A. van der Est

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.

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Q, in Photosystem I (PS I) and reaction centres (RCs) of Rhodobacter Sphaeroide-R26 in which, the non-heme iron has been replaced by zinc (Zn-bRCs) is studied using transient EPR spectroscopy.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 Native samples of the two RCs as well as those in which A1 and QA have been replaced by perdeuterated napthoquinone (NQ-d6) and duroquinone (DQ-d12) are compared.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.

TRANSIENT ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY ON GREEN-SULFUR BACTERIA AND HELIOBACTERIA AT TWO MICROWAVE FREQUENCIES

Spin polarized transient EPR spectra taken at X-band (9 GHz) and K-band (24 GHz) of membrane fragments of Chlorobium tepidum and Heliobacillus mobilis are presented along with the spectra of two fractions obtained in the purification of reaction centers (RC) from C. tepidum. The lifetime of P+. is determined by measuring the decay of the EPR signals following relaxation of the initial spin polarization. All samples except one of the RC fractions show evidence of light induced charge separation and formation of chlorophyll triplet states. The lifetime of P+. is found to be biexponential with components of 1.5 ms and 30 ms for C. tepidum and 1.0 and 4.5 ms for Hc. mobilis at 100 K. In both cases, the rates are assigned to recombination from F-X. The spin polarized radical pair spectra for both species are similar and those from Hc. mobilis at room temperature and 100 K are identical. In all cases, an emission/absorption polarization pattern with a net absorption is observed. A slight narrowing of the spectra and a larger absorptive net polarization is found at K-band. No out-of-phase echo modulation is observed. Taken together, the recombination kinetics, the frequency dependence of the spin polarization and the absence of an out-of-phase echo signal lead to the assignment of the spectra to the contribution from P+. to the state P+.F-X. The origin of the net polarization and its frequency dependence are discussed in terms of singlet-triplet mixing in the precursor. It is shown that the field-dependent polarization expected to develop during the 600-700 ps lifetime of P+.A-.0 is in qualitative agreement with the observed spectra. The identity that the acceptor preceding FX and the conflicting evidence from EPR, optical methods and chemical analyses of the samples are discussed.

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.

Electron spin polarization in consecutive spin-correlated radical Pairs: Application to short-lived and long-lived precursors in type 1 photosynthetic reaction centres

An analytical treatment of the spin dynamics in sequential photoinduced correlated coupled radical pairs is presented and applied to the spectra of the states P+A1− and P+Fx− in type 1 photo-synthetic reaction centres. Expressions for the spin polarized spectra are derived for the specific limiting cases of a very short-lived and very long-lived primary radical pair which correspond to the situation found in heliobacteria and photosystem I (PSI), respectively. The inhomogeneous line-broadening due to the unresolved hyperfine couplings is taken explicitly into account. It is shown that the density matrix of the secondary pair ρ2 can be written as the sum of two terms corresponding to (i) the part which is independent of the spin dynamics in the precursor, (ii) the additional spin polarization which is generated during the lifetime of the precursor and transferred to the secondary pair. The latter term contains two contributions which arise from the difference of the Zeeman interactions of the radicals in the primary pair and from the inhomogeneous line broadening. The predicted polarization patterns are compared to those established for chemically induced dynamic electron polarization (CIDEP) when uncoupled radicals are generated from a radical pair precursor. The expressions are then used to simulate the experimental spectra of the consecutive pairs P+A1− and P+Fx− in PSI using parameters derived entirely from independent experimental data. Excellent agreement with the experimental results is obtained. The spectra of P+Fx− in heliobacteria at X- and K-band are also simulated and it is shown that the observed polarization patterns can be reproduced assuming direct electron transfer from A0 to Fx with a time constant ofτ = 600 ps.

The transient EPR spectra and spin dynamics of coupled three-spin systems in photosynthetic reaction centres

The concept of introducing an additional, stable paramagnetic species into photosynthetic reaction centres to increase the information content of their spin polarized transient EPR spectra is investigated theoretically. The light-induced electron transfer in such systems generates a series of coupled three-spin states consisting of sequential photoinduced radical pairs coupled to the stable spin which acts as an “observer”. The spin polarized transient EPR spectra are investigated using the coupled three-spin system P+I−Q−A in pre-reduced bacterial reaction centres as a specific example which has been studied experimentally. The evolution of the spin system and the spin polarized EPR spectra of P+I−Q−A and Q−A following recombination of the radical pair (P = primary donor, I = primary acceptor, QA = quinone acceptor) are calculated numerically by solving the equations of motion for the density matrix. The net polarization of the observer spin is also calculated analytically by perturbation theory for the case of a single, short-lived, charge-separated state. The result bears a close resemblance to the chemically induced nuclear polarization (CIDNP) generated in photolysis reactions in which a nuclear spin plays the role of the observer interacting with the radical pair intermediates. However, because the Zeeman frequencies of the three electron spins involved are usually quite similar, the polarization of the electron observer spin in strong magnetic fields can reflect features of the CIDNP effect in both, high and low magnetic fields. The dependence of the quinone spin polarization on the exchange couplings in the three-spin system is investigated by numerical simulations, and it is shown that the observed emissive polarization pattern is compatible with either sign, positive or negative, for a range of exchange couplings, JPI, in the primary pair. The microwave frequency and orientation dependence of the spectra are discussed as two of several possible criteria for determining the sign of JPI.

