Search

The X-ray structure analysis of photosystem (PS) I single crystals showed that the primary electron donor P700 is a heterodimer formed by one chlorophyll (Chl) a and one Chl a′ [Nature 411 (2001) 909]. The electronic structure of the cation radical P700+ of the primary donor, which is created in the charge separation process, has been probed by semiempirical molecular orbital calculations including spin polarization effects (RHF-INDO/SP). The calculations, which were based on the X-ray structure, clearly show that P700 is a supermolecule formed by two chlorophyll species. They furthermore predict an asymmetrical charge and spin density distribution in favor of the monomeric Chl a half of this dimer in accordance with results from earlier EPR and ENDOR studies [J. Phys. Chem. B 105 (2000) 1225]. The stepwise inclusion of various electrostatic interactions of the dimer with its nearest surrounding (one threonine forming a hydrogen bond to the keto group of Chl a′ and two histidines liganding the Mg atoms of the two chlorophylls) leads to a systematic enhancement of this electronic asymmetry yielding a spin density ratio of almost 5:1 as also found experimentally. A large part of this value is caused by spin polarization effects. This result is only weakly affected by the electrostatic field of more remote amino acid residues and other pigment molecules (‘accessory’ Chl a molecules) present in PS I. A separate group of calculations involving local geometry optimizations by energy minimization techniques yields a further enhancement of the spin density asymmetry. A particularly strong effect is obtained by allowing for variations of the geometry of the vinyl groups on both chlorophylls of the P700 dimer. Theoretical results for individual isotropic proton and nitrogen hyperfine coupling constants, showing a satisfactory agreement with experimental findings, are also presented.

The frozen solution electron paramagnetic resonance spectrum of the primary donor cation P + in reaction centers of site-directed mutants of Rhodobacter ( Rb. ) sphaeroides has been obtained at a microwave frequency ν =360 GHz and a magnetic field B 0 =12.8 T. Due to the high Zeeman resolution of the powder pattern, all three principal components of the rhombic g -tensors at T =160 K could be determined with high accuracy. We compare spectra of the site-directed mutants, in which the axial ligand histidine M202 of the primary donor is replaced by glutamic acid (HE(M202)) or leucine (HL(M202)), with those of the strain R26, whose primary donor is similar to that of the wild type and only lacks the carotenoid. For HE(M202), this is the first determination of its g -tensor with the principal components g xx =2.00335(3), g yy =2.00236(2) and g zz =2.00191(2). While in R26 the primary donor is a bacteriochlorophyll a dimer, the HL(M202) and HE(M202) mutants have previously been shown to be bacteriochlorophyll:bacteriopheophytin heterodimers. Their g -tensor anisotropy Δ g = g xx − g zz shows significant variations in opposite directions when compared with R26, with an increased anisotropy for HE(M202) and a decreased one for HL(M202). Calculations employing Density Functional Theory suggest that the observed shifts originate in different torsional angles of the acetyl group attached to the spin-carrying bacteriochlorophyll half L of the dimer.

The conversion of solar light by the reaction center (RC) of purple bacteria proceeds through a construct of two identical pigment branches and a ferrous ion. The imperfect C 2 symmetry of the two branches has been a major issue in trying to understand the preference of only one branch for the energy migration process. In view of the significance of this imperfection, it is desirable to express this structural property on a quantitative scale. For this purpose we have applied on six different RC structures the continuous symmetry measure methodology, an approach which allows one to measure the degree of any symmetry group in any structure. For each structure, the C 2 content of the whole RC was calculated, as well as of the top special pair where it is believed that the initial charge separation occurs. Full analysis for all pairs was carried out for one of the RCs. It was found that the top pair of cofactors is the most symmetric and that the second pair is of the highest deviation.

We present a model by which the efficiency of T 0 -S and T −1 -S mixing in a polyphyrin-quinone radical pair can be calculated. Rotational diffusion of the quinone relative to the porphyrin, causing a stochastic modulation of the exchange integral J , is included and contributes to the efficiency of triplet-singlet mixing. The model calculations can reproduce the mixing efficiencies extracted from time-resolved X-band EPR measurements.

