Peter Gast

ESEEM study of spin-spin interactions in spin-polarised P+QA− pairs in the photosynthetic purple bacterium Rhodobacter sphaeroides R26

Abstract Electron spin echo envelope modulation (ESEEM) of the transient spin-polarised P+QA− pairs has been studied at 20 and 220 K. The observed strong out-of-phase modulation is interpreted as resulting from electron-electron dipolar and exchange interactions. Fourier-transformed echo envelopes are consistent with the theory developed recently by Tang, Thurnauer and Norris and allow us to obtain readily the values of dipolar and exchange couplings. The results are insensitive to 15N enrichment.

Asymmetric binding of the 1- and 4-C=O groups of QA in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10.

Using 1‐, 2‐, 3‐ and 4‐13C site‐specifically labelled ubiquinone‐10, reconstituted at the QA site of Rhodobacter sphaeroides R26 reaction centres, the infra‐red bands dominated by the 1‐ and 4‐C = O vibration of QA are assigned in the QA(‐)‐QA difference spectra. The mode dominated by the 4‐C = O vibration is drastically downshifted in the reaction centres as compared with its absorption frequency in free ubiquinone‐10. In contrast, the mode dominated by the 1‐C = O vibration absorbs at similar frequencies in the free and the bound forms. The frequency shift of the 4‐C = O vibration is due to a large decrease in bond order and indicates a strong interaction with the protein microenvironment in the ground state. In the charge‐separated state the mode dominated by the semiