Absolute signs of hyperfine coupling constants as determined by pulse ENDOR of polarized radical pairs

A novel method that allows the determination of absolute signs of hyperfine coupling constants in polarized radical pair (RP) pulse electron-nuclear double resonance (ENDOR) spectra is presented, The variable mixing time (VMT) ENDOR method used here leads to a separation of ENDOR transitions originating from different electron spin manifolds by employing their dependence on the time-dependent parameters of the pulse sequence. The simple kinetic model of the RP VMT ENDOR experiment shows very good agreement with the experiments performed on the P ₇₀₀ ^.+ A ₁ ^.- RP in photosystem I. This method relies on the selective excitation of absorptive or emissive lines of one radical in the RP EPR spectrum and therefore requires high spectral resolution. This condition was fulfilled for the system studied at the low-field edge of the RP EPR spectrum obtained at Q-band. The method presented here has a very high sensitivity and does not require any equipment additional to the one used for RP pulse ENDOR. The VMT ENDOR method offers the possibility for selective suppression of signals from different electron spin manifolds.     

Applications of pulsed ELDOR-detected NMR measurements to studies of photosystem II: Magnetic characterization of YD tyrosine radical and Mn2+ bound to the high-affinity site

The pulsed electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (NMR) technique has been applied to the investigation of the hyperfine structure of the oxidized radical of Y_D tyrosine (Y _D ^· ) and Mn²⁺ ion bound to the high-affinity site in photosystem II. The resulting ELDOR spectrum of Y _D ^· was found to correspond with the pulsed electron-nuclear double resonance (ENDOR) spectrum except for a slightly larger linewidth (by a factor of 1.7). The spectrum showed marked anisotropy and revealed three peaks which can be assigned to matrix protons, C-3 and ?5 protons and one of the β-protons in the tyrosine molecule. The results demonstrate that the pulsed ELDOR-detected NMR technique is applicable to the study of organic radicals in biological systems. The Mn²⁺ ion bound to the high-affinity site in photosystem II yielded well-resolved ELDOR signals spreading over ±1000 MHz. The magnetic properties of the Mn²⁺ were characterized on the basis of the calculation of the ENDOR transitions and the anisotropy of the ELDOR spectrum.     

High-field EPR, ENDOR and ELDOR on bacterial photosynthetic reaction centers

We report on recent 95 and 360 GHz high-field electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and pulsed electron-electron double resonance (PELDOR) studies of wild-type and mutant reaction centers (RCs) from the photosynthetic bacteriumRhodobacter sphaeroides. Taking advantage of the excellent spectral and temporal resolution of EPR at 95 and 360 GHz, the electron-transfer (ET) cofactors radical ions and spin-correlated radical pairs were characterized by theirg- and hyperfine-tensor components, their anisotropicT ₂ relaxation as well as by the dipolar interaction between P ₈₆₅ ^?+ Q _A ^?? radical pairs. The goal of these studies is to better understand the dominant factors determining the specificity and directionality of transmembrane ET processes in photosynthetic RC proteins. In particular, our multifrequency experiments elucidate the subtle cofactor-protein interactions, which are essential for fine-tuning the ET characteristics, e.g., the unidirectionality of the light-induced ET pathways along the A branch of the RC protein. By our high-field techniques, frozen-solution RCs of novel site-specific single and double mutants ofR. sphaeroides were studied to modulate the ET characteristics, e.g., even to the extent that dominant B branch ET prevails. The presented multifrequency EPR work culminates in first 360 GHz ENDOR results from organic nitroxide radicals as well as in first 95 GHz high-field PELDOR results from orientationally selected spin-polarized radical pairs P ₈₆₅ ^?+ Q _A ^?? , which allow to determine the full geometrical structure of the pairs even in frozen-solution RCs.     

