ENDOR Results Research Articles

Möbius-Hückel topology switching in an expanded porphyrin cation radical as studied by EPR and ENDOR spectroscopy.

The symmetry of the arrangement of objects has fascinated philosophers, artists and scientists for a long time, and still does. Symmetries often exist in nature, but are also created artificially, for instance by chemical synthesis of novel molecules and materials. The one-sided, non-orientable Möbius band topology is a paradigm of such a symmetry-based fascination. In the early 1960s, in synthetic organic chemistry the interest in molecules with Möbius symmetry was greatly stimulated by a short paper by Edgar Heilbronner. He predicted that sufficiently large [n]annulenes with a closed-shell electron configuration of 4n π-electrons should allow for sufficient π-overlap stabilization to be synthesizable by twisting them with a 180° phase change into the Möbius symmetry of their hydrocarbon skeleton. In 2007, the group of Lechosław Latos-Grażyński succeeded in synthesizing the compound di-p-benzi[28]hexa-phyrin(1.1.1.1.1.1), compound 1, which can dynamically switch between Hückel and Möbius conjugation depending, in a complex manner, on the polarity and temperature of the surrounding solvent. This discovery of "topology switching" between the two-sided (Hückel) and one-sided (Möbius) molecular state with closed-shell electronic configuration was based primarily on the results of NMR spectroscopy and DFT calculations. The present EPR and ENDOR work on the radical cation state of compound 1 is the first study of a ground-state open-shell system which exhibits a Hückel-Möbius topology switch that is controlled by temperature, like in the case of the closed-shell precursor. The unpaired electron interacting with magnetic nuclei in the molecule is used as a sensitive probe for the electronic structure and its symmetry properties. For a Hückel conformer with its higher symmetry, we expect - and observe - fewer ENDOR lines than for a Möbius conformer. The ENDOR results are supplemented by and in accordance with theoretical calculations based on density functional theory at the ORCA level.

ChemInform Abstract: Conformational and Substituent Effects on Spin Distributions in Porphyrin Cation Radicals

Optical, redox, EPR, and ENDOR results are presented for zinc meso-tetraphenyltetrabenzoporphyrin (ZnTPTBP). The molecule is a hybrid of the common synthetic porphyrins zinc tetraphenylporphyrin (ZnTPPP) and zinc tetrabenzoporphyrin (ZnTBP). The consequences of the additional peripheral substituents in the hybrid are evident in a previously obtained x-ray structure of ZnTPTBP that shows the macrocycle to be severely puckered to minimize steric interactions between the substituents. The cumulative effects of conformational changes and substituent addition are reflected in the red-shifted optical spectrum and ease of oxidation of the neutral compound, and in a shift of unpaired spin density from a{sub 1u} (ZnTBP) to a{sub 2u} (ZnTPP) character in the radical. Significant spin density is found to delocalize onto the phenyl substituents of ZnTPTBP{sup {sm bullet}+} and illustrates the shortening of the effective distance between donor and acceptor that may occur in biomimetic complexes used to investigate the distance and energy dependence of electron transfer.

Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG)

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe(4)S(4) cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using (2)H, (13)C and (17)O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an eta(2)-alkenyl pi- (or pi/sigma) complex with the 4th Fe in the Fe(4)S(4) cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K(i) approximately 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a (13)C hyperfine coupling of approximately 7 MHz), as well as literature precedent, we propose that the alkyne forms another pi/sigma metallacycle, an eta(2)-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.

What Affects the Quartet−Doublet Energy Splitting in Peroxidase Enzymes?

Density functional theory calculations have been performed on the active species (Compound I) of cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX) models. We have calculated a large model containing oxo-iron porphyrin plus a hydrogen-bonded network of the axial bound imidazole ligand connected to an acetic acid and an indole group, which mimic the His(175), Asp(235), and Trp(191) amino acids in cytochrome c peroxidase. Our optimized geometries are in good agreement with X-ray and crystallographic structures and give an electronic ground state in agreement with EPR and ENDOR results. We show that the quartet-doublet state ordering and the charge distribution within the model are dependent on small external perturbations. In particular, a single point charge at a distance of 8.7 A is shown to cause delocalization of the charge and radical characters within the model, thereby creating either a pure porphyrin cation radical state or a tryptophan cation radical state. Thus, our calculations show that small external perturbations are sufficient to change the electronic state of the active species and subsequently its catalytic properties. Similar effects are possible with the addition of an electric field strength along a specific coordination axis of the system. The differences between the electronic ground states of CcP and APX Cpd I are analyzed on the basis of external perturbations.

Conformational substates of the oxyheme centers in alpha and beta subunits of hemoglobin as disclosed by EPR and ENDOR studies of cryoreduced protein.

Exposure of frozen solutions of oxyhemoglobin to gamma-irradiation at 77 K yields EPR- and ENDOR-active, one-electron-reduced oxyheme centers which retain the conformation of the diamagnetic precursor. EPR spectra have been collected for the centers produced in human HbO(2) and isolated alphaO(2) and betaO(2) chains, as well as alphaO(2)beta(Zn), alpha(Zn)betaO(2), and alphaO(2)beta(Fe(3+)) hybrids, each in frozen buffer and in frozen glasses that form in the presence of glycols and sugars and also in the presence of IHP. These reveal two spectroscopically distinct classes of such ferriheme centers (g(1) <or= 2.25), denoted A and B. Averaged over many similar sites, the A-center has a rhombic EPR signal with a g-tensor, g(A) = [2.248(4), 2.146(1), 1.966(1)]; the B-center exhibits a less anisotropic EPR signal, g(B) = [2.216(3), 2.118(2), 1.966(1)]. Early measurement had suggested that, in the cryoreduced HbO(2) tetramer, the two centers corresponded to the two different chains [Symons, M. C. R., and Petersen, R. L. (1978) Proc. R. Soc. London, Ser. B 201, 285-300]. However, the present EPR and ENDOR results show that the two signals instead reflect the fact that the parent oxyhemes exist in two major conformational substates and that this is true for both alphaO(2) and betaO(2) subunits: alphaO(2)(A) (minor species) and alphaO(2)(B) (major species); betaO(2)(A)(major species) and betaO(2)(B) (minor species). Similar behavior is seen for MbO(2) [Kappl, R., Höhn-Berlage, M., Hüttermann, J., Bartlett, N., and Symons, M. C. R. (1985) Biochim. Biophys. Acta 827, 327-343]. The A/B g-tensors of alphaO(2) and betaO(2) chains vary little with the environment of the chains, while the relative populations of the substates depend greatly on glycols and IHP. These results suggest a quaternary influence on the oxyheme distal pocket of alpha chains and that the glycol-induced changes in the substate populations of the R-state HbO(2) tetramer are largely associated with the alphaO(2) subunit. (1)H ENDOR spectra from the distal histidine proton hydrogen-bonded to the peroxo ligand show very different isotropic coupling for the A- and B-centers. Analysis of the spectroscopic data suggests that the A- and B-centers represent different orientations of the oxyheme O(2) ligand relative to the distal histidine. It is likely that the A and B conformational substates in the alphaO(2) and betaO(2) subunits differ not only in their tertiary structures but in their affinities for O(2).

EPR and ENDOR studies of single crystals of 2-oxazolidinone X-irradiated at 295 K

When single crystals of the 5-membered heterocyclic ring structure 2-oxazolidinone (C3O2NH5) are irradiated at room temperature, the major radical formed (R1) decays during a period of a few hours, leaving a broad, unstructured EPR spectrum not amenable to analysis. Cooling the crystals to about 140 K immediately after irradiation at room temperature allows analysis of the R1 radical by EPR and ENDOR. The radical is formed by a net H-abstraction from one of the two methylene groups of the molecule. A full EPR and ENDOR analysis of four proton interactions (one α-coupling, and three β-couplings) together with ESR evidence for a small nitrogen hyperfine interaction allowed for a precise identification of R1. The results show that the carbon-centered radical R1 is puckered at the radical center. Using DFT calculations together with the experimental EPR and ENDOR results, the torsion angle of the C1–H bond with respect to the N–C1–C2 plane is estimated to be 13–15° (bending angle θ ≈ 7°). The DFT calculations reproduced the carbon-bonded proton hyperfine coupling constants satisfactorily but failed to reproduce the experimental results for the nitrogen and nitrogen-bonded proton hyperfine interactions.

