1H Electron Nuclear Double Resonance Research Articles

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

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.

Solution Tetrahydrobiopterin Radical vs. the Enzyme-Bound Radical: A Paramagnetic Reconciliation

Background: Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) and citrulline from L-arginine, dioxygen (O2), and nicotinamide adenine dinucleotide phosphate (NADPH) in a two-step reaction, with the enzyme-bound intermediate Nω-hydroxy-L-arginine (NHA). Previous electron paramagnetic resonance (EPR) studies of NOS reaction have shown that (6R, 1'R, 2'S)-6-(l',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin (H4B) acts as a single electron donor in both steps of the reaction, resulting in the transient generation of a tetrahydropterin cation radical (H4B•+). Results: H4B•+can also be chemically generated in strongly acidic solutions. EPR studies of chemically generated H4B•+and similar pterin radicals date back to the 1960s. However, the reported paramagnetic parameters of H4B•+in NOS do not seem to match the corresponding reported parameters for either H4B•+or other pterin centered radicals chemically generated in solution. In particular, the rather isotropic hyperfine coupling of ca. 45 MHz for 1H6 of H4B•+in NOS is at least 15 MHz larger than that of H4B•+ or any other previously studies pterin solution radical. In the work reported here, a combination of 9.5 - 9.8 GHz contentious wave (cw-) EPR, 34GHz 1H electron nuclear double resonance (ENDOR), spectral simulation and Density Functional Theory (DFT) calculations were used to investigate this seeming discrepancy. Conclusion: We demonstrated that the differences in the paramagnetic parameters of the chemically generated H4B radicals in solutions and those of the H4B radicals in NOS are consistent with the presence of two different conformers of the same cation radical in the two media.

Vanadyl Porphyrin Speciation Based on Submegahertz Ligand Proton Hyperfine Couplings

The speciation of vanadyl (VO2+) porphyrins in crude oil (vanadyl petroporphyrins) is an area of ongoing interest in petroleomics. In this paper, we describe a method for the speciation of vanadyl porphyrins that uses electron nuclear double resonance (ENDOR), a high-resolution electron paramagnetic resonance (EPR) spectroscopic technique. We use 1H ENDOR to measure hyperfine couplings between ligand protons and the paramagnetic vanadyl ion as small as about 0.15 MHz. From the measured hyperfine couplings, we directly determine all vanadium–ligand proton distances up to 8 A. This information differentiates porphyrin ligands by their ring substitution pattern and substituent nature. We demonstrate this using a series of vanadyl porphyrin model compounds. Additionally, we demonstrate that the composition of binary vanadyl porphyrins mixtures can be determined. The ability of ENDOR to differentiate types of ligand protons in vanadyl porphyrin mixtures provides a basis for analyzing more complex mixtures of v...

1H high field electron-nuclear double resonance spectroscopy at 263 GHz/9.4 T.

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.

Pulse EPR and ENDOR Study of Manganese Doped [(CH3)2NH2][Zn(HCOO)3] Hybrid Perovskite Framework

We present a pulse electron paramagnetic resonance (EPR) and electron–nuclear double resonance (ENDOR) study of a manganese-doped [(CH3)2NH2][Zn(HCOO)3] dense metal–organic framework which exhibits a structural phase transition at 163 K. The echo-detected field sweep Mn2+ EPR spectrum of the low-temperature phase is in a perfect agreement with the previous continuous-wave EPR results, while the spectrum of the disordered phase reveals a significant EPR transition-dependent relaxation. The 1H ENDOR pattern indicates several protons in the vicinity of the Mn2+ ion. The experimental ENDOR spectrum is successfully simulated using the proton hyperfine tensors calculated by the density functional theory. A multifrequency electron spin echo envelope modulation (ESEEM) spectroscopy shows a peculiar signal which is unaffected by the external magnetic field. The modulation depth of this signal starts to decrease above 40 K, coinciding with the temperature at which the methyl groups of the (CH3)2NH2+ cations start t...

Understanding the Coordination Modes of [Cu(acac)2(imidazole)n=1,2] Adducts by EPR, ENDOR, HYSCORE, and DFT Analysis.

The interaction of imidazole with a [Cu(acac)2] complex was studied using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), hyperfine sublevel correlation spectroscopy (HYSCORE), and density functional theory (DFT). At low Im ratios (Cu:Im 1:10), a 5-coordinate [Cu(acac)2Imn=1] monoadduct is formed in frozen solution with the spin Hamiltonian parameters g1 = 2.063, g2 = 2.063, g3 = 2.307, A1 = 26, A2 = 15, and A3 = 472 MHz with Im coordinating along the axial direction. At higher Im concentrations (Cu:Im 1:50), a 6-coordinate [Cu(acac)2Imn=2] bis-adduct is formed with the spin Hamiltonian parameters g1 = 2.059, g2 = 2.059, g3 = 2.288, A1 = 30, A2 = 30, and A3 = 498 MHz with a poorly resolved 14N superhyperfine pattern. The isotropic EPR spectra revealed a distribution of species ([Cu(acac)2], [Cu(acac)2Imn=1], and [Cu(acac)2Imn=2]) at Cu:Im ratios of 1:0, 1:10, and 1:50. The superhyperfine pattern originates from two strongly coordinating N3 imino nitrogens of the Im ring. Angular selective 14N ENDOR analysis revealed the NA tensor of [34.8, 43.5, 34.0] MHz, with e2qQ/h = 2.2 MHz and η = 0.2 for N3. The hyperfine and quadrupole values for the remote N1 amine nitrogens (from HYSCORE) were found to be [1.5, 1.4, 2.5] MHz with e2qQ/h = 1.4 MHz and η = 0.9. 1H ENDOR also revealed three sets of HA tensors corresponding to the nearly equivalent H2/H4 protons in addition to the H5 and H1 protons of the Im ring. The spin Hamiltonian parameters for the geometry optimized structures of [Cu(acac)2Imn=2], including cis-mixed plane, trans-axial, and trans-equatorial, were calculated. The best agreement between theory and experiment indicated the preferred coordination is trans-equatorial [Cu(acac)2Imn=2]. A number of other Im derivatives were also investigated. 4(5)-methyl-imidazole forms a [Cu(acac)2(Im-3)n=2] trans-equatorial adduct, whereas the bulkier 2-methyl-imidazole (Im-2) and benzimidazole (Im-4) form the [Cu(acac)2(Im-2,4)n=1] monoadduct only. Our data therefore show that subtle changes in the substrate structure lead to controllable changes in coordination behavior, which could in turn lead to rational design of complexes for use in catalysis, imaging, and medicine.

Influence of Ring-Expanded N-Heterocyclic Carbenes on the Structures of Half-Sandwich Ni(I) Complexes: An X-ray, Electron Paramagnetic Resonance (EPR), and Electron Nuclear Double Resonance (ENDOR) Study.

Potassium graphite reduction of the half-sandwich Ni(II) ring-expanded diamino/diamidocarbene complexes CpNi(RE-NHC)Br gave the Ni(I) derivatives CpNi(RE-NHC) (where RE-NHC = 6-Mes (1), 7-Mes (2), 6-MesDAC (3)) in yields of 40%-50%. The electronic structures of paramagnetic 1-3 were investigated by CW X-/Q-band electron paramagnetic resonance (EPR) and Q-band 1H electron nuclear double resonance (ENDOR) spectroscopy. While small variations in the g-values were observed between the diaminocarbene complexes 1 and 2, pronounced changes in the g-values were detected between the almost isostructural species (1) and diamidocarbene species (3). These results highlight the sensitivity of the EPR g-tensor to changes in the electronic structure of the Ni(I) centers generated by incorporation of heteroatom substituents onto the backbone ring positions. Variable-temperature EPR analysis also revealed the presence of a second Ni(I) site in 3. The experimental g-values for these two Ni(I) sites detected by EPR in frozen solutions of 3 are consistent with resolution on the EPR time scale of the disordered components evident in the X-ray crystallographically determined structure and the corresponding density functional theory (DFT)-calculated g-tensor. Q-band 1H ENDOR measurements revealed a small amount of unpaired electron spin density on the Cp rings, consistent with the calculated SOMO of complexes 1-3. The magnitude of the 1H A values for 3 were also notably larger, compared to 1 and 2, again highlighting the influence of the diamidocarbene on the electronic properties of 3.