Pulse ENDOR studies on the radical pair P 700 +· A 1 ·− and the photoaccumulated quinone acceptor A 1 ·− of photosystem I

The secondary acceptor A₁ of the electron transport chain(s) of photosystem (PS) I is a phylloquinone (vitamin K₁, VK₁). Pulse electron paramagnetic resonance and electron nuclear double resonance (ENDOR) experiments at X-band frequencies were performed on the photoaccumulated acceptor radical A ₁ ^·? and the radical pair state P ₇₀₀ ^·+ A ₁ ^·? in PS I ofThermosynechococcus elongatus. The data obtained were compared with data from the respective radical anion of VK₁ in organic solvents. The unusualg tensor magnitude of A ₁ ^·? is explained by the hydrophobic binding pocket of this radical. The hyperfine couplings and the spin (and charge) density distribution is very different for A ₁ ^.? in PS I and VK ₁ ^·? in frozen alcoholic solution. This is attributed to a rather strong one-sided hydrogen bond to A ₁ ^·? . The presence of a hydrogen bond to A ₁ ^·? has only a minor effect ong. The hyperfine coupling constants of A ₁ ^·? determined from the radical pair spectra deviate only slightly from those derived from photoaccumulated A ₁ ^·? in PS I treated with dithionite at high pH. ENDOR resonances of the proton in a H bond were detected and an estimate of the strength and geometry of this bond to A ₁ ^·? was obtained. The significance of the hydrogen bond and other (hydrophobic) interactions of A₁ with the surrounding are briefly discussed.     

Comparison of electron spin polarization of P 870 + Qa a − observed in Zn2+-containing and Fe2+-depleted bacterial reaction centers

Recently, a general model has been developed to explain electron spin polarized (ESP) electron paramagnetic resonance (EPR) signals found in systems where radical pairs are formed sequentially. The photosynthetic bacterial reaction center (RC) is such a system in which we can experimentally vary parameters (lifetime, structure, and magnetic interactions in the sequentially formed radical pairs) that affect ESP development in order to test this model. In Fe²⁺-depleted transfer step from intermediate radical pair, P ₈₇₀ ⁺ Q _a ^? which is produced in an electron transfer step from intermediate radical pair, P ₈₇₀ ⁺ I^?. (P ₈₇₀ ⁺ is the oxidized primary donor, a special pair of bacteriochlorophyll molecules, I^? is the reduced bacteriopheophytin acceptor, and Q _a ^? is the reduced primary quinone acceptor.) The lifetime of P ₈₇₀ ⁺ I^? can be shortened relative to the lifetime of P ₈₇₀ ⁺ I^? in Fe²⁺-depleted RCs by substitution of Zn²⁺. We report the first observation of X-band and Q-band ESP EPR signals due to P ₈₇₀ ⁺ Q^? from bacterial reaction centers that contain Zn²⁺. Comparison of these signals to those observed from Fe²⁺-depleted bacterial reaction centers shows intensity differences and g-factor shifts. The results are discussed in terms of the general sequential radical pair model.     

31P and1H powder ENDOR study of ozonide radicals in carbonated apatites,synthesized from aqueous solutions

An O ₃ ^? defect in Na⁺ CO ₃ ^2? containing apatite powder has been investigated with ENDOR after X-irradiation. The powder, synthesized by a hydrolysis of octo-calciumphosphate (OCP) and Na₂CO₃ was dried at 25°C until constant weight was reached. At low temperatures, both³¹P and¹H ENDOR spectra were recorded for different settings of the magnetic field (i.e., when the magnetic field is swept through the EPR O ₃ ^? spectrum). The ENDOR powder spectra were thoroughly analyzed using computer simulations based on the “orientation selection principle”. Interactions with two types of protons and two types of³¹P nuclei could be resolved. In this way, a detailed model could be established for the O ₃ ^? ion in the hydroxyapatite lattice. The defect is located between two successive vacant hydroxyl sites. The axis connecting the two outer oxygen atoms (g _y-axis) of the O ₃ ^? ion is found to be along the hexagonalc-axis of the lattice. The twofold axis of the defect ion (g _z-axis) is parallel to theb-axis of the lattice.     

Optically detected electron nuclear double resonance of anion antisite defects in GaP

Anion antisite defects in semi-insulating GaP single crystals (doped with group V atoms) are investigated with optically detected electron paramagnetic resonance and electron nuclear double resonance (ODEPR and ODENDOR) ODEPR and ODENDOR are measured as microwave- and microwave- and rf-induced changes of the magnetic circular dichroism of the optical absorption (MCDA), respectively. In GaP: V two P antisite defects are found in about equal concentration which within ENDOR precision have tetrahedral site symmetry with respect to the nearest P ligands. The two P_GaP₄ defects have only slightly differentg-factors and³¹P superhyperfine interactions, but differ by one order of magnitude in their spin-lattice relaxation times. Their optical properties are also different from those of the P_GaP₄ defects reported earlier in GaP: Cr. It is concluded that there is no experimental prove yet. that any of the P_GaP₄ defects hitherto observed in GaP in an isolated P_GaP₄ defect which is not associated with another defect to form an antisite related defect complex.     

High-frequency EPR studies on cofactor radicals in photosystem I

Electron paramagnetic resonance (EPR) spectroscopy at W-band (94 GHz) is used to resolve theg-tensors of the radical ions of the primary chlorophyll donor P ₇₀₀ ^+? and the quinone acceptor A ₁ ^?? in photosystem I. The obtainedg-tensor principal values are compared with those of the isolated pigment radicals in organic solvents and the shifts are related to an impact of the protein environment. P ₇₀₀ ^+? has been investigated in photosystem I single crystals at 94 GHz. W-band EPR applied to the photoinduced radical pair P ₇₀₀ ^+? A ₁ ^?? is used to correctly assign theg-tensor axes of P ₇₀₀ ^+? to the molecular structure of the primary donor. Density functional theory calculations on a model of A ₁ ^?? in its binding pocket derived from the recent crystal structure of photosystem I were utilized to correlate experimental magnetic resonance parameters with structural elements of the protein.     

The primary and secondary acceptors in bacterial photosynthesis III. Characterization of the quinone radicals Q A − ⋅ and Q B − ⋅ by EPR and ENDOR

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) techniques were used to investigate the electronic structure of the primary (Q _A ^?? ) and secondary (Q _B ^?? ) ubiquinone electron acceptors in reaction centers (RCs) of the photosynthetic bacteriumRhodobacter sphaeroides. To reduce the EPR linewidth, the high-spin Fe²⁺ present in native RCs was replaced by diamagnetic Zn²⁺. Experiments were performed both on frozen solutions and single crystals at microwave frequencies of 9, 35 and 94 GHz. Differences in the EPR/ENDOR spectra were observed for Q _A ^?? and Q _B ^?? , which are attributed to different environments of the quinones in the RC. The differences exhibited themselves in: (i) the g-tensors, (ii) the¹⁷O and¹³C hyperfine coupling (hfc) constants of the quinones labeled at the carbonyl group, (iii) the¹H-hfcs of the quinone ring and (iv) the exchangeable protons hydrogen bonded to the carbonyl oxygens. From these results and from H/D exchange experiments, the following conclusions were drawn: both Q _A ^?? and Q _B ^?? have at least two hydrogen bonds of different strengths to the carbonyl oxygens. The hydrogen bonds for Q _A ^?? are stronger and more asymmetric than for Q _B ^?? . For Q _A ^?? the stronger bond (to O₄) was assigned to His(M219) and the weaker (to O₁) to Ala(M260). For Q _B ^?? the stronger bond (to O₄) was assigned to His(L190), with several weaker bonds (to O₁) to Ser(L223), Ile(224) and Gly(L225). From the temperature dependence of the hfcs of the exchangeable protons some dynamic properties of the RC were deduced. Hfcs with more distant nitrogens were observed by electron spin echo envelope modulation (ESEEM). For Q _A ^?? they were assigned to N_δ of His(M219) and to the peptide backbone nitrogen of Ala(M260) and for Q _B ^?? to N_δ of His(L190). These interactions indicate the extent of the electron wave function, which is important for the understanding of the electron transfer mechanism. Based on the magnetic resonance results, the function of the quinone acceptors in the reaction center is discussed.     

A study of hydroxyapatite nanocrystals by the multifrequency EPR and ENDOR spectroscopy methods

Specimens of powders of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) with average crystallite sizes in the range of 20–50 nm synthesized by the wet precipitation method have been investigated by the multifrequency (9 and 94 GHz) electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) methods. In specimens subjected to X-ray irradiation at room temperature, EPR signals that are caused by nitrogen compounds have been observed. Numerical calculations performed in terms of the density functional theory show that the observed EPR signal is caused by the occurrence of paramagnetic centers, the structure of which is NO ₃ ^2? and which replace the positions of PO ₄ ^3? in the hydroxyapatite structure.     

Hyperfeinstruktur des 82P3/2-Terms von Cäsium 134

The electric quadrupole interaction constantB of the 8p²P_3/2 state of Cs¹³⁴ has been determined by an optical double resonance measurement of the hyperfine structure transition v_F=11/2?F=9/2=47.84(12) Mc/s. The results are: B _8P ¹³⁴ =8.06(20) Mc/s and Q _8P ¹³⁴ =+ 0.427(8) · 10^?24cm². Comparison is made between the measurements in the 7p and 8p electronic states: Q _8P ¹³⁴ /Q _7P ¹³⁴ =0.977(20). The ratio of the corresponding Sternheimer correction factors yields the value C_7p/C_8P=0.982.     

Hyperfine structure of EPR spectra of Gd3+ odd isotopes in PbMoO4, Pb5Ge3O11, YVO4, and quadrupole interaction (temperature dependence)

A hyperfine structure of EPR signals of odd isotopes Gd³⁺ in Pb₅Ge₃O₁₁, PbMoO₄, and YVO₄ single crystals has been investigated at different temperatures. The observation of forbidden (with the nuclear spin flip) transitions has made it possible to determine quadrupole interaction P ₂ ⁰ associated with the gradient of the electric field of ligands at the impurity. It has been shown for the first time that, under the condition |P ₂ ⁰ | ≥ |A _{x, y} | (A _i are the tensor components), not only the magnitudes of splitting but also the observed asymmetry in a hyperfine structure (in perpendicular orientations of the magnetic field) depends on mutual signs of parameters of initial splitting b ₂ ⁰ and P ₂ ⁰ . Results of studying the spectra have demonstrated that |b ₂ ⁰ (T)|/|P ₂ ⁰ (T)| ～ const for a concrete single crystal, which assumes the similarity of physical mechanisms determining these parameters.     

Lebensdauer- und Hyperfeinstrukturmessungen an einigen angeregten Zuständen der Konfiguration 4d 9 5p im Pd I-Spektrum mit der Level-crossing-Methode

Lifetimes and hfs coupling constants of some excited states of the 4d ⁹ 5p configuration of Pd I have been determined in a level crossing experiment by observing the field dependence of the polarization of the fluorescence radiation in a magnetic field. From the halfwidths of the measured zero field level crossing signals one obtains the mean lifetimes of the following fine structure states:τ(³P ₁ ⁰ )=(7.46±0.32)nsec;τ(³ P ₂ ⁰ )=(6.9±0.76)nsecτ(³P ₁ ⁰ )=(4.99±0.35)nsec;τ(³ D ₁ ⁰ )=(4.89±0.40)nsecτ((³D ₃ ⁰ )=(6.99±0.49)nsec;τ(³ F ₄ ⁰ )=(7.09±0.46)nsec.Δm=2 crossing signals were detected in the³ P ₁ ⁰ ,³D ₃ ⁰ and³F ₄ ⁰ -states of the odd isotope¹⁰⁵Pd. A detailed analysis of the experimental curves yields the hfs coupling constantsA andB of these states:A(³P ₁ ⁰ )=?(133±2) Mc/sec;B(³ P ₁ ⁰ )=(140±30) Mc/secA(³D ₃ ⁰ )=?(120±10) Mc/sec;B(³ D ₃ ⁰ )=?(660±100) Mc/secA(³F ₄ ⁰ )=?(87±2) Mc/sec;B(³ F ₄ ⁰ )=?(330±30) Mc/sec. A theoretical calculation of the hfs constants is given on the basis of reduced matrix elements. Within the limit of the errors these values agree with the experimental ones. The nuclear electric quadrupole moment deduced from the measuredB values isQ (¹⁰⁵Pd)=(0.8±0.3)·10^?24 cm² (without corrections).     

