum asphaltenes are considered the most irritating components of various oil systems, complicating the extraction, transportation, and processing of hydrocarbons. Despite the fact that the paramagnetic properties of asphaltenes and their aggregates have been studied since the 1950s, there is still no clear understanding of the structure of stable paramagnetic centers in petroleum systems. The paper considers the possibilities of various electron paramagnetic resonance (EPR) techniques to study petroleum asphaltenes and their solubility fractions using a carbon-centered stable free radical (FR) as an intrinsic probe. The dilution of asphaltenes with deuterated toluene made it possible to refine the change in the structure at the initial stage of asphaltene disaggregation. From the measurements of samples of bitumen, a planar circumcoronene-like model of FR structure and FR-centered asphaltenes is proposed. The results show that EPR-based approaches can serve as sensitive numerical tools to follow asphaltenes' structure and their disaggregation.

Electron nuclear interactions in spin-3/2 color centers in silicon carbide: A high-field pulse EPR and ENDOR study

High-frequency pulsed electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) were used to determine electron nuclear interactions on remote ligand shells of silicon and carbon in spin-3/2 color centers with an optically induced high-temperature spin alignment in hexagonal $4H$-, $6H$-, and rhombic $15R$-silicon carbide (SiC) polytypes. The EPR and ENDOR experimental data relate unambiguously to spin-3/2 centers in which the optically induced alignment of the spin-level populations occurs. The identification is based on resolved ligand hyperfine interactions with carbon and silicon nearest, next-nearest, and the more distant neighbors and on the determination of the unpaired electron spin densities. The hyperfine interactions with $^{29}\mathrm{Si}$ and $^{13}\mathrm{C}$ nuclei are unambiguously separated due to the selective population of the fine-structure levels with certain values of ${\mathrm{M}}_{\mathrm{S}}$. The signs of these interactions and, as a result, the signs of oscillating spin density at $^{29}\mathrm{Si}$ and $^{13}\mathrm{C}$ nuclei, are determined. On the basis of the EPR and optically induced ENDOR measurements, signs of the fine-structure splitting for all the centers were demonstrated, which made it possible to establish the character of optically induced spin alignment, including the inverse populations of the spin levels for these centers. The values of hyperfine interaction with $^{29}\mathrm{Si}$ and $^{13}\mathrm{C}$ nuclei, including those remote from the localization of the spin-3/2 center, are tabulated, which can be used by a number of algorithms in quantum information processing as long-term memory.

Radiation-Induced Stable Radicals in Calcium Phosphates: Results of Multifrequency EPR, EDNMR, ESEEM, and ENDOR Studies

This article presents the results of a study of radiation-induced defects in various synthetic calcium phosphate (CP) powder materials (hydroxyapatite—HA and octacalcium phosphate—OCP) by electron paramagnetic resonance (EPR) spectroscopy at the X, Q, and W-bands (9, 34, 95 GHz for the microwave frequencies, respectively). Currently, CP materials are widely used in orthopedics and dentistry owing to their high biocompatibility and physico-chemical similarity with human hard tissue. It is shown that in addition to the classical EPR techniques, other experimental approaches such as ELDOR-detected NMR (EDNMR), electron spin echo envelope modulation (ESEEM), and electron-nuclear double resonance (ENDOR) can be used to analyze the electron–nuclear interactions of CP powders. We demonstrated that the value and angular dependence of the quadrupole interaction for 14N nuclei of a nitrate radical can be determined by the EDNMR method at room temperature. The ESEEM technique has allowed for a rapid analysis of the nuclear environment and estimation of the structural positions of radiation-induced centers in various crystal matrices. ENDOR spectra can provide information about the distribution of the nitrate radicals in the OCP structure.

Metal ion fluxes controlling amphibian fertilization.

Mammalian oocytes undergo major changes in zinc content and localization in order to be fertilized, the most striking being the rapid exocytosis of over ten billion zinc ions, known as zinc sparks. Here we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese: synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through Electron-Nuclear Double-Resonance (ENDOR) studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.

Structures of Impurity Defects in Lithium Niobate and Tantalate Derived from Electron Paramagnetic and Electron Nuclear Double Resonance Data

Point intrinsic and extrinsic defects, especially paramagnetic ions of transition metals and rare-earth elements, have essential influence on properties of lithium niobate, LN and tantalate, LT, and often determine their suitability for numerous applications. Discussions about structures of the defects in LN/LT have lasted for decades. Many experimental methods facilitate progress in determining the structures of impurity centers. This paper gives current bird’s eye view on contributions of Electron Paramagnetic Resonance (EPR), and Electron Nuclear Double Resonance (ENDOR) studies to the determination of impurity defect structures in LN and LT crystals for a broad audience of researchers and students. Symmetry and charge compensation considerations restrict a number of possible structures. Comparison of measured angular dependences of ENDOR frequencies with calculated ones for Li and Nb substitution using dipole–dipole approximation allows unambiguously to determine the exact location of paramagnetic impurities. Models with two lithium vacancies explain angular dependencies of EPR spectra for Me3+ ions substituting for Li+ like Cr, Er, Fe, Gd, Nd, and Yb. Self-compensation of excessive charges through equalization of concentrations of Me3+(Li+) and Me3+(Nb5+) and appearance of interstitial Li+ in the structural vacancy near Me3+(Nb5+) take place in stoichiometric LN/LT due to lack of intrinsic defects.

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.

Murine Calprotectin Coordinates Mn(II) at a Hexahistidine Site with Ca(II)-Dependent Affinity.

Manganese is an essential metal ion that bacterial pathogens need to acquire from the vertebrate host during infection. In the mammalian nutritional immunity strategy to combat bacterial infection, the host restricts bacterial access to Mn(II) by sequestering this metal nutrient using the protein calprotectin (CP). The role of murine calprotectin (mCP) in Mn(II) sequestration has been demonstrated in vivo, but the molecular basis of this function has not been evaluated. Herein, biochemical assays and electron paramagnetic resonance (EPR) spectroscopy are employed to characterize the Mn(II) binding properties of mCP. We report that mCP has one high-affinity Mn(II) binding site. This site is a His6 site composed of His17 and His27 of mS100A8 and His92, His97, His105, and His107 of mS100A9. Similar to the human ortholog (hCP), Ca(II) binding to the EF-hand domains of mCP enhances the Mn(II) affinity of the protein; however, this effect requires ≈10-fold more Ca(II) than was previously observed for hCP. Mn(II) coordination to the His6 site also promotes self-association of two mCP heterodimers to form a heterotetramer. Low-temperature X-band EPR spectroscopy revealed a nearly octahedral Mn(II) coordination sphere for the Mn(II)-His6 site characterized by the zero-field splitting parameters D = 525 MHz and E/D = 0.3. Further electron-nuclear double resonance studies with globally 15N-labeled mCP provided hyperfine couplings from the coordinating ε-nitrogen atoms of the His ligands (aiso = 4.3 MHz) as well as the distal δ-nitrogen atoms (aiso = 0.25 MHz). Mn(II) competition assays between mCP and two bacterial Mn(II) solute-binding proteins, staphylococcal MntC and streptococcal PsaA, showed that mCP outcompetes both proteins for Mn(II) under conditions of excess Ca(II). In total, this work provides the first coordination chemistry study of mCP and reveals striking similarities in the Mn(II) coordination sphere as well as notable differences in the Ca(II) sensitivity and oligomerization behavior between hCP and mCP.

HYSCORE Insights into the Distribution of the Unpaired Spin Density in an Engineered CuA Site in Azurin and Its His120Gly Variant.

A comparative study of the 1H and 14N hyperfine interactions between the CuA site in an engineered CuA center in azurin (WT-CuAAz) and its His120Gly variant (H120G-CuAAz) using the two-dimensional ESEEM technique, HYSCORE, is reported. HYSCORE spectroscopy has clarified conflicting results in previous electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies and found clear differences between the two CuA azurins. Specifically, a hyperfine coupling AN⊥ of 15.3 MHz was determined for the first time from the frequencies of double-quantum transitions of 14N histidine nitrogens coordinated to CuA in WT-CuAAz. In contrast, such coupling was not observed in the spectra of H120G-CuAAz, indicating at least a several megahertz increase in AN⊥ for the coordinated nitrogen in this variant. In addition, 14N HYSCORE spectra of WT-CuAAz show interaction with only one type of weakly coupled nitrogen assigned to the remote Nε atom of coordinated imidazole residues based on the quadrupole coupling constant ( e2 Qq/4 h) of ∼0.4 MHz. The spectrum of H120G-CuAAz resolves additional features typical for backbone peptide nitrogens with larger e2 Qq/4 h values of ∼0.7 MHz. Hyperfine couplings with these nitrogens vary between ∼0.4 and 0.7 MHz. In addition, the two resolved cross-peaks from Cβ protons in H120G-CuAAz display only ∼1 MHz shifts relative to the corresponding peaks in WT-CuAAz. These new findings have provided the first experimental evidence of the previous density functional theory analysis that predicted changes in the delocalized electron spin population of ∼0.02-0.03 (i.e., ∼10%) on copper and sulfur atoms of the CuA center in H120 variants relative to WT-CuAAz and resolved contradicting results between EPR and ENDOR studies of the valence distribution in CuAAz and its variants.

Radicals initiated by gamma rays in selected amino acids and collagen

Abstract Calf skin collagen and three amino acids essential for its structure, namely glycine, L-proline and 4-hydroxyl-L-proline, were irradiated with gamma rays up to a dose of 10 kGy. Conversion of radicals over time or after thermal annealing to selected temperatures was monitored by X-band electron paramagnetic resonance (EPR) spectroscopy. Some experimental spectra were compared with signals simulated based on literature data from the electron nuclear double resonance (ENDOR) studies. The following phenomena were confirmed in the tested amino acids: abstraction of hydrogen atom (glycine, proline, hydroxyproline, collagen), deamination (glycine, hydroxyproline), decarboxylation (hydroxyproline). Chain scission at glycine residues, radiation-induced decomposition of side groups and oxidative degradation were observed in irradiated collagen. The decay of radicals in collagen saturated with water occurred at lower temperatures than in macromolecules having only structural water. The paramagnetic centres were the most stable in an oxygen-free atmosphere (vacuum). Radical processes deteriorated the structure of collagen; hence, radiation sterilization of skin grafts requires careful pros and cons analysis.

Lanthanide Ions as Local Probes in Ionic Hydrides: A Pulsed Electron Nuclear Double Resonance and Thermoluminescence Study of Eu2+-Doped Hydride Perovskites

A model study on the use of pulsed electron paramagnetic resonance spectroscopy and optical spectroscopy of Eu2+ as a local probe in hydrides is presented for the Eu2+-doped (and codoped) perovskites LiMH3 and LiMD3 (M = Sr or Ba). Two-pulse field-swept electron spin echo, Davies and Mims electron nuclear double resonance, Davies-type difference triple resonance experiments, and thermoluminescence spectroscopy, as well as density functional calculations, were carried out. Electron nuclear double resonance spectroscopy allows for an unambiguous determination of the position occupied by the dopant ion, which is important information necessary for the correct interpretation of optical spectra. With regard to the current importance of hydrides and mixed anionic hydrides, doping with rare-earth or transition-metal ions and the combination of pulsed EPR and optical spectroscopy may serve as a promising and convenient tool for studying such materials.

Biomolecular EPR Meets NMR at High Magnetic Fields

In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.

EPR and ENDOR in manganese doped SrTiO3: Electric quadrupole interaction and local disorder

Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies were performed on heavily manganese doped SrTiO3 crystals. At low temperatures (T≈6K), the ENDOR measurements on MnTi4+ show the presence of electric field gradient associated with the electrical polarization. The observation of EPR signals of rhombic Mn2+ complexes remotely compensated in the SrTiO3 lattice is discussed in connection with local disorder in which Mn2+ appears due to the reduction process.

Electron paramagnetic resonance of a copper doped [(CH3)2NH2][Zn(HCOO)3] hybrid perovskite framework.

We report a continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) as well as pulse electron nuclear double resonance (ENDOR) study of Cu2+ doped [(CH3)2NH2][Zn(HCOO)3] hybrid perovskite which exhibits a structural phase transition. The multifrequency (X, Q and W-band) CW EPR measurements allow the temperature evolution of the Cu2+ ion local environment to be studied. The spectrum of the ordered (low-temperature) phase reveals an axially distorted octahedral Cu2+ site confirming the successful replacement of the Zn2+ ions and formation of the CuO6 octahedra. The CW EPR spectrum of the disordered (high-temperature) phase shows an additional broad line which gradually diminishes on cooling. The EPR linewidth of the axially symmetric Cu2+ ion site exhibits an anomaly at the phase transition point and Arrhenius-type behavior in the disordered phase. The temperature dependent Cu2+ spin Hamiltonian parameters change abruptly at the phase transition point indicating a strong first-order character of the transition. The X-band pulse ENDOR spectrum of the ordered phase reveals several protons in the vicinity of the Cu2+ center.

In Quest of the Alanine R3 Radical: Multivariate EPR Spectral Analyses of X-Irradiated Alanine in the Solid State.

The amino acid l-α-alanine is the most commonly used material for solid-state electron paramagnetic resonance (EPR) dosimetry, due to the formation of highly stable radicals upon irradiation, with yields proportional to the radiation dose. Two major alanine radical components designated R1 and R2 have previously been uniquely characterized from EPR and electron-nuclear double resonance (ENDOR) studies as well as from quantum chemical calculations. There is also convincing experimental evidence of a third minor radical component R3, and a tentative radical structure has been suggested, even though no well-defined spectral signature has been observed experimentally. In the present study, temperature dependent EPR spectra of X-ray irradiated polycrystalline alanine were analyzed using five multivariate methods in further attempts to understand the composite nature of the alanine dosimeter EPR spectrum. Principal component analysis (PCA), maximum likelihood common factor analysis (MLCFA), independent component analysis (ICA), self-modeling mixture analysis (SMA), and multivariate curve resolution (MCR) were used to extract pure radical spectra and their fractional contributions from the experimental EPR spectra. All methods yielded spectral estimates resembling the established R1 spectrum. Furthermore, SMA and MCR consistently predicted both the established R2 spectrum and the shape of the R3 spectrum. The predicted shape of the R3 spectrum corresponded well with the proposed tentative spectrum derived from spectrum simulations. Thus, results from two independent multivariate data analysis techniques strongly support the previous evidence that three radicals are indeed present in irradiated alanine samples.

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na+ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M+ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron–sulfur cluster. ENDOR studies of the PFL-AE/[13C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little–no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li+ to Cs+, PFL-AE activity sharply maximizes at K+, with NH4+ closely matching the efficacy of K+. PFL-AE is thus a type I M+-activated enzyme whose M+ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron–sulfur cluster.

Multi-frequency (S, X, Q and W-band) EPR and ENDOR Study of Vanadium(IV) Incorporation in the Aluminium Metal-Organic Framework MIL-53.