Separation of the exchange and dipolar contributions to the spin-spin coupling in the donor-acceptor complex TAPD-ZnP-NQ

The donor-acceptor complex, 2-tetraalkylphenylenediamine-zinc porphyrin-2-naphthoquinone (TAPD-ZnP-NQ), is studied using transient EPR at K-band (24 GHz). Spin polarized spectra of the radical pair TAPD+.NQ−. and of the triplet state TAPD-3ZnP-NQ are observed simultaneously following light excitation of the complex at 30 K in frozen solutions 2-methyl-tetrahydrofuran and the liquid crystal BDH E7. In the liquid crystal, the complex is partially ordered and the spectra depend on the orientation of the sample with respect to the magnetic field. The orientational distribution function is obtained independently by simulating the spectra of the triplet state and assuming that the principal axes of the order matrix coincide with those of the inertia tensor as has been found for solutes in nematic phases. The spectra of TAPD+.NQ−. are analyzed on the basis of the coupled correlated radical pair (CCRP) model using an estimate of the geometry based on the X-ray crystal structures of the components and the independently obtained orientational distribution function. It is shown that the experimental spectra are consistent with the assumed geometry and that the exchange coupling,J, is approximately 0.1 mT. This value is at least two order of magnitude larger than that found in the analogous radical pair P+.Q−. in photosynthetic reaction centres. This difference is rationalized in terms of different mechanisms for the exchange coupling.

The application of time resolved EPR to the study of photoreactive molecules in solid and liquid crystalline environments

The use of transient EPR spectroscopy for the study of photoreactions in solid and liquid crystalline is demonstrated. The method is applied to an intramolecular photoreaction in 2-(4-methylpentyl)-7-nonanoylfluorene (MPNOF) and to an intermolecular photoreaction in 4-cyano-4′-octylbiphenyl (8CB) doped with phenazine. It is shown that both MPNOF and phenazine undergo photoreactions via their first excited triplet state. Orientational ordering in the solid and liquid crystalline phase is studied via the excited triplet powder spectrum. Spectra of precursor and products can be identified and separated by their kinetics. The possible nature of the products of reaction is discussed.

Liquid Crystalline Side Chain Polymers with Fluorene as Mesogenic Group

Abstract The properties of some liquid crystalline side chain polysiloxanes with 2,7-disubstituted fluorene as mesogenic unit are reported. The polymers exhibit smectic A and smectic E like textures. Beside DSC and polarizing microscopy 2H NMR spectroscopy was used to characterize the phase transitions. Anthracene-d10 was used as a solute probe molecule and its order parameters were calculated in the liquid crystalline polymer phases. Two other low molecular weight liquid crystals (8CB. MPNOF) were studied by the same method. The general use of anthracene-d10 for 2H NMR spectroscopy as probe molecule for liquid crystalline polymers is discussed.

Structural and Functional Properties of the State P+· Q-·from Transient EPR Spectroscopy

The structure of the charge separated state P+· Q-· reaction centres (RC’s) of Rhodobacter sphaeroides R-26 in which, the non-heme iron has been replaced by zinc (Zn-bRC’s) is studied using transient EPR spectroscopy. A qualitative description of the spin polarized transient EPR spectra of P+·Q-· is presented and used to draw conclusions about the relative orientations of P 865 +. and Q A -. . Experimental and calculated spectra are presented for fully deuterated and protonated Zn-bRC’s at three microwave frequencies: X-band (9 GHz), K-band (24 GHz) and W-band (95 GHz). The simulated spectra are calculated on the basis of independently determined experimental parameters. From the good agreement between the experimental spectra and those calculated using the ground state x-ray structure, it is concluded that no significant light induced structural changes occur. K-band spectra at 300 K and 50 K are compared and it is suggested that the narrowing of the spectrum at room temperature is a result of changes in the hyperfine couplings due to motion of the chromophores as well as a change in the charge distribution on (Math).