Recent improvements of a 3 mm high-field/high-frequency CW EPR/ENDOR spectrometer [O. Burghaus, M. Rohrer, T. Gotzinger, M. Plate, and K. Mobius, Meas. Sci. Technol.3, 765 (1992)] are presented, making it possible for ENDOR measurements to be performed with NMR radiofrequeney fields of more than 2.5 mT (rotating frame) over a frequency range of 40 MHz. Applications of this setup to plastosemiquinone anion radicals are described which serve as a model for the secondary electron acceptor in plant photosystem II. The orientation-selected high-field ENDOR spectra of the plastoquinone anion radicals, recorded at different EPR field positions, are analyzed with the aid of computer simulations. The determination of hyperfine interactions of the radical in a perdeuterated solvent is described. The experiments demonstrate the potential of high-field/high-frequency EPR/ENDOR in structure determination including anisotropic interactions with the environment.

Semiempirical molecular orbital calculations of the electronic g-tensor have been performed on the primary donor cation radical P865+· of Rb. sphaeroides R-26 reaction centers (RCs). These calculations are based on Stone's theory for free radical doublet states and RHF-INDO wave functions and orbital energies. The result is in qualitative agreement with recent W-band (95 GHz) EPR measurements of the g-tensor in RC single crystals which show a breaking of C2 symmetry in the primary donor electronic structure.

Cation radicals of the primary electron donor (D + ) in bacterial photosynthesis were investigated by high field, high frequency (95 GHz) EPR. Measurements on frozen solutions of D + , a dimeric π-cation radical, of various organisms ( Rps. viridis, Rb. sphaeroides, Chloroflexus aurantiacus and a heterodimer mutant) are reported, revealing differences in the principal values of the G -tensor. Elements of a theory relating the magnitudes of G principal values to the electronic structure are discussed.

Liquid solution ENDOR and TRIPLE resonance experiments have been performed on texaphyrin radical anions generated by sodium reduction in tetrahydrofuran. Six proton and two nitrogen hyperfine couplings could be determined, including their signs. The results were compared with theoretical isotropic hyperfine couplings. They were calculated for energy-minimized texaphyrin structures, including the sodium metallo-derivative, by state-of-the-art SCF-MO methods (RHF-INDO/SP). This comparison suggests that in the course of the alkali metal reduction of texaphyrin the acidic hydrogen in the macrocycle is replaced by sodium.

The electron spin density distribution of the cation radical of the primary donor, D+, a bacteriochlorophyll a dimer was determined by ENDOR and TRIPLE resonance experiments performed on single crystals of reaction centers (RCs) of Rhodobacter sphaeroides R-26. Nine isotropic proton hyperfine coupling constants (hfc's) were obtained and from the angular dependence of the hfc's in three crystallographic planes, five complete hyperfine (hf) tensors were determined. Theoretical hf tensors were calculated by the all-valence-electron SCF molecular orbital method RHF-INDO/SP using the X-ray structure data of the dimer D and its amino acid environment. A comparison of the directions of the principal axes of the experimental and calculated hf tensors enabled us to identify the hfc's with specific protons on the two bacteriochlorophyll halves DL and DM of the dimer. The result shows that the unpaired valence electron is unequally distributed over the dimer halves, favoring DL by approx. 2:1. This ratio has been obtained from the proton hfc's of rotating methyl groups, which directly reflect the π-spin densities at the corresponding positions in the two macrocycles, DL and DM. It was further confirmed by recent 15N-ENDOR experiments on RC single crystals (Lendzian, F., Bonigk, B., Plato, M., Mobius, K. and Lubitz, W. (1992) in The Photosynthetic Bacterial Reaction Center II (Breton, J. and Vermeglio, A., eds.), pp. 89–97, Plenum Press, New York). The observed asymmetry of D+ is attributed to the difference in energies of the highest filled molecular π-orbitals of the monomeric halves, DL and DM, which is caused by differences in the structure of the two bacteriochlorophylls and/or their environment. Possible implications of this asymmetry for the electron transfer in the RC are discussed.

Austin model 1 (AMI) geometry-optimized coordinates were used as input to an INDO and an RHF-INDO/SP molecular orbital calculation. Comparison to ENDOR-derived methyl proton hyperfine coupling of the carotenoid cation radical formed photolytically on Nafion film and the second-moment-deduced EPR linewidth of the electrochemically prepared carotene cation in solution showed excellent agreement with the RHF-INDO/SP-derived proton hyperfine couplings. The INDO-derived solution linewidth was calculated to be 60% larger than that found experimentally due to the calculated INDO methyl protons being larger by 64%.

The EPR g -factor rotation pattern of the primary donor cation radical in the x  y plane of illuminated reaction center single crystals of Rb. sphaeroides R-26 near room temperature was measured at 3 mm wavelength (W-band, 95 GHz). In contrast to earlier X-band (9.5 GHz) EPR experiments, the two magnetically inequivalent sites of the four reaction centers per unit cell were well resolved. in frozen reaction center solution was also measured by 3 mm EPR. The analysis of both the frozen-solution spectrum and the single-crystal rotation pattern yields the principal values of the g -tensor and the phase angle between the two magnetically inequivalent sites in the x  y plane.

ENDOR measurements have been performed on 13 C labelled biradicals, namely Yang's biradical and p -phenylenebisgalvinoxyl, in frozen solutions. Besides intense ENDOR lines at the free nuclear Zeeman frequencies, hyperfine shifted 1 H and 13 ENDOR lines could be detected with excellent signal-to-noise ratios. From these spectra not only the magnitudes, but also the signs of the hyperfine splitting components relative to the zero field splitting parameter D could be derived. It is demonstrated that the sign of D in Yang's biradical must be negative. This may be due to a spin-orbit coupling contribution to the zero field splitting parameter.

Three stable dimer configurations of bacteriochlorophyll a are predicted by a self-consistent-field molecular orbital calculation (all valence electrons, restricted Hartree-Fock, INDO-parametrization, perturbational treatment of spin polarization) including geometry optimization by energy minimization. Spin-density distributions of the cation radicals are also calculated and compared with results from magnetic resonance experiments. The largest binding energy is obtained from a strong overlap of the π-systems combined with the formation of two symmetrical MgO bonds between the monomeric components. This structure is compatible with the experimental spin density distribution and very similar to the recently determined X-ray structure of the bacteriochlorophyll b dimer in the photosynthetic reaction center of Rps. viridis. Some possible perturbations by the protein environment are discussed.

Time-resolved spectroscopy in conjunction with X-ray structural data for reaction centers of Rhodopseudomonas viridis and Rhodobacter sphaeroides reveal a branching ratio a > 5 for the primary electron-transfer rates, favouring one of the two, almost symmetrical pigment/protein branches, L and M. In this paper we explore the origins of this unidirectionality of electron transfer between the excited singlet state of the bacteriochlorophyll dimer ( 1 P∗) and the bacteriopheophytin (H) along the L protein subunit. Nonadiabatic electron-transfer theory is applied to analyze the asymmetry of the electron-transfer rates, k L and k M across the L and M branches. The asymmetry originates from the cumulative contributions of the nuclear Franck-Condon factor and the electronic coupling, both of which enhance the electron transfer rate across the L branch. The nuclear Frank-Condon factors are modified by the energy difference ΔE LM between the states P + H − L and P + H − M , which is induced by the electrostatic interactions of these ion-pair states with the protein polar groups, as well as by asymmetric Coulomb and medium polarization interactions. The computation results in ΔE LM = −(0.09 ± 0.04) eV, which yields a nuclear enhancement contribution at 300 K of 1.5 (+0.8, −0.3) to k L k M and therefore is insufficient to explain alone the observed asymmetry in reaction centers of Rps. viridis . Another contribution to the unidirectionality originates from electronic superexchange coupling for 1 P∗-B-H via the virtual states of the accessory bacteriochlorophyll (B). The ratio of the intermolecular 1 P∗-B L and 1 P∗-B M electronic interaction terms was evaluated utilizing the tight-binding approximation with SCF-MO wavefunctions, together with the structural data for the prosthetic groups and for the polar amino acid side chains of the protein in reaction centers of Rps. viridis . The contribution to the enhancement of k L k M by the electronic superexchange is approx. 8 ± 4. This asymmetry was traced to the combination of an excess negative charge density on the M-dimer component P M , together with structural asymmetry, which enhances the P M -B L electronic overlap. Consequently, the 1 P∗-B L -H L superexchange is favoured over the 1 P∗-B M -H M interaction. The combined effects of asymmetric nuclear Franck-Condon factors and electronic couplings yield a branching ratio of the electron-transfer rates along the two pigment branches in reaction centers of Rps. viridis of an approx. 12 (−7, +15). This is sufficiently large to explain the experimentally observed unidirectionality.

A phosphorescence line-narrowing technique involving direct quartet ground state to doublet excited state pumping, is used to resolve completely optical—Zeeman components at field strengths compatible with Q-band EPR spectroscopy. By incorporating this technique, ODMR of the lowest doublet excited state of Cr3+ ions in an organometallic host crystal (tris(acetylacetonato)aluminum(III)) has been obtained for several magnetic field orientations. Selective, direct optical pumping shows that the three prominent, close-lying phosphorescence bands at 773, 776 and 778 nm correspond to three zero-phonon origins. ODMR performed on the 778 nm origins yields the values|g⊥|=1.921 ± 0.003, |{g∥|}|=1.60 ± 0.03 for the excited-state g-tensor and provides an order of magnitude estimate of ≈ 10 μs for the excited-state spin—lattice relaxation time.

In the ENDOR spectra of some silacyclopentadiene anion radicals in solution, temperature-dependent line widths and line positions were observed. This dynamic behaviour could be interpreted as being due to hindered rotation of substituted phenyl rings. It was possible to determine activation energies and entropies, from which it is concluded that the main part of the potential barrier stems from π conjugation effects. The observed dynamic effects were also exploited for assignment of ENDOR lines to molecular positions.

Solution 25 Mg ENDOR and TRIPLE resonance experiments have been performed on the bacteriochlorophyll a (BChl a) anion and cation radicals enriched in 25 Mg. The 25 Mg isotropic hyperfine coupling constants for BChl a + · and BChl a − · have been determined. The values are in good agreement with those obtained from recent MO calculations.

From chemically generated anion radicals of anti[2.2](1,4)naphthalenophane and 1,4-dimethylnaphthalene, hyperfine coupling constants including relative signs are determined by ENDOR and TRIPLE resonance. The data are compared with spin density distributions obtained by MO calculations. The results are discussed with respect to the spin density distribution in the “special pair” primary donor of bacterial photosynthesis.

The radical cation of bacterioehlorophyll b (BChl b) is investigated by ENDOR and TRIPLE resonance in liquid solution. The experimental hyperfine coupling constants, ten proton and three nitrogen couplings, are compared with the predictions from advanced molecular-orbital calculations (RHF INDO/SP). The detailed picture obtained of the spin density distribution is a prerequisite for the investigation of the primary electron donor radical cation in BChl b containing photosynthetic bacteria.

Zusammenfassung Die Synthese biradikalischer Kohlenwasserstoffe mit unterschiedlich verbrukten Tetraarylcyclopentadienyl-Halften (‘Bis-ZIEGLER-Radikalen”) wird beschrieben. Aus den EPR-Pulverspektren lassen sich bei den regellos orientierten Zweispinsystemen intramolekulare Elektronen-Spin-Spin-Kopplungen zu Triplettzustanden erkennen. Aus der Grosse der Nullfeldaufspaltung wird zunachst anhand einfacher Molekulmodellbetrachtungen auf die Molekulkonformation geschlossen. Insbesondere werden jedoch mit Hilfe quantenmechanischer Modellrechnungen und durch Vergleich der Rechenergebnisse mit den experimentellen Daten detailliertere Strukturuntersuchungen durchgefuhrt. Dabei wird der Einfluss der Verdrillung zwischen den einzelnen Ringsystemen und damit auch ggf. der cis- oder trans-Orientierung der halftigen Spindichtezentren auf die Grosse der D-Parameter ermittelt. Schliesslich wird das Verhalten der vier hochsten besetzten, nahezu entarteten Molekulorbitale gegenuber strukturellen Veranderungen diskutiert.

