The spin distribution in low-spin iron porphyrins

In many low-spin (S = 1/2) iron porphyrin derivatives, electron spin resonance (ESR) spectra indicate that one of the d(pi) orbitals of iron, either a d(xz) or d(yz), depending on the axial ligands of the porphyrin complex as well as their orientation, is essentially singly occupied; the unpaired electron is almost completely located at the metal. In contrast, nuclear magnetic resonance (NMR) and electron nuclear double resonance (ENDOR) spectroscopy convincingly show that a significant share of the unpaired electron is delocalized. This apparent contradiction is explained by the present density-functional-theory (DFT) calculations performed on a heme a model as well as on bis-imidazole-ligated iron porphyrin without substituents. The calculations show that the integrated spin density at the iron atom is nearly one, in agreement with the ESR measurements. However, significant areas with opposite (beta) spin are found along the Fe-N bond axes, thus evoking a need for additional alpha-spin density to be present in the porphyrin ring, ring substituents, and the axial ligands to keep the net amount of unpaired spin exactly one. The gross spin density, that is, the sum of unpaired alpha and beta spins, amounts to about 1.3 electrons. It seems that the degree to which alpha and beta spin dominate in different regions of the heme structure, as evidenced in these calculations, has not been previously observed.     

Davies electron-nuclear double resonance revisited: enhanced sensitivity and nuclear spin relaxation

Over the past 50 years, electron-nuclear double resonance (ENDOR) has become a fairly ubiquitous spectroscopic technique, allowing the study of spin transitions for nuclei which are coupled to electron spins. However, the low spin number sensitivity of the technique continues to pose serious limitations. Here we demonstrate that signal intensity in a pulsed Davies ENDOR experiment depends strongly on the nuclear relaxation time T(1n), and can be severely reduced for long T(1n). We suggest a development of the original Davies ENDOR sequence that overcomes this limitation, thus offering dramatically enhanced signal intensity and spectral resolution. Finally, we observe that the sensitivity of the original Davies method to T(1n) can be exploited to measure nuclear relaxation, as we demonstrate for phosphorous donors in silicon and for endohedral fullerenes N@C(60) in CS(2).     

Separation and complete analyses of the overlapped and unresolved 1H NMR spectra of enantiomers by spin selected correlation experiments

NMR spectroscopic discrimination of optical enantiomers is most often carried out using (2)H and (13)C spectra of chiral molecules aligned in a chiral liquid crystalline solvent. The use of proton NMR for such a purpose is severely hindered due to the spectral complexity and the significant loss of resolution arising from numerous short- and long-distance couplings and the indistinguishable overlap of spectra from both R and S enantiomers. The determination of all the spectral parameters by the analyses of such intricate NMR spectra poses challenges, such as, unraveling of the resonances for each enantiomer, spectral resolution, and simplification of the multiplet pattern. The present study exploits the spin state selection achieved by the two-dimensional (1)H NMR correlation of selectively excited isolated coupled spins (Soft-COSY) of the molecules to overcome these problems. The experiment provides the relative signs and magnitudes of all of the proton-proton couplings, which are otherwise not possible to determine from the broad and featureless one-dimensional (1)H spectra. The utilization of the method for quantification of enantiomeric excess has been demonstrated. The studies on different chiral molecules, each having a chiral center, whose spectral complexity increases with the increasing number of interacting spins, and the advantages and limitations of the method over SERF and DQ-SERF experiments have been reported in this work.     

Electron transfer pathways and protein response to charge separation in photosynthetic reaction centers: time-resolved high-field ENDOR of the spin-correlated radical pair P865(+)QA(-)

Recently we reported the first observation of time-resolved (TR) high-frequency (HF) electron nuclear double resonance (ENDOR) of the transient charge separated state P865(+)Q(-)A in purple photosynthetic bacterial reaction centers (RC) (Poluektov, O. G., et al. J. Am. Chem. Soc. 2004, 126, 1644-1645). The high resolution and orientational selectivity of HF ENDOR allows us to directly probe protein environments by spectrally selecting specific nuclei in isotopically labeled samples. A new phenomenon associated with the spin correlated radical pair (SCRP) nature of P865(+)Q(-)A was observed. The TR-HF ENDOR spectra of protein nuclei (protons) surrounding deuterated QA(-) exhibit a derivative-like, complicated line shape, which differs considerably from the HF ENDOR spectrum of the protein nuclei surrounding thermally equilibrated QA(-). Here, a theoretical analysis of these observations is presented that shows that the positions and amplitudes of ENDOR lines contain information on hyperfine interactions (HFI) of a particular nucleus (a proton of the protein) with both correlated electron spins. Thus, spin density delocalization in the protein environment between the SCRP donor and acceptor molecules can be revealed via HF ENDOR. Novel approaches for acquiring and analyzing SCRP ENDOR that simplify interpretation of the spectra are discussed. Furthermore, we report here that the positions of the ENDOR lines of the SCRP shift with an increase in the time after laser flash, which initiates electron transfer. These shifts provide direct spectroscopic evidence of reorganization of the protein environment to accommodate the donor-acceptor charge-separated state P865(+)QA(-).     

Integrated paramagnetic resonance of high-spin Co(II) in axial symmetry: chemical separation of dipolar and contact electron-nuclear couplings

Integrated paramagnetic resonance, utilizing electron paramagnetic resonance (EPR), NMR, and electron-nuclear double resonance (ENDOR), of a series of cobalt bis-trispyrazolylborates, Co(Tp ( x )) 2, are reported. Systematic substitutions at the ring carbons and on the apical boron provide a unique opportunity to separate through-bond and through-space contributions to the NMR hyperfine shifts for the parent, unsubstituted Tp complex. A simple relationship between the chemical shift difference (delta H - delta Me) and the contact shift of the proton in that position is developed. This approach allows independent extraction of the isotropic hyperfine coupling, A iso, for each proton in the molecule. The Co..H contact coupling energies derived from the NMR, together with the known metrics of the compounds, were used to predict the ENDOR couplings at g perpendicular. Proton ENDOR data is presented that shows good agreement with the NMR-derived model. ENDOR signals from all other magnetic nuclei in the complex ( (14)N, coordinating and noncoordinating, (11)B and (13)C) are also reported.     

ENDOR of spin-correlated radical pairs in photosynthesis at high magnetic field: a tool for mapping electron transfer pathways

A new phenomenon has been detected in the time-resolved electron-nuclear double resonance (ENDOR) spectra of the spin-correlated radical pairs in photosynthetic reaction center proteins. The observed effects result from both increased resolution and orientational selectivity provided by high magnetic field EPR and are manifest as specific, derivative-type lines in the ENDOR spectrum. Importantly, the positions and amplitudes of these lines contain information on the interaction of a particular nucleus with both correlated electron spins. Thus, spin density delocalization in the protein environment between the donor and acceptor in the SCRP can be revealed via SCRP ENDOR, providing a unique opportunity to probe the electron-transfer pathways in natural and artificial photosynthetic assemblies.     

31P NMR as a tool for studying incorporation of Ni, Co, Fe, and Mn into aluminophosphate zeotypes

The incorporation of moderate amounts of Ni(II), Co(II), Fe(II/III), and Mn(II/III) into aluminophosphate zeotype AlPO4-34 and Fe(II/III) into aluminophosphate zeotype AlPO4-36 was studied by broadline 31P NMR. The technique provided direct evidence on isomorphous substitution of framework aluminum by transition metals and allowed us to determine the extent of the substitution. 31P NMR proved to be complementary to other spectroscopic techniques such as X-ray absorption spectroscopy (XAS), M?ssbauer, electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR) spectroscopies. The position of the NMR signal belonging to phosphorus in the P(OAl)3(OMe) environment depended mostly on the magnitude of the hyperfine interaction between a phosphorus nucleus and an unpaired electron, which was delocalized from the transition metal atom Me by covalent bonding. The width of the NMR signal was dominated by dipolar coupling among phosphorus nuclei and nearest paramagnetic centers. In addition, broadline NMR of ethylenediamine-templated manganese phosphate (C2H10N2)[Mn2(HPO4)3(H2O)], which was used as a model compound, showed that on the basis of line positions and line widths different 31P signals could easily be assigned to different phosphorus crystallographic sites. The technique could thus be applied to extract valuable structural information about metal phosphates as well.     

Electronic structure of the lowest excited triplet state of 5,12-naphthacenequinone

Continuous-wave time-resolved EPR (cw-TREPR) and pulsed electron nuclear double resonance (ENDOR) studies have been carried out to clarify the electronic structure of the lowest excited triplet (Tl) state of 5,12-naphthacenequinone (5,12-NpQ) as well as 1,4-anthraquinone (1,4-AQ) and 6,13-pentacenequinone (6,13-PeQ). The Tl energy level and the D value of the zero-field splitting (ZFS) parameters only slightly decreased with the increasing pi-conjugated system. The Tl states of these linear para-acenequinones were assigned to the pi pi* character. In triplet 5,12-naphthacenequinone, more than 80% of the unpaired electron spins are localized on the naphthalene aromatic sub-system.     

Magnetic resonance study of Rh complexes in AgCl microcrystals

X-ray or UV irradiation at room temperature of Rh3+ doped AgCl emulsion powders leads to the production of three paramagnetic Rh2+ related centres, labeled R4, R5 and R6. A combined X and Q band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) study allowed the determination of a nearly complete structural model for these centres. In the X band ENDOR spectra of R4 and R5 interactions of the unpaired electron with nearby protons have been identified, indicating that for these centres Cl- ligands have been exchanged by H2O or OH-. The R6 centre, identified as a (RhCl6)4- complex, has been found to be fundamentally different from the dominant centre in large Rh2+ doped AgCl single crystals grown from the melt. The results are compared with recent work by other researchers in the same field.     

Cross‐Polarization Electron‐Nuclear Double Resonance Spectroscopy

Magnetic nuclei in the proximity of a paramagnetic center can be polarized through electron‐nuclear cross‐polarization and detected in electron‐nuclear double resonance (ENDOR) spectroscopy. This principle is demonstrated in a single‐crystal model sample as well as on a protein, the β2 subunit of E.coli ribonucleotide reductase (RNR), which contains an essential tyrosyl radical. ENDOR is a fundamental technique to detect magnetic nuclei coupled to paramagnetic centers. It is widely employed in biological and materials sciences. Despite its utility, its sensitivity in real samples is about one to two orders of magnitude lower than conventional electron paramagnetic resonance, thus restricting its application potential. Herein, we report the performance of a recently introduced concept to polarize nuclear spins and detect their ENDOR spectrum, which is based on electron‐nuclear cross polarization (eNCP). A single‐crystal study permits us to disentangle eNCP conditions and CP‐ENDOR intensities, providing the experimental foundation in agreement with the theoretical prediction. The CP‐ENDOR performance on a real protein sample is best demonstrated with the spectra of the essential tyrosyl radical in the β2 subunit of E.coli RNR.     

Measurement of Angstrom to Nanometer Molecular Distances with 19F 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.     

Dynamic Nuclear Polarization in Some Aliphatic and Aromatic Solutions as Studied by Fluorine‐Electron Double Resonance

Dynamic nuclear polarization experiments were performed to study the solutions of the stable free radical α,γ‐Bisdiphenylene‐β‐phenyl allyl complex with benzene (1∶1) in some highly fluorinated aliphatic and aromatic solvents. In solutions examined in this study, the Overhauser effect, which normally arises due to both dipolar and scalar interactions between the unpaired electrons of the free radical molecules and fluorine nuclei of solvent molecules occurs mainly. 1‐Iodotridecafluorohexane, 2,2,3,4,4,4‐Hexafluoro‐1‐butanol, N‐methyl‐bis‐trifluoroacetamide, hexafluoroacetylacetone, octafluorotoluene, and hexafluorobenzene were used as the solvents. The experiments were performed at a low field double resonance NMR spectrometer, which operates at 1.53 mT. The NMR enhancements depend on competition between intermolecular magnetic interactions. The measurements were performed at four different temperatures to test the dipolar and the scalar part of the coupling between the fluorine nucleus (¹⁹F) and the unpaired electron. It was found that the dipolar interactions are more effective for the aliphatic solvents, while the scalar interactions are more effective for the aromatics. The nuclear‐electron coupling parameter varies between 0.018 and 0.157 in all aliphatic solvents and between ?0.063 and ?0.035 in aromatic solvents.     