Interpretation of multifrequency transient EPR spectra of the P 700 + A0Q K − state in photosystem I complexes with a sequential correlated radical pair model: Wild type versus A0 mutants

Transient electron paramagnetic resonance (TR-EPR) spectra of the electron-hole pair state P ₇₀₀ ⁺ A₀Q _K ^? in photosystem I are numerically calculated. Parameter variation concerns mainly the exchange integralJ of the precursor spin pair state P ₇₀₀ ⁺ A ₀ ^? Q_K and its lifetime τ. A prominent emissive feature in the high-field region (P ₇₀₀ ⁺ part) of the EPR spectrum turns out to be diminished with increasing lifetime τ of the precursor pair state in the case of positive exchange couplingJ>0 (ferromagnetic type). Correspondingly, the emissive feature becomes more pronounced with increasing lifetime τ in the case of negative exchange couplingJ<0 (antiferromagnetic type). These results can be used to interpret the changes in the pattern observed in TR-EPR spectra comparing wild-type and specific A₀ mutants. The central ligating amino acid residue to the A₀ chlorophyll cofactor is mutated from native methionine (M) to leucine (L) in either the PsaA or the PsaB branch. Changes are observed only for the A-side mutant: M688L(PsaA). They are consistent with the following parameters in the precursor pair P ₇₀₀ ⁺ A ₀ ^? :J≈0.5÷1.0 mT and τ=1.5÷2 ns (as compared to τ～0.05 ns in the wild type).     

Threshold electron impact excitation of Cl2

A high resolution electron impact threshold spectroscopy technique was used to examine the excitation of Cl₂ in the 2–14 eV region. This study complements previous photon absorption and emission measurements, because it is capable of detecting transitions which are optically forbidden. In the region up to 7.5 eV, broad dissociative structures are correlated with optically active valence states, although relative intensities in the threshold spectrum differ considerably and indicate a substantial contribution from the optically forbidden transitions. At 7.46 eV a series of 5 equidistant sharp peaks is detected and interpreted as arising from the² π _g Feshbach resonance, which differs from the ground state positive ion Cl ₂ ⁺ by a pair of Rydberg electrons: (4sσ)². The decay channels responsible for the appearance of the resonance in a threshold spectrum are discussed and it is suggested that they include several valence states of the (2431) and (2341) configurations, whose potential energy curves cross the Cl ₂ ^? ,²π_g curve in the region of energy at which the resonance state is formed. At higher incident electron energies and up to ionisation, Rydberg states predominate, starting with (2430) 4s^3,1 π _g states detected for the first time. The absence of broad peaks above 8 eV and the irregular appearance of Rydberg bands is consistent with the strong Rydberg-valence configuration mixing proposed by Peyerimhoff and Buenker. Where our resolution permits comparison, good general agreement is found with recent synchrotron radiation absorption measurements of optically allowed transitions.     

Impurity magneto-optical absorption with the participation of resonance states of D 2 − centers in quantum wells

The dependence of the average binding energy of the resonance g-state of a D ₂ ^? center on the induction of an external magnetic field in a quantum well with a parabolic confining potential is studied using the zero-range potential method. It has been shown that with an increasing exchange interaction, the character of the dependence of the average binding energy of the resonance g-state of the D ₂ ^? center on the induction of the external magnetic field changes. It has been assumed that in GaAs/AlGaAs quantum wells alloyed with small Si donors, resonance D ₂ ^? states can exist under conditions of exchange interaction. It has been found that in spectra of impurity magneto-optical absorption in multiwall quantum structures, exchange interaction manifests itself as oscillations of interference origin.     