A theory of stimulated nuclear polarization (SNP) is proposed for the first time. The calculations are performed by the method of summation of radical pair (RP) re-encounter contributions to the recombination of neutral radicals in high magnetic fields in the framework of a continuous diffusion model. Some rules for the sign of the SNP effect are formulated. Based on the calculations for model systems, we have studied the effect of different parameters of RPs on the SNP effects: the rate constants of RP recombination from singlet and triplet states, the rates of RP decay, e.g. due to the reaction of radicals with acceptors, and the effective in-cage RP lifetime. It is shown that in SNP the phenomenon of spin-looking manifests itself: when the amplitude of an applied microwave field exceeds the hf splitting in the ESR spectrum, the sign of polarization changes. The mutual effect of nuclei on the magnitude and sign of SNP is studied. For non-viscous liquids (η ≈ 1 cP), the analytical expressions for the SNP effects are obtained by perturbation theory. It is demonstrated that over a wide range of RP parameters, the results for SNP obtained on the basis of these analytical expressions are in good agreement with numerical results. The comparison of theoretical and experimental results exhibits their full conformity.

ODMR and ODENDOR spectra were obtained for quinoxaline and p-dichlorobenzene in their excited triplet states. These molecules were studied in mixed crystals using heavy atom containing hosts. For protons the complete hyperfine tensors could be evaluated, yielding information about the symmetry and geometry of these molecules in their triplet states. For quinoxaline no deviations from planarity have been observed whereas triplet p-dichlorobenzene turned out to be non-planar. The measured π-spin densities in p-dichlorobenzene confirm the 3B1u symmetry of this molecule in its first excited triplet state.

The light-induced radical cation of the primary electron donor P960+• in photosynthetic reaction centers from Rhodopseudomonas viridis has been investigated by ESR, ENDOR and TRIPLE techniques. Both the comparison with the cation radical of monomeric bacteriochlorophyll b (BChl b) and with molecular-orbital calculations performed on P960+• using the results of an X-ray structure analysis, consistently show an asymmetric distribution of the unpaired electron over the two BChl b molecules which constitute P960+•. The possible relevance of this result for the primary electron transfer step in the reaction center is briefly discussed.

Die Synthese von Schlenk'schen und Tschitschibabin'schen Kohlenwasserstoffanaloga mit unterschiedlich verbru¨ckten Dibiphenylmethyl-Ha¨lften wird beschrieben. Mit Hilfe von EPR-Pulverspektren lassen sich bei den regellos orientierten Zweispinsystemen intramolekulare Elektronen-Spin-Spin-Kopplungen zu Triplettzusta¨nden erkennen. Aus der Gro¨sse der Nullfeldaufspaltung wird zuna¨chst anhand einfacher Moleku¨lmodellbetrachtungen auf die Moleku¨lkonformation geschlossen. Insbesondere werden jedoch mit Hilfe quantenmechanischer Modellrechnungen und durch Vergleich der Rechenergebnisse mit den experimentellen Daten detailliertere Strukturuntersuchungen durchgefu¨hrt. Dabei wird der Einfluss der Verdrillung zwischen den einzelnen Phenylringen und damit auch der cis- oder trans-Orientierung der ha¨lftigen Spindichtezentren auf die Gro¨sse der D-Parameter ermittelt. Eine Klassifizierung der einzelnen Zweispinsysteme nach ihren magnetischen Eigenschaften wird diskutiert.

A quantitative test of ENDOR theory for free radicals in solution is performed on the 14N nucleus of the nitrobenzene radical anion in dimethoxyethane. Dominant relaxation contributions are caused by modulation of the electron nuclear dipolar interaction. They can be calculated from the known G and 14N hyperfine tensors of the nitrobenzene radical anion. The effects of spin-rotation interaction and Heisenberg spin exchange at different concentrations are investigated experimentally and theoretically. The concentration and temperature dependences of the 14N ENDOR amplitudes are found to agree well with theoretical predictions.

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.

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.