超极化核磁共振方法的原理和应用

核磁共振(NMR)技术凭借其高空间分辨率,宽时间响应尺度和非侵入检测等特点,在化学分析和医疗诊断中发挥着重要的作用。但是原子核的低极化使现阶段NMR技术的灵敏度较低。超极化技术是一类可以有效提高NMR灵敏度的方法。其通过物理或化学过程把原子核自旋态推向一个偏离热力学平衡的状态,使NMR信号强度得到几个数量级的提升,极大地改善了灵敏度。多种超极化技术已经在各个领域崭露头角。本文用较为形象的描述对几种常见的超极化技术包括:动态核极化、光泵、光核极化、化学诱导动态核极化、仲氢诱导极化。从其精巧的原理和广泛的应用进行介绍,有助于人们对超极化技术的认知。     

Study of relaxation rates of stable paramagnetic centers in gamma-irradiated alanine

The stable L-alanine radical induced by gamma-irradiation was examined by electron paramagnetic resonance (EPR), transfer saturation EPR and electron nuclear double resonance (ENDOR) in the temperature region of fast motion of the methyl group (180-320 K). From the obtained spectral line broadening and spectral intensity the correlation time for the methyl rotation was estimated. The complex processes determining the relaxation rate were examined in the same temperature interval. It was shown that important contributions to the relaxation rate arise from non-secular and pseudo-secular types of contributions. The non-secular contribution involves intramolecular dynamics while the pseudo-secular contribution originates from intermolecular motions. The obtained values for the dynamical parameters have been compared with those obtained by pulse EPR methods and by proton nuclear magnetic resonance (NMR) on undamaged crystals.     

Pulsed EPR and NMR spectroscopy of paramagnetic iron porphyrinates and related iron macrocycles: how to understand patterns of spin delocalization and recognize macrocycle radicals

Pulsed EPR spectroscopic techniques, including ESEEM (electron spin echo envelope modulation) and pulsed ENDOR (electron-nuclear double resonance), are extremely useful for determining the magnitudes of the hyperfine couplings of macrocycle and axial ligand nuclei to the unpaired electron(s) on the metal as a function of magnetic field orientation relative to the complex. These data can frequently be used to determine the orientation of the g-tensor and the distribution of spin density over the macrocycle, and to determine the metal orbital(s) containing unpaired electrons and the macrocycle orbital(s) involved in spin delocalization. However, these studies cannot be carried out on metal complexes that do not have resolved EPR signals, as in the case of paramagnetic even-electron metal complexes. In addition, the signs of the hyperfine couplings, which are not determined directly in either ESEEM or pulsed ENDOR experiments, are often needed in order to translate hyperfine couplings into spin densities. In these cases, NMR isotropic (hyperfine) shifts are extremely useful in determining the amount and sign of the spin density at each nucleus probed. For metal complexes of aromatic macrocycles such as porphyrins, chlorins, or corroles, simple rules allow prediction of whether spin delocalization occurs through sigma or pi bonds, and whether spin density on the ligands is of the same or opposite sign as that on the metal. In cases where the amount of spin density on the macrocycle and axial ligands is found to be too large for simple metal-ligand spin delocalization, a macrocycle radical may be suspected. Large spin density on the macrocycle that is of the same sign as that on the metal provides clear evidence of either no coupling or weak ferromagnetic coupling of a macrocycle radical to the unpaired electron(s) on the metal, while large spin density on the macrocycle that is of opposite sign to that on the metal provides clear evidence of antiferromagnetic coupling. The latter is found in a few iron porphyrinates and in most iron corrolates that have been reported thus far. It is now clear that iron corrolates are remarkably noninnocent complexes, with both negative and positive spin density on the macrocycle: for all chloroiron corrolates reported thus far, the balance of positive and negative spin density yields -0.65 to -0.79 spin on the macrocycle. On the other hand, for phenyliron corrolates, the balance of spin density on the macrocycle is zero, to within the accuracy of the calculations (Zakharieva, O.; Schünemann, V.; Gerdan, M.; Licoccia, S.; Cai, S.; Walker, F. A.; Trautwein, A. X. J. Am. Chem. Soc. 2002, 124, 6636-6648), although both negative and positive spin densities are found on the individual atoms. DFT calculations are invaluable in providing calculated spin densities at positions that can be probed by (1)H NMR spectroscopy, and the good agreement between calculated spin densities and measured hyperfine shifts at these positions leads to increased confidence in the calculated spin densities at positions that cannot be directly probed by (1)H NMR spectroscopy. (13)C NMR spectroscopic investigations of these complexes should be carried out to probe experimentally the nonprotonated carbon spin densities.     