Doping the well-known metal-organic framework MIL-53(Al) with vanadium(IV) ions leads to significant changes in the breathing behaviour and might have repercussions on the catalytic behaviour as well. To understand the properties of such a doped framework, it is necessary to determine where dopant ions are actually incorporated. Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) are applied to reveal the nearest environment of the paramagnetic vanadium(IV) dopant ions. EPR spectra of as-synthesised vanadium-doped MIL-53 are recorded at S-, X-, Q- and W-band microwave frequencies. The EPR spectra suggest that at low dopant concentrations (1.0-2.6 mol %) the vanadium(IV) ions are well dispersed in the matrix. Varying the vanadium dopant concentration within this range or the dopant salt leads to the same dominant EPR component. In the ENDOR spectra, hyperfine (HF) interactions with (1) H, (27) Al and (51) V nuclei are observed. The HF parameters extracted from simulations strongly suggest that the vanadium(IV) ions substitute Al in the framework.

Spectroscopic studies reveal that the heme regulatory motifs of heme oxygenase-2 are dynamically disordered and exhibit redox-dependent interaction with heme.

Heme oxygenase (HO) catalyzes a key step in heme homeostasis: the O2- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. Mammals contain two isoforms of this enzyme, HO2 and HO1, which share the same α-helical fold forming the catalytic core and heme binding site, as well as a membrane spanning helix at their C-termini. However, unlike HO1, HO2 has an additional 30-residue N-terminus as well as two cysteine-proline sequences near the C-terminus that reside in heme regulatory motifs (HRMs). While the role of the additional N-terminal residues of HO2 is not yet understood, the HRMs have been proposed to reversibly form a thiol/disulfide redox switch that modulates the affinity of HO2 for ferric heme as a function of cellular redox poise. To further define the roles of the N- and C-terminal regions unique to HO2, we used multiple spectroscopic techniques to characterize these regions of the human HO2. Nuclear magnetic resonance spectroscopic experiments with HO2 demonstrate that, when the HRMs are in the oxidized state (HO2O), both the extra N-terminal and the C-terminal HRM-containing regions are disordered. However, protein NMR experiments illustrate that, under reducing conditions, the C-terminal region gains some structure as the Cys residues in the HRMs undergo reduction (HO2R) and, in experiments employing a diamagnetic protoporphyrin, suggest a redox-dependent interaction between the core and the HRM domains. Further, electron nuclear double resonance and X-ray absorption spectroscopic studies demonstrate that, upon reduction of the HRMs to the sulfhydryl form, a cysteine residue from the HRM region ligates to a ferric heme. Taken together with EPR measurements, which show the appearance of a new low-spin heme signal in reduced HO2, it appears that a cysteine residue(s) in the HRMs directly interacts with a second bound heme.

EPR, ESE, and pulsed ENDOR study of the nitrogen donors in 15R SiC grown under carbon‐rich conditions

X‐band field‐sweep electron spin echo and pulsed electron nuclear double resonance (ENDOR) spectroscopy were used to study n‐type 15R SiC wafers grown under carbon (C)‐rich conditions with the aim to verify the recently proposed concept that nitrogen (N) donors substitute both carbon (C) and silicon (Si) sites and may occupy nonequivalent positions at Si sites. It was found that besides the 14N ENDOR spectra of the C substituting quasicubic “k1”, “k2”, and “k3” positions five doublet lines due to 14N nuclei at other lattice positions were observed in the ENDOR spectrum of highly compensated C‐rich n‐type 15R SiC. Three of them with the hyperfine interaction (HFI) constants 34.66, 32.20, and 29.77 MHz were attributed to the N donors substituting “k1”, “k2”, and “k3” positions at Si sites. Two other additional ENDOR spectra were explained by the presence of the C antisite (CSi) defects in C‐rich 15R SiC, which leads to the formation of CSiNC complexes with donor levels close to the conduction‐band minimum and isotropic 14N HFI constants of 21.58 and 25.94 MHz. The energy levels of N donors have been evaluated by comparing the number of ENDOR spectra observed in C‐rich 15R SiC with different degrees of compensation.