Conformational disorder in the propagating radical of dimethacrylate polymers

Photopolymerization of methacrylic monomers yields samples with trapped radicals that are easily detected by electron paramagnetic resonance (EPR) spectroscopy. Despite its simplicity, there is no general agreement about the interpretation of this spectrum, in particular, about the role of methylene β protons. An extensive ENDOR study of the propagating radical in photopolymerized dimethacrylates has been carried out in order to obtain detailed information about methylene hyperfine couplings and, thus, about radical conformation. It is shown that literature models are not able to reproduce the ENDOR results and that only accurate fitting of ENDOR spectra obtained by saturating the EPR spectrum at different positions gives reliable information about radical conformation, thanks to the exploitation of conformational selectivity. It turns out that most radicals are in the minimum energy conformation, but any possible conformation is assumed by non negligible fractions of radical.

Structure Characterization of the Copper(II) Complex of Poly(4-vinylpyridine) by a Combination of EPR, ENDOR, and Molecular Modeling Techniques

A combination of EPR and orientation-selective pulse ENDOR methods provides enough constraints to characterize the structure of the metal center in the copper(II) complex of poly(4-vinylpyridine). The structure derives from the propeller structure of the tetrapyridino copper(II) complex in pyridine glass determined by the same methods. It features more disorder with respect to the angle between the pyridine ring and complex plane but only moderate tetrahedral distortion. The enhanced resolution of high-field EPR allows one to detect another metal center at higher copper loading of the polymer that probably involves only three pyridyl side group ligands. No evidence is found for an inhomogeneous metal distribution in the polymer if care is taken to equilibrate the structure during preparation. The EPR and ENDOR results are supported by molecular modeling based on density functional and force-field calculations. This combination of experimental and theoretical techniques provides a more detailed account of ...

VOH center in magnesium oxide: an ab initio supercell study

The V OH trapped-electron-hole center in magnesium oxide is studied with the periodic ab initio Hartree–Fock program crystal by using the supercell scheme. The H atom that formally is substituted for a Mg atom at its lattice position migrates towards one of the neighboring O atoms, forming a strong covalent bond with it, whereas the hole localizes at the opposite O atom completely. The electronic structure of the defect is discussed in terms of electron charge and spin density maps and band structure. The isotropic and anisotropic components of the hyperfine coupling constant between the H nucleus and the unpaired electron are calculated and are in quite good agreement with ENDOR results.

And centres in single crystals characterized by ESR and ENDOR spectroscopy

ESR and ENDOR methods are used to characterize a new centre with symmetry in reduced single crystals. Present ENDOR results also confirm that for the recently reported centre in UV- or -irradiated crystal an observed superhyperfine interaction results from a nearby hydrogen nucleus. Comparison of spin-Hamiltonian parameters shows the similarity of and centres in crystals.

Lithium trapped-hole centre in magnesium oxide. An ab initio supercell study

The lithium trapped-hole centre in magnesium oxide is studied with the periodic ab initio Hartree Fock program CRYSTAL by using the supercell scheme. Trends in the defect-defect interaction are investigated by considering supercells containing 16, 32, 64 and 128 atoms, and it is shown that full relaxation requires the use of the largest supercell. The hole is very localized on an oxygen atom (O −). It turns out that Li + and O − relax in opposite directions, in agreement with experimental ENDOR results. The electronic structure of the defect is discussed in terms of electron charge and spin density maps and band structure.