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli.

The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz' = 11.8 MHz, whereas for H2, Tz' = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.

Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.

Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.

The Use of Deuterated Camphor as a Substrate in 1H ENDOR Studies of Hydroxylation by Cryoreduced Oxy P450cam Provides New Evidence of the Involvement of Compound I

Electron paramagnetic resonance and (1)H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1R)-camphor (H(2)-camphor) and (1R)-5,5-dideuterocamphor (D(2)-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam-O(2)-substrate complex. Hydroxylation of H(2)-camphor produced a primary product state in which 5-exo-hydroxycamphor is coordinated with Fe(III). ENDOR spectra contained signals derived from two protons [Fe(III)-bound C5-OH(exo) and C5-H(endo)] from camphor. When D(2)-camphor was hydroxylated under the same condition in H(2)O or D(2)O buffer, both ENDOR H(exo) and H(endo) signals are absent. For D(2)-camphor in H(2)O buffer, H/D exchange causes the C5-OH(exo) signal to reappear during relaxation upon annealing to 230 K; for H(2)-camphor in D(2)O, the magnitude of the C5-OH(exo) signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam.

Solvation of Small Disulfonate Anions in Water/Methanol Mixtures Characterized by High-Field Pulse Electron Nuclear Double Resonance and Molecular Dynamics Simulations

The solvation of Fremy's salt, the paramagnetic nitrosodisulfonate anion ON(SO(3)(-))(2), in binary solvent mixtures was investigated by means of pulse (Mims- and Davies-type) electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) simulations. (1)H and (2)H pulse ENDOR measurements were performed on small Fremy's salt radicals in isotope-substituted solvent mixtures of methanol and water in frozen solution. We were able to obtain well-resolved, orientation-selective, high-field/high-frequency pulse ENDOR spectra of methyl protons from the alcohol moiety and exchangeable protons from the alcohol-hydroxyl group and water. In the studied solvent systems (volume ratio v/v = 30:70, 50:50, 70:30), the solvation of 2.5 mM Fremy's salt by methyl protons was found to be almost identical. From the analysis of the dependence of pulse ENDOR spectra on the observer field position and spectral simulations, we obtained the principal components of the hyperfine coupling (hfc) tensor for each class of protons. The combination of Mims- and Davies-type pulse ENDOR measurements was necessary to obtain blind spot free information on hfc that spans a broad range of 0.25-6 MHz. Using the point-dipole approximation, the dipolar hfc component yields a prominent electron-nuclear distance of 3.5 A between Fremy's salt and methyl protons, which was found along the molecular z-axis (perpendicular to the approximate plane spanned by ON(S)(2)) of the probe molecule. Exchangeable protons were found to be distributed nearly isotropically, forming a hydrogen-bonded network around the sulfonate groups. The distribution of exchangeable and methyl protons found in MD simulations is in very good agreement with the pulse ENDOR results, and we find that solvation is dominated by an interplay of H-bond (electrostatic) interactions and steric properties. The elucidation of the microscopic solvation of a small probe molecule in binary solvent mixtures represents the first step for understanding the interactions in more complex biochemical systems. In particular, this includes the potential perturbation of the H-bond network due to the presence of a spin probe or other polar molecules.

Air-stable Curved π-Radical Based on Corannulene: Dynamic Electronic-spin Structure Induced by Temperature-dependent Conformational Changes

A new corannulene-based curved neutral π-radical bearing a tert-butylnitroxide moiety has been synthesized and isolated as an air-stable solid. Direct attachment of the spin centre to the corannulene skeleton gives rise to an extensive spin-delocalization onto the curved π-conjugated system from the nitroxide moiety. This salient electronic feature in the curved neutral radical system and its high stability have allowed us to find a dynamic electronic-spin behaviour induced by the temperature-dependent conformational change of the nitroxide moiety, as studied by liquid-phase variable-temperature electron spin resonance and 1H electron-nuclear double resonance and electron-nuclear-nuclear triple resonance spectroscopies with the help of density functional theory calculations.

The catalytic Mn2+ sites in the enolase-inhibitor complex: crystallography, single-crystal EPR, and DFT calculations.

Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 A, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and 1H ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the 1H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the Mn-O bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites.

Determination of the Hydration Number of Gadolinium(III) Complexes by High‐Field Pulsed 17O ENDOR Spectroscopy

Pulsed 17O Mims electron-nuclear double resonance (ENDOR) spectroscopy at the W band (95 GHz) and D band (130 GHz) is used for the direct determination of the water coordination number (q) of gadolinium-based magnetic resonance imaging (MRI) contrast agents. Spectra of metal complexes in frozen aqueous solutions at approximately physiological concentrations can be obtained either in the presence or absence of protein targets. This method is an improvement over the 1H ENDOR method described previously, which involved the difference ENDOR spectrum of exchangeable protons from spectra taken in H2O and D2O. In addition to exchangeable water protons, the 1H ENDOR method is also sensitive to other exchangeable protons, and it is shown here that this method can overestimate hydration numbers for complexes with exchangeable protons at GdH distances similar to that of the coordinated water, for example, from NH groups. The 17O method does not suffer from this limitation. 17O ENDOR spectroscopy is applied to Gd(III) complexes containing zero, one, or two inner-sphere water molecules. In addition, 13C and 1H ENDOR studies were performed to assess the extent of methanol coordination, since methanol is used to produce a glass in these experiments. Under the experimental conditions used for the hydration number determination (30 mol % methanol), fewer than 15 % of the coordination sites were found to be occupied by methanol.

Middle Macroradicals and Their Influence on the Strength of Oriented Polymers

The macroradicals formed through the detachment of hydrogen atoms in amorphous-crystalline polymers are studied using electron-nuclear double resonance (ENDOR) spectroscopy. The 1H ENDOR spectra are analyzed, and the hyperfine interaction constants of the nearest neighbor and remote protons are measured. The conformational structure of radicals in poly(ethylene) (PE), poly(propylene) (PP), and poly(caproamide) (PCA) is determined. The ENDOR and EPR spectra of fibers subjected to tensile loading are recorded. It is revealed that torsional strain arises in the radicals with a nonplanar structure. The results of mechanical testing of irradiated oriented polymers demonstrate that the weakening of β bonds in the radicals has an effect on the macroscopic strength.

Preservation of endometallofullerene La@C 82 in polycarbonate: EPR, ENDOR, and NMR studies

On embedding in poly(bisphenol A carbonate) film, La@C 82 has featured the exclusive stability against oxygen for more than 1 year's storage of the polymer films on air. The EPR spectral parameters of La@C 82 have occurred to be sensitive to the segmental mobility and the phase transition of the polymer from the glassy to the high-elasticity state. Furthermore, the intense matrix 1H-electron nuclear double resonance (ENDOR) signal discovered in this system testifies the partial transfer of the electron spin density outside the fullerene cage onto the polymer protons. The 139La NMR spectrum of the same suggests that La@C 82 forms nanoparticles, in which the metal atoms strongly interact with the delocalized electrons. The “spin-leakage” of the fullerene shell, along with the tendency of endohedral fullerenes (EMF) to form nanoparticles, is worth consideration in sight of their applications in material sciences.