High-field ENDOR and the sign of the hyperfine coupling

Two different approaches for assigning electron nuclear double resonance (ENDOR) signals to their respectiveM _s manifolds by a controlled generation of asymmetric ENDOR spectra, are described and applied to a number of systems. This assignment then allows a straightforward determination of the sign of the hyperfine coupling. Both approaches rely on a high thermal polarization that is easily achieved at high fields and low temperatures. For high-spin systems, such asS = 5/2 the assignment is afforded by the selective inversion of the | ?3/2〉 → | ?1/2〉 electron paramagnetic resonance (EPR) transition which is highly populated as compared to its symmetric counterpart, the |1/2〉 → |3/2〉 EPR transition, and therefore is easily identified. ForS = 1/2 the determination of the sign of the hyperfine coupling becomes possible when the cross- and nuclear-spin relaxation rates are much slower than the electron-spin relaxation rate and variable mixing time pulse ENDOR is used to measure the spectrum. Under these conditions the signals of theM _s = 1/2 (α) manifold become negative when the mixing time is on order of the electron-spin relaxation time, whereas those of theM _s =?1/2 (β) manifold remain positive. Under partial saturation of the nuclear transitions and short mixing time the opposite behavior is observed. Pulse W-band¹H ENDOR experiments demonstrating these approaches were applied and the signs of the hyperfine couplings of the water ligands in Mn(H₂O) ₆ ²⁺ , the H_α and H_β histidine protons in the Cu(histidine)₂ complex, the imidazole protons in Cu(imidazole) ₄ ²⁺ and the cysteine β-protons in nitrous oxide reductase were determined.     

High-frequency EPR approach to the electron spin-polarization effects observed in the photosynthetic reaction centers

Time-resolved high-frequency electron paramagnetic resonance (EPR) spectroscopy was applied to study the structure and dynamics of the electron transfer pathways in the photosynthetic RC proteins. When the spin-polarized EPR spectra are recorded at the high field, the singlet-triplet mixing in the radical pairs becomes faster due to the increase of Zeeman interaction, and a sequential electron transfer polarization model, which includes both the primary and secondary radical pairs, should be considered. Application of the sequential electron transfer polarization model for the interpretation of the bacterial RC proteins with a “slow” electron transfer rate reveals the importance of the protein dynamics. It was shown that the reorganization energy for the electron transfer process between P ₈₆₅ ⁺ H^?Q_A and P ₈₆₅ ⁺ HQ _A ^? , but not the change in the structure of the donor-acceptor complex, is a dominant factor that alters the electron transfer rate. The relaxation data, obtained in the delay after laser flash experiment, have been used to estimate the magnetic interaction in the weakly coupled radical pair. High-frequency spin-polarized EPR spectra allow the quantitative characterization of isotopically labeled quinone exchange in the PS I reaction center proteins.     

151,153Eu electron paramagnetic resonance in SrMoO4 and determination of signs of spin Hamiltonian parameters at different temperatures

The electron paramagnetic resonance (EPR) spectra of Eu²⁺ impurity centers in SrMoO₄ crystals have been studied in the temperature range of 1.8, 100–300 K. A hyperfine structure has been simulated for ^151,153Eu of different EPR transitions observed experimentally at different temperatures and external magnetic field orientations. A unique set of all parameters of the spin Hamiltonian for the known sign of the hyperfine interaction parameters A _i has been determined. It has been found that the diagonal parameters |b _n ⁰ | of the spin Hamiltonian decrease with increasing temperature; however, the parameter b ₄ ⁴ increases. The results of the study have demonstrated that |b ₂ ⁰ (T)/P ₂ ⁰ (T)| ～ const for ^151,153Eu in this single crystal.