EPR and ENDOR studies of carotenoid–solid Lewis acid interactions

EPR and ENDOR results provide strong support for the formation of carotenoid radical cations on activated alumina and silica–alumina as a consequence of electron transfer from the adsorbed carotenoid molecules to the Lewis acid sites on these surfaces. This assignment is confirmed by the correlation between the AlIII Lewis acid site content on the activated surfaces and the maximum concentration of the generated carotenoid radical cations. The results are compared with those obtained for a solution of carotenoid and AlCl3, which serves as a model system. The ENDOR line at ca. 6.5 MHz obtained for the carotenoids adsorbed on solid supports and carotenoid–AlCl3 solution, is attributed to the high-frequency feature of a hyperfine doublet, centred about the 27Al Larmor frequency. ENDOR detection of the hyperfine doublet, instead of the single line at the Larmor frequency, indicates the formation of strong complexes between carotenoid molecules and Lewis acid sites on the surface.

Resonance Raman study of carotenoid cation radicals

The resonance Raman (RR) spectra of the neutral and the cation radical of 8′-apo-β-caroten-8′-al (I), 7′,7′-dicyano-7′-apo-β-carotene (II) and canthaxanthin (III) were measured using 752.6 nm laser excitation. Optical and RR spectra of the electrochemically prepared cation radicals of I–III were measured in situ at different time intervals during the electrolysis. The RR spectra of the cation radicals are considerably different from those of the neutral species. The frequencies of the CC stretching vibrations are decreased by 30–40 cm −1, whereas those of CC stretching vibrations are increased by 15–30 cm −1. These results suggest delocalization of the unpaired electron density throughout the entire carbon backbone which is consistent with our previous EPR and ENDOR results.

Study of Water Binding to Low-Spin Fe(III) in Cytochrome P450 by Pulsed ENDOR and Four-Pulse ESEEM Spectroscopies

Cytochrome P450cam (CP450cam) was studied by pulsed ENDOR and two- and four-pulse ESEEM spectroscopies. Spectra were recorded and simulated at the three principal g-values of the rhombic EPR spectrum. The four-pulse ESEEM experiment gave a direct measure of the anisotropic hyperfine interaction for the protons. Using the point dipole approximation this gives a Fe−H distance of 2.6 A. The measured anisotropic hyperfine interaction reduced the number of hyperfine interaction parameters required to simulate the ENDOR line shapes. Both the four-pulse ESEEM frequencies and the ENDOR spectra at all three principal g-values could be satisfactorily simulated using two magnetically equivalent protons and a water orientation similar to that obtained in our previous 17O ESEEM study. Thus, the pulsed ENDOR and four-pulse ESEEM results are self-consistent with the 17O ESEEM data and indicate that the axial ligand is a water molecule rather than an OH- ligand. The isotropic hyperfine value derived from the numerical si...

An ENDOR study of RbI:O2-

In this work, an ENDOR analysis of the O2- defect in RbI is presented. A complete angular variation of one set of 85Rb and 87Rb ENDOR transitions is shown. The corresponding hyperfine and nuclear quadrupole coupling tensors are determined. The orientation of the principal axes of these tensors is in good agreement with the overall D2h symmetry of the O2- ion in the crystal. The ENDOR results can only be explained assuming a monovacancy model, wherein the O2- molecule is replacing a single I- ion. A comparison with a previous ENDOR study of the S2- centre in RbCl is presented. The linewidth of the EPR signals can be explained using the ENDOR results.

The lowest energy unoccupied molecular orbitals of the cyclopolysilanes: experimental and theoretical studies

Inner-shell electron energy loss spectra at the Si2p edge have been measured for [Si(CH 3 2] n, n=5 and 6, and have been assigned using ab initio SCF MO calculations for the related molecules (SiH 2) n , n=2–6, and [Si(CH 3) 2] inn, n=3 and 4. Ou calculations indicate that the lowest energy ISEELS feature, which lies about 2 eV below the Si2p ionization threshold, arises from excitation of a Si2p electron to an empty MO which is of π symmetry and has appreciable Si3p character. Calculations of vertical and adibatic electron attachment energies establish that the gas-phase anions of all the cyclopolysilanes considered will be unstable with respect to electron detachment and will show electron scattering resonances in the region around 2 eV. We have also measured the total elastic electron scattering cross sections of [Si(CH 3) 2] inn, n=5 and 6, and have indeed found an increase in cross section at an energy of about 2 eV. We have calculated the 29Si NMR shielding of (SiH 2) inn, n=3,4, using the RPA LORG method and have reproduced the large shielding anisotropy recently measured for some [SiR 2] 3 species by solid-state NMR. Calculation of the trace of the square of the anisotropic hyperfine interaction for the Si 3H 6 − anion gives a value reasonably consistent with experiment, but much smaller than that expectred for a single unpaired atomic Si3p electron, due to the delocalization of electron density onto the Hs. Thus, π symmetry and substantial Si3p character are not inconsistent with the small experimental value of the hyperfine anisotropy in such anions, and the apparent inconsistency of ESR and UV data versus ENDOR results is removed.