Distance measurements between paramagnetic centers and a planar object by matrix Mims electron nuclear double resonance

Frequency-domain electron nuclear double resonance (ENDOR), two time-domain electron nuclear double resonance techniques, and electron spin echo envelope modulation spectroscopy are compared with respect to their merit in measurements of small hyperfine couplings to nuclei with intermediate gyromagnetic ratio such as 31P. The frequency-domain Mims ENDOR experiment is found to provide the most faithful line shapes. In the limit of long electron-nuclear distances of more than 0.5 nm, sensitivity of this experiment is optimized by matching the first interpulse delay to the transverse relaxation time of the electron spins. In the same limit, Mims ENDOR efficiency scales inversely with the sixth power of distance. Hyperfine splittings as small as 33 kHz can be detected, corresponding to an electron-31P distance of 1 nm. In systems, where a certain kind of nuclei is distributed in a plane, measurements of intermolecular hyperfine couplings can be analyzed in terms of a distance of closest approach of a paramagnetic center to that plane. By applying this technique to spin-labeled lipids in a fully hydrated lipid bilayer it is found that for a fraction of lipids, chain tilt angles can be 25 degrees larger than the mean tilt angle of the lipid chains. This model of all-trans hydrocarbon chains with a broad distribution of tilt angles is also consistent with orientation selection effects in high-field ENDOR spectra.     

Antiferromagnetic interactions in the quarter-filled organic conductor (EDO-TTF)2PF6

The ground state electronic structure of the high-temperature (HT) and the low-temperature (LT) phases of (EDO-TTF)(2)PF(6) is investigated using the embedded cluster approach in combination with the density functional method designed to describe the strong non-dynamic electron correlation. It is found that, in the HT phase, the unpaired electron spins located on pairs of neighbouring EDO-TTF molecules are antiferromagnetically coupled along the stacking direction with the Heisenberg exchange integral J = -655 cm(-1). In the LT phase, the unpaired spins located on the cationic EDO-TTF molecules are coupled antiferromagnetically with J values strongly alternating along the stacking axis of the crystal thus rendering it diamagnetic. The parameters of the extended Hubbard model are evaluated and the conductance properties of the two phases are estimated using these parameters. It is suggested to investigate the charge and spin excitations in the two phases of (EDO-TTF)(2)PF(6) with the use of angle-resolved photoemission spectroscopy.     

EPR and ENDOR study of the frozen ammoniated electron at low alkali-metal concentrations

Ammoniated electrons in dilute frozen solutions are examined using EPR spectroscopy under conditions where the formation of metallic nanoparticles is avoided. Two signals from two different species have been observed. One signal is metastable and decays irreversibly upon annealing. The metastable species saturates at a spin concentration of 10 nM. The annealing temperature for this species amounts to 60 K for frozen solutions of sodium in neat ammonia and is raised upon addition of metal iodide. The observed g value is smaller than the free electron g value and is compatible with a cluster-anion radical rather than with a cavity electron. The wave function of the unpaired electron contains about 6%-10% of 2p character at nitrogen. The observed g shift is fully compatible with previously reported theoretical calculations (Shkrob, I. A. J. Phys. Chem. A 2006, 110, 3967-3976). The second signal cannot be annealed in the frozen state. The line shape is homogeneous, and its width depends on the identity of the metal and at large metal concentrations on the metal concentration itself. Upon increasing alkali metal concentration above 0.15 MPM, the line shape changes from Lorentzian to Dysonian, indicating the presence of metal nanoparticles. A new ENDOR pulse sequence is introduced to investigate the presence of weakly coupled nuclear spins for homogeneous EPR lines. The observations are critically compared with available literature data.     

Recovering Invisible Signals by Two‐Field NMR Spectroscopy

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two‐field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low‐field dimension. Two‐field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.