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Two representative porphyrin dimers, each containing meso-tetraphenylporphyrin (TPP) monomers as constituents, have been studied by resonance Raman (RR) spectroscopy. In both cases the monomer units are linked through one common phenyl ring. The first incorporates a para-phenylene bridge thereby retaining a centre of inversion; the second incorporates a metaphenylene bridge and has no centre of inversion. Modes giving rise to distinguishing features (monomer versus dimer and the p -dimer versus the m -dimer) in the 300–420 cm −1 region are qualitatively assigned. It is concluded that in each dimer the componentmonomers are weakly interacting in their Soret excited states. The excitation may be considered monomer-localised on the RR time scale of ≈ 1 ps.

First measurements of deuterium quadrupole coupling constants of a polyatomic doublet state radical are reported. The quadrupole couplings were determined by ENDOR and electron–nuclear–nuclear TRIPLE resonance in liquid crystals. For the partially deuterated perinaphthenyl radical the out‐of‐plane component of the deuterium quadrupole coupling was measured to be e2q33Q/h =−(94±3) kHz for both molecular sites. Within experimental error these values are equal to those measured earlier for deuterated alternant hydrocarbons in the diamagnetic singlet ground state.

Quadrupole splittings of organic nitroxide radicals in solution have been observed by ENDOR in a liquid crystal. The analysis of the line shifts yields e 2 q ′ 33 Q/h = −2.5 MHz. This result could be rationalized by a population analysis of the nitrogen bonds. The theory predicts that the main contribution to the observed splitting comes from the unpaired π-electron.

The nuclear-nuclear coherence effect in electron-nuclear double resonance (ENDOR) experiments is studied in the case of the perinaphthenyl radical in solution. It is shown that in certain cases this coherence effect can be used to determine the number of protons contributing to an ENDOR line. In the case of ion-radicals it turns out that NMR fields as high as 35 gauss amplitude (rotating frame) are necessary to produce significant effects. By this method it was possible to determine all the 12 proton hyperfine splitting constants for the ion-radicals 1-phenylnaphthalene and 2-phenylnaphthalene. They are interpreted in terms of π-electron theories of Huckel, McLachlan, and Amos-Snyder. The large discrepancies between experimental and calculated splitting constants indicate that π-σ separability is no longer permissible for these nonplanar molecules.

Electrolytically generated semiquinone radical ions have been studied in the nematic mesophase of p-azoxyanisole by EPR methods. The degree of molecular ordering of benzo-, naphtho-, and anthrasemiquinones varied from 0.085, 0.18 to 0.20, respectively. The observed shifts of the hfs splitting constants and the g factors are in reasonable agreement with theoretical predictions. The negative sign of the C1 13 splitting constant of benzosemiquinone has been confirmed.

The E.S.R. spectrum of the o-dimesitoylbenzene anion-alkali cation radical shows unusually large isotropic alkali hyperfine splitting constants. We report a solution ENDOR study of this radical in which both alkali (85,87Rb) and proton ENDOR spectra were recorded. Both the alkali and proton intensities showed a strong dependence on the metal ion nuclear spin quantum number of the E.S.R. line being saturated. This dependence is attributed to strong flip-flop cross-relaxation induced by modulation of the isotropic alkali hyperfine splitting. The powder E.S.R. spectrum of the complex reveals a small anisotropy of the Rb hyperfine splitting tensor. This indicates a small metal non-s-contribution to the half-filled molecular orbital, which is consistent with the observed relaxation behaviour and the small g shift. The intensity variations in the alkali and proton ENDOR spectra were used to determine the relative signs of all hyperfine splitting constants, and the absolute signs of the hyperfine splitting const...

In the nematic and isotropic phases of the liquid crystal p-azoxyanisole (PAA) the neutral radicals perinaphthenyl (PNT), triphenylmethyl (TPM), and pentaphenylcyclopentadienyl (PPCPD) have been studied by electron spin resonance techniques. A method is discussed for measuring the degree of ordering of the radicals by means of the observed g factors in the different phases. For PNT and TPM the components of the axially symmetric g tensor are determined and compared with the values predicted by STONE’S g factor theory. The shift of the hyperfine splitting constants (C13 and H1) of PNT and TPM agree rather well with the theoretical shifts calculated by the dipol-dipol interaction theory of Mc CONNELL and STRATHDEE. For TPM, however, the agreement is only satisfactory when all the long range contributions are considered. For PNT and TPM also the signs of the spin densities could be determined. PPCPD shows drastically that the validity of both g factor theory and hfs theory in nematic mesophases breaks down for unplanar molecules with large twist angles.

In the course of an investigation of electrolytically generated radical ions in the nematic mesophase of liquid crystals, we studied the influence of supporting electrolyte on the degree of molecular ordering. In the case of the radical anion of tetracyanoethylene (TCNE-) electrolytic generation results in a lower degree of ordering than chemical generation with sodium iodide. By using the neutral radical perinaphthenyl as a probe it was assured that the degree of ordering of the solvent is not disturbed by adding 0.1-m. tetrapropylammoniumperchlorate. A pronounced linewidth variation is observed for the chemically generated TCNE- which is attributed to spin density modulation by dynamic interaction with the Na+ counter ion.

Zusammenfassung Von den negativen und positiven Radikalionen des Rubrens (Tetraphenyl tetracen) wurden ENDOR-Spektren in Losung aufgenommen. Die eindeutige Zuordnung der Protonen -hfs-Kopplungskonstanten wurde durch Vergleich mit den ENDOR-Spektren verschiedener teildeuterierter Rubrene ermoglicht. Das positive Vorzeichen einer Phenylkopplungskonstante wurde mit NMR nachgewiesen. Die gemessene Reihenfolge der Phenylkopplungskonstanten |aHmeta|⪢ |aHpara| = |aHortho| widerspricht den Voraussagen von reinen π-MO-Theorien. Die Ergebnisse lassen sich mit einer direkten Delokalisation des ungepaarten π-Elektrons in das σ-System der Phenyl- substituenten deuten. Die Synthese der teildeuterierten Verbindungen wird beschrieben.

From the ENDOR spectra of the mononegative ions of biphenyl and ortho-, meta- and para-terphenyl in solution complete sets of hyperfine splitting constants could be determined. The splittings are interpreted in terms of SCF MO spin densities obtained from McLachlan type calculations. It turns out that π-σ spin delocalization effects do not play a significant role in the spin density distribution in these radicals although some of them deviate considerably from coplanarity.

In the liquid crystal “Phase IV” which exhibits its nematic range between 16 and 76 °C ENDOR spectra of perinaphthenyl and tri- t -butylphenoxyl radicals could be recorded. It is demonstrated that the combined use of the ENDOR and liquid crystal methods yeilds valuable structural information.

This study aims to investigate the g xx heterogeneity of the g-tensor commonly observed in high-field electron paramagnetic resonance (EPR) spectra of nitroxide spin-labeled sites in proteins. This heterogeneity is addressed in terms of spin-label populations characterized by different polarity and H-bonding properties of the nitroxide micro-environment. The g xx value for each population is determined from the fit of continuous-wave high-field spectra obtained at 95, 275 and 360 GHz with a series of nitroxide spin-labels covalently attached to different sites in both membrane and water-soluble proteins. The spin-labeled proteins investigated include sensory rhodopsin II and its cognate transducer molecule (HtrII) from Natronomonas pharaonis both in micelles and membranes, bacteriorhodopsin from Halobacterium salinarum in native purple membrane lipid bilayers and water-soluble colicin A from Escherichia coli. To avoid contributions to the g xx spectral features of the nitroxide label due to nuclear quadrupole interactions arising from 14N nuclei, and to simplify the nitrogen hyperfine pattern, methanethiosulfonate spin labels, containing the 15N isotope (I = 1/2) in some experiments, were employed. A consistent analysis of all multi-frequency EPR spectra revealed three distinct g xx values, g , for each investigated position of the labeled proteins. In contrast, distinctly different nitrogen hyperfine splittings A zz of the nitroxides in the various labeled proteins could not be resolved, but rather an average hyperfine splitting $$\bar{A}_{{zz}}$$ was obtained. The g values as well as the fractions of the different nitroxide populations were found to be correlated with the average hyperfine constant $$\bar{A}_{{zz}},$$ a parameter which likewise is known to be sensitive to the local polarity of the spin-label micro-environment. Plotting the different g values obtained for each EPR spectrum versus $$\bar{A}_{{zz}}$$ of the labeled proteins reveals new interesting aspects of the nitroxide label micro-environment in terms of polarity and H-bonding propensity (proticity). Linear approximations of the different regions of the plot g versus $$\bar{A}_{{zz}}$$ are presented and compared with theoretical and experimental data available from the literature.

Experiments have been performed on a model guest molecule, phenazine, in biphenyl single crystal and n-heptane polycrystalline host matrices to illustrate the two main contributions to inhomogeneous line broadening in ODMR spectroscopy. These arise from (a) inhomogeneous crystal fields experienced by the guest molecule in a host lattice and (b) unresolved hyperfine splitting. It is shown that the former may be reduced by making optical site selection during both excitation and detection. In the latter category, the most offensive group of nuclei is shown to be the guest protons and to a lesser extent the near-lying host protons. This contribution may be drastically reduced by fully deuterating both guest and host systems. Performing ODMR spectroscopy under these conditions allows the typical linewidths of 1–10 MHz to be reduced to ca. 200 kHz. The advantages of such high-resolution spectroscopy are manifold, not the least of which being that it forms a prerequisite for highly specific multiresonance experiments such as the recently developed ODNQR method.

In molecular spectroscopy, the introduction of more than one resonant electromagnetic field is a well-established technique applied in the full spectral range between radiofrequency (rf) and ultraviolet.(1) The main motivation for extending single resonance to double or multiple resonance techniques can be twofold: (1) either one wants to increase the sensitivity of detection by “quantum transformation” from low-frequency absorbed quanta to high-frequency detected quanta, or (2) one wants to enhance the resolution of the spectra, i.e., reduce the number of spectral lines in a given frequency range by introducing specific coherent time-dependent interactions that average out unwanted static interactions in the Hamiltonian,(2) by imposing additional “selection rules,”(2) or by exploiting degeneracies of certain transitions.(3)

For magnetic resonance studies of paramagnetic species electron paramagnetic resonance (EPR) is a well-established method. However, when trying to elucidate the electronic structure of large and lowsymmetry radicals, as they typically occur in biological systems, one is often hampered by problems of spectral resolution. It was as early as 1956 when Feher (1956) demonstrated that by electron nuclear double resonance (ENDOR) the spectral resolution can be greatly improved. ENDOR signals are obtained by monitoring the changes of the amplitude of a saturated EPR line that occur when sweeping the frequency of an additionally applied rf field through the nuclear (NMR) region. This first ENDOR experiment was technically feasible only because the sample—phosphorus doped silicon—was studied at low temperature, where all the relaxation times are sufficiently long to easily obtain saturation. For radicals in liquid solution, however, these relaxation times are much shorter—on the order of 10-5-10-7 sec—and, consequently, ENDOR-msolution experiments are technically much more sophisticated since much larger saturating microwave and rf fields have to be applied. This probably explains why the first ENDOR-in-solution experiments required many more years before they could be successfully performed by Cederquist (1963) and by Hyde and Maki (1964).

The structure of the primary donor P in bacterial photosynthesis is of considerable interest due to the unique role this species plays in the very first events of the charge-separation process in the reaction center (RC). It could be shown that P is a specialized bacteriochlorophyll (BChl) [l] which was proposed by NORRIS et al. [2] to be a BChl-dimer. This “special pair” model was a matter of considerable controversy in recent years. Different dimer models [3, 4, 5] and perturbed monomer models [6, 7] have been proposed for the primary donor. The existence of a dimer has recently been demonstrated, at least for Rhodopseudomonas (Rp.) viridis RC’s by an X-ray structure analysis of RC single crystals [8]. An alternative approach to the characterization of the primary donor in its cationic state is provided by EPR and electron nuclear doubleresonance (ENDOR) spectroscopy [4, 5, 9–12]. In this paper we summarize the most recent contributions of EPR and ENDOR in elucidating the electronic structure details of the primary donor cation radicals. In particular P+.865in RC’s of Rp. sphaeroides and Rhodospirillum (R.) rubrum, and P+.960 in RC’s of Rp. viridis are studied. The isotropic hyperfine coupling constants (hfc’s) extracted by ENDOR and TRIPLE resonance [13, 14] are compared with values from advanced MO calculations, thereby relating the experimental values to structural details of the dimers.

One outstanding question in bacterial photosynthesis research concerns the relevance of the dimeric nature of the primary donor P for the fast electron transfer step from the first excited singlet state 1P*H to the charge separated state P+H−, where H stands for the intermediate acceptor bacteriopheophytin. This problem can only be attacked by performing a detailed study of the electronic structure of the primary donor itself and of the electronic interactions between all involved cofactors in their various active states. This requires the combined effort of high resolution X-ray crystallography, powerful spectroscopic methods and sufficiently advanced quantum-chemical calculations.

The light-induced cation radical of the primary electron donor, P 870 +· , in photosynthetic reaction centers from Rhodospirillum rubrum G-9, has been investigated by electron-nuclear double resonance (ENDOR) in liquid aqueous solution. The measured hyperfine coupling constants are assigned to specific molecular positions by partial deuteration. Comparison with the bacteriochlorophyll a cation radical shows different reduction factors of the individual coupling constants deviating from the value 2.0 reported in earlier investigations in frozen solutions. The average of the coupling constants is, however, reduced by a factor very close to 2.0. EPR simulations using the ENDOR coupling constants support a dimer model for P 870 +· with C 2 symmetry, where the two macrocycles are close enough to form a supermolecular orbital resulting in a different distribution of the unpaired electron, compared with the monomeric bacteriochlorophyll a cation radical. Molecular orbital calculations were used to obtain structural information about this dimer.

A field-jump device for fast stepping the electron paramagnetic resonance magnetic field around 3.4 T during pulsed electron-electron double resonance experiments at W-band (95 GHz) is described. Field jumps up to ±160 G and submicrosecond times for the full field-jump cycle allow precession frequency transfer experiments to be made for the determination of the nanometer distance and the orientation of nitroxide spin-label pairs in disordered samples.

In the last decade joint efforts of biologists, chemists, and physicists were made to understand the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. Characteristic examples of such factors are time varying specific H-bonding patterns and/or polarity effects of the microenvironment. In this overview, a few large paradigm biosystems are surveyed which have been explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three transfer proteins in action are characterized with respect to structure and dynamics: (1) light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from photosynthetic bacteria Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales.

Light-driven electron transfer (ET) and energy transfer (EnT) in a self-assembled via axial coordination Zn-porphyrin-pyridylfullerene (ZnP-PyrF) complex were studied by time-resolved electron paramagnetic resonance (TREPR) spectroscopy at 9.5 GHz (X-band) and 95 GHz (W-band). The studies over a wide temperature range were carried out in media of different polarity, including isotropic toluene and tetrahydrofuran (THF), and anisotropic nematic liquid crystals (LCs), E-7 and ZLI-4389. At low temperatures (frozen matrices), photoexcitation of the ZnP donor results mainly in singlet-singlet EnT to the pyridine-appended fullerene acceptor. In fluid phases ET is the dominant process. Specifically, in isotropic solvents the generated radical pairs (RPs) are long-lived, with lifetimes exceeding that observed for covalently linked donor-acceptor systems. It is concluded that in liquid phases of both polar and nonpolar solvents the separation of the tightly bound complex into the more loosely bound structure slows down the back ET (BET) process. Photoexcitation of the donor in fluid phases of LCs does not result in the creation of the long-lived RPs, since the ordered LC matrix hinders the separation of the complex constituents. As a result, fast intramolecular BET takes place in the tightly bound complex. Contrarily to the behavior of covalently linked donor-acceptor systems in different LCs, the polarity of the LC matrix affects the ET process. Moreover, in contrast to covalently linked D-s-A systems, utilization of LCs for the coordinatively linked D-s-A complexes does not reduce the ET rates significantly.

A time-resolved EPR and ENDOR study of the photoexcited triplet state of the free-base tetraphenylchlorin has been made in a polycrystalline toluene matrix at 120 K. Crystallization of the toluene results in a partially aligned sample. The nature of the orientation of the solute molecules is investigated by time-resolved EPR spectroscopy using the anisotropy of the zero-field splitting tensor of the triplet state as the observable parameter. It is determined that 55% of the triplet molecules are oriented in a single crystal-like domain with the triplet z-axes oriented within 15° of the magnetic field. In the ENDOR study selective excitation, of only those molecules which have their triplet z-axes parallel to the magnetic field, has permitted the measurement of the Azzcomponent of the hyperfine coupling tensor of protons, in the reference frame of the zero-field splitting tensor. The sign and magnitude of the matrix couplings are also determined. The use of the partially oriented sample drastically enhanced the signal intensity over that achieved in a randomly oriented sample by increasing the number of molecules with their triplet z-axes parallel to the magnetic field. Additionally, time-resolved ENDOR spectroscopy allowed the hyperfine interactions to be determined at far higher temperatures than usual for the study of triplet states.

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.

The addition rate constants of five differently substituted benzoyl radicals to butyl acrylate were determined by continuous-wave time-resolved EPR (TR-EPR at X-band (9 GHz) and at W-band (95 GHz)) spectroscopy. Remarkably, the reactivity of the benzoyl radicals, is divided into two domains: One is established at low (

Time-resolved electron paramagnetic resonance (TREPR) spectra of spin-polarized transient radicals in liquid solution, generated in a continuous flow-system of a W-band (95 GHz) high-field (3.4 T) EPR spectrometer, are reported. The organic free radicals are created by laser flash photolysis of ω,ω-dimethoxy-ω-phenylacetophenone (DMPA) and diphenyl-2,4,6-trimethylbenzoil phosphine oxide (TMDPO) inside the microwave cavity, and are observed at 10 ns to 20 μs delay times after the laser pulse. The analysis of the positions of the well-separated EPR signals at W-band yields the g-values of the observable transients with high accuracy. The chemically induced dynamic electron polarization (CIDEP) patterns are different from those in conventional X-band (9.5 GHz) EPR. This is due to different spin relaxation times at different magnetic fields, to field-dependent CIDEP mechanisms operating in the studied systems, and to the increased Boltzmann polarization at high fields.

The detection of hyperfine couplings in large and low-symmetry molecules is often hampered by problems of sensitivity and/or resolution. In such situations solutions to the problem are offered by simultaneously driving n.m.r., e.s.r. and optical transitions leading to well characterized electron–nuclear double and triple resonance (ENDOR, TRIPLE) and optically detected quadrupole resonance (ODNQR) spectra. The basic strategies of these multiple resonance techniques are discussed, together with applications to photosynthetic systems and spin-polarized aromatic molecules.