W. Lubitz

The electronic structure of the primary donor cation radical in Rhodobacter sphaeroides R-26: ENDOR and TRIPLE resonance studies in single crystals of reaction centers

Abstract 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 (hfcs) were obtained and from the angular dependence of the hfcs 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 hfcs 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 hfcs 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.

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 (QA−⋅) and secondary (QB−⋅) 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 QA−⋅ and QB−⋅, 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 QA−⋅ and QB−⋅ have at least two hydrogen bonds of different strengths to the carbonyl oxygens. The hydrogen bonds for QA−⋅ are stronger and more asymmetric than for QB−⋅. For QA−⋅ the stronger bond (to O4) was assigned to His(M219) and the weaker (to O1) to Ala(M260). For QB−⋅ 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 QA−⋅ they were assigned to Nδ of His(M219) and to the peptide backbone nitrogen of Ala(M260) and for QB−⋅ to Nδ 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.

Time-resolved W-band (95 GHz) EPR spectroscopy of Zn-substituted reaction centers of Rhodobacter sphaeroides R-26

Abstract Transient EPR spectra of protonated and deuterated Zn-substituted reaction centres of Rhodobacter sphaeroides R-26, measured at a microwave frequency of 95 GHz and an external magnetic field of 3.5 T, are presented. The high-field/high-frequency spin-polarized spectrum of the correlated coupled radical pair P 865 +. Q A −. after laser flash excitation is a very sensitive monitor of the relative orientation of the two g -matrices and the dipolar tensor with respect to each other. Therefore, detailed structural information of the RC concerning the relative orientation of the primary donor P 865 +. and the acceptor Q A −. with respect to the axis connecting the two molecules in the RC can be derived. Together with the information obtained by high-field cw-EPR on single crystals, the enhanced resolution of the polarized high-field spectra allows an unambiguous assignment of the g -matrix of the donor P 865 +. to the molecular axis system. The experimental results are compared with earlier X-band (9 GHz) and K-band (24 GHz) EPR experiments.

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: the tyrosine radical Y(D)(*).

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P212121, with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{D}^{{\bullet}}}}\end{equation*}\end{document} in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.

DFT calculations of the electronic structure of the paramagnetic states Ni-A, Ni-B and Ni-C of [NiFe] hydrogenase

Structural parameters and atomic spin densities of the active centre of [NiFe] hydrogenases in the paramagnetic states Ni-A, Ni-B and Ni-C are reported. The DFT (BLYP/DZVP) calculated spin density distribution is found to be in good agreement with experimental data when the bridging ligand is OH− in the Ni-B and O2− in the Ni-A states of the enzyme. For the reduced enzyme (Ni-C) it is postulated that a hydride ligand bridges the Ni and Fe atoms. The atomic spin densities for the proposed paramagnetic states are in good agreement with experimental electron magnetic resonance data. Based on the deduced composition of the states Ni-A, Ni-B and Ni-C a model for the reaction mechanism is proposed.

Differences in the binding of the primary quinone receptor in Photosystem I and reaction centres of Rhodobacter sphaeroides-R26 studied with transient EPR spectroscopy

Abstract The binding of the primary quinone acceptor, Q, in Photosystem I (PS I) and reaction centres (RCs) of Rhodobacter Sphaeroide-R26 in which, the non-heme iron has been replaced by zinc (Zn-bRCs) is studied using transient EPR spectroscopy. In PS I, Q is phylloquinone (vitamin K1, VK1) and is referred to as A1. In Zn-bRCs, it is ubiquinone-10 (UQ10) and called QA. Native samples of the two RCs as well as those in which A1 and QA have been replaced by perdeuterated napthoquinone (NQ-d6) and duroquinone (DQ-d12) are compared. The spin polarized K-band (24 GHz) spectra of the charge separated state P+.Q−. (P = primary chlorophyll donor) in Zn-bRCs show that substitution of QA, with NQ-d6 and DQ-d12 does not have a measurable effect on the quinone orientation in the QA site. In contrast, large differences in the orientation of VK1, NQ-d6 and DQ-d12 in the A1 site in PS I are found. In addition, all three quinones in PS I are oriented differently than QA in Zn-bRCs. Further, the x and y principal values of the g-tensors of VK1−., NQ−. and DQ−. in PS I are shown to be significantly larger than in frozen alcohol and Zn-bRCs. It is suggested that the differences in the orientation and a g-values of the quinones in the two RCs arise from a weaker binding to the protein in PS I.