ESR and ENDOR experiments using a disc-shaped resonator working in the Whispering Gallery Mode (WGM)

Different instrumental apparatuses are presented employing disc-shaped dielectric resonators working in the Whispering Gallery Mode (WGM). The conditions suitable to take the best from the microwave power obtained from a trade klystron in the X-band have been verified for the ESR part of the experiments. It is well known that a good ESR saturation condition is an essential prerequisite to have satisfactory ENDOR results. The WGM dielectric resonators seem to be very suitable for multiple irradiation purposes and in particular for ENDOR experiments which is the aspect we wanted to exploit here. Various radiofrequency ENDOR irradiation circuits have been tested and the obtained spectra of a γ-irradiated single crystal are presented and criticized. Some results for very high frequency ESR applications are also reported.

Radical Ions and Their Reactions in DNA Constituents: ESR/ENDOR Studies of Radiation Damage in the Solid State

This updating review contains the results of recent ESR/ENDOR experiments on DNA constituents in the solid state. The compounds reviewed include single crystals of the DNA bases, nucleosides, and nucleotides. The emphasis is on low-temperature ENDOR results. Typical experiments involve irradiations at or near helium temperatures in attempts to determine the primary radiation-induced oxidation and reduction products. The use of the ENDOR technique allows one to determine the protonation state of the initial products. Subsequent warming of the sample facilitates a study of the reactions that these primary products undergo. A critical review of previous ESR experiments is considered to see if these results conflict with the newer ENDOR results. Finally, a summary is provided to show which of the numerous results discussed may have some relevance to understanding the radiation chemistry of DNA.

The [2H8]THF radical cation in CF3CCl3 and CFCl3. An EPR and ENDOR study

The structure and dynamics of the radical cation of deuteriated tetrahydrofuran ([2H8]THF) stabilized in the freon matrices CFCl3 and CF3CCl3 have been investigated by means of EPR and ENDOR spectroscopy. The EPR and ENDOR results of the rigid structure give two pairs of strongly interacting β-deuterium hf splittings [aD= 1.36 (2 D) and aD= 0.60 mT (2 D)] consistent with the protonated analogue [Kubodera et al., J. Phys. Chem., 1981, 85, 2583 (ref. 1)]. An additional hf splitting (0.9–1.1 mT) assigned to the matrix fluorine was resolved above ca. 110 K in CFCl3. The temperature dependent EPR lineshapes (77–145 K) involving the exchange between four coupled (I= 1) nuclei have been analysed. A two-site model explained qualitatively the alterating line-widths at intermediate temperatures and resulted in an activation energy of ca. 1.7 kcal mol–1 A non-perturbative approach adopting a four-site model to account for puckering motion was tested. The dynamics were also manifested as a reversible temperature effect for the ENDOR. The relative abundance of resonances due to an averaged (hf) structure become predominant as the temperature is increased.

Solvent structure around cations determined by proton ENDOR spectroscopy and molecular dynamics simulation

Classical molecular dynamics simulations of metal ion solvation have been found to confirm the experimental 1 H ENDOR and EPR spectroscopic results on the coordination structure of water molecules surrounding paramagnetic Mn 2+ ions in frozen aqueous solutions. The well-known simplification and intensification of the six-line Mn 2+ EPR signal in frozen solutions upon reducing the pH was found to coincide with the appearance of three strong 1 H ENDOR resonances, reflecting symmetrization of the first two solvation shells