Effects of substrates (methyl isocyanide, C 2H 2) and inhibitor (CO) on resting-state wild-type and NifV −Klebsiella pneumoniae MoFe proteins

We report the use of electron nuclear double resonance (ENDOR) spectroscopy to examine how the metal sites in the FeMo-cofactor cluster of the resting nitrogenase MoFe protein respond to addition of the substrates acetylene and methyl isocyanide and the inhibitor carbon monoxide. 1H, 57Fe and 95Mo ENDOR measurements were performed on the wild-type and the NifV −proteins from Klebsiella pneumoniae. Among the molecules tested, only the addition of acetylene to either protein induced widespread changes in the 57Fe ENDOR spectra. Acetylene also induced increases in intensity from unresolved protons in the proton ENDOR spectra. Thus we conclude that acetylene may bind to the resting-state MoFe protein to perturb the FeMo-cofactor environment. On the other hand, the present results show that methyl isocyanide and carbon monoxide do not substantially alter the FeMo cofactor’s geometric and electronic structures. We interpret this as lack of interaction between those two molecules and the FeMo cofactor in the resting state MoFe protein. Thus, although it is generally accepted that substrates or inhibitors bind to the FeMo-cofactor only under turnover condition, this work provides evidence that at least one substrate can perturb the active site of nitrogenase under non-catalytic conditions.

Heterogeneous distribution of paramagnetic radicals in insoluble organic matter from the Orgueil and Murchison meteorites

An electron paramagnetic resonance (EPR) investigation was performed on the insoluble organic matter from the Orgueil and Murchison meteorites and on terrestrial coals with similar elemental composition. A complementary electron nuclear double resonance (ENDOR) study was also carried out. The measured g-factors of the observed paramagnetic radicals in the meteoritic organic matter exhibit a similar correlation with the chemical composition as for the type III (i.e., hydrogen-poor) terrestrial coals. The main result, obtained from EPR saturation and 1H ENDOR enhancement measurements, showed that the effective local concentration in radicals of about 3 to 4 × 10 19 spin.g −1 in the meteoritic organic matter is much higher than the average concentration, hence the occurrence of radical-rich regions accounting for 5% and 20% of the total volume for Murchison and Orgueil, respectively. This heterogeneity of concentration seems to be unique among natural organic macromolecules. It is proposed that these radical-rich regions correspond to pristine parts of the organic matter synthesized in conditions close to those prevailing in the interstellar medium, and which have survived the hydrothermal process on the parent body.

The Process of Mn(II) Incorporation into Aluminophosphate Zeotypes through High-Field ENDOR Spectroscopy and DFT Calculations

High-field EPR and pulsed electron−nuclear double resonance (ENDOR) spectroscopies were used to investigate the formation of Mn−AlPO4-11, Mn−AlPO4-5, and Mn−SAPO-5. Samples recovered from reaction mixtures quenched at different times were subjected to EPR, ENDOR and X-ray diffraction (XRD) measurements, and the variations in the 31P and 1H hyperfine couplings, which are sensitive probes to the Mn−P interaction and the Mn(II) hydration, respectively, were followed. The intensity of the 1H ENDOR signal decreased with reaction time, showing that the amount of both water ligands and solvent water in the Mn(II) vicinity decreased. A relatively large isotropic 31P hyperfine coupling (Aiso(31P) ≈ 7 MHz), confirming the formation of Mn(II) framework sites, was found in all final products, whereas a smaller Aiso(31P), 4−5 MHz, was detected in samples quenched at early stages of the reaction. The latter was assigned to Mn(II) incorporated into a network of disordered aluminophosphate precursors. These precursors are formed prior to the detection of an XRD pattern, and are gradually transformed to the final three-dimensional crystalline structures. The changes in Aiso(31P) were attributed to transformations occurring both in the bonding topology and in the coordination sphere of Mn(II), where water ligands are gradually replaced by −O−P linkages. This interpretation was supported by the decrease in the intensity of the 1H ENDOR signals, and by a series of DFT cluster model optimizations on intermediates of the form [Mn(H2O)x(OP(OH)3)y]2+, where x + y = 6, 5 or 4, followed by calculations of hyperfine coupling constants. Although the theoretical hyperfine values were overestimated with respect to the experimental ones, a satisfactory correlation was found between the trends within the calculated Aiso(55Mn,31P), and the experimental trends observed during the molecular sieves formation.