Time-resolved EPR of the radical pair P865+.QA−. in bacterial reaction centers. Observations of transient nutations, quantum beats and envelope modulation effects

Abstract The time evolution of the spin-polarized transient EPR signals of the radical pair state P 865 +. Q A −. is studied in perdeuterated reaction centers of Rhodobacter sphaeroides R-26 in which the non-heme iron has been replaced by zinc. The transients show modulations of the signal amplitude on several time scales. Fast quantum beat oscillations with frequencies around 15 MHz occur within the first 100 ns after the light excitation because the radical pair is generated in a coherent superposition of its eigenstates. Transient nutations also occur as a result of the precession of the magnetization about the applied microwave field B 1 . At the maximum B 1 field available (0.125 mT) a nutation frequency of ≈ 3.5 MHz is obtained. Small variations in the nutation frequency throughout the spectrum occur as a result of the dipolar coupling in the radical pair. Additional oscillations in the frequency ranges 1.5 to 2.0 MHz and 2.5 to 3.0 MHz are also observed which are attributed to the interaction between the nuclear spins and the unpaired electron spins. Analogies and differences to well-known nuclear modulation phenomena observed in pulsed EPR experiments are discussed.

Transient EPR spectroscopy of the charge separated state P+Q− in photosynthetic reaction centers. Comparison of Zn-substituted Rhodobacter sphaeroides R-26 and Photosystem I

Abstract Transient EPR spectra measured at 24 GHz and room temperature are compared for: (i) bacterial reaction centers (bRC) of Rhodobacter sphaeroides R-26 in which the non-heme iron has been replaced by zinc, (ii) Photosystem I (PS I) particles from Synechocystis 6803 and (iii) PS I in perdeuterated Synechococcus lividus algae. A comparison of Zn-bRC spectra at 9 GHz and 24 GHz in liquid and frozen solution is also presented. The spectra are assigned to the charge separated state P+·Q−· (P = primary chlorophyll donor, Q = primary quinone acceptor) and show no evidence of motional narrowing thus confirming the condition of slow molecular motion of the RC complex on the microsecond time scale of the experiment. The spectra are interpreted on the basis of the correlated coupled radical pair concept and are dependent upon the relative orientation of the two radical ions as well as their magnetic interaction parameters which are available from independent experiments. For PS I, simulations using independently measured g-tensors confirm that the phylloquinone x-axis (along the C=O bonds) lies roughly parallel to the axis connecting P+· and Q−· (dipole axis). For the Zn-bRCs the spectra show that these two axes make an angle of ∼ 60° with each other in agreement with the two independent sets of atomic coordinates which are available for the ground state structure of Rhodobacter sphaeroides R-26. The two sets of coordinates differ in the orientation of Q which results in large changes in the simulated spectra, primarily because of a shift in the effective g-factor along the dipolar axis. Starting from one of the two sets of atomic coordinates, it is shown that a rotation of Q−· through 12° about its x-axis results in a change in the sign of the polarization of the X-band spectrum. The orientation of Q−· (and P+·) in the charge separated state can thus be determined with a high degree of accuracy by transient EPR, if the magnetic interaction tensors are sufficiently well known from independent measurements.

ENDOR and ESEEM of the 15N labelled radical cations of chlorophyll a and the primary donor P700 in photosystem I

Abstract The hyperfine couplings of the nitrogen nuclei in the radical cations of both 15N-labelled chlorophyll a and the primary donor P700 in Photosystem I of Synechococcus elongatus and spinach (Spinacea oleracea) in frozen solutions were investigated by ENDOR and, for confirmation, by two-dimensional ESEEM techniques. In addition, 1H ENDOR experiments were performed on these compounds. The experimental 15N hyperfine couplings of the chlorophyll a radical cation are compared with theoretical ones obtained by RHF-INDO/SP calculations and with the respective hyperfine couplings in the closely related 15N-bacteriochlorophyll a radical cation. Based on the observed 15N and 1H hyperfine couplings two possible models are discussed for P700+: (a) the special pair model with a strongly asymmetric spin density distribution over the dimer halves; (b) the model of a strongly perturbed chlorophyll a monomer.

Transient EPR spectroscopy of perdeuterated Zn-substituted reaction centres of Rhodobacter sphaeroides R-26

Spin polarized transient EPR spectra measured in frozen solution at 9 and 24 GHz are presented for fully deuterated reaction centres of Rhodobacter sphaeroides R-26 in which the non-heme iron has been replaced by zinc. The spectra, which are assigned to the charge separated state P +. 865 Q -. A (P 865 =primary chlorophyll donor, Q A = primary quinone acceptor), are simulated entirel on the basis of independent experimental data using the correlated coupled radical pair concept. From the simulations, it is shown that it is possible to distinguish between the four possible orientations of the g tensor of P +. 865 obtained from high-field EPR on single crystals (Klette, Torring, Plato, Mobius, Bonigk and Lubitz). From the excellent agreement obtained between the simulations and the experimental results it is concluded that within the current accuracy of the X-ray structure no change in the orientation of the chromophores is induced by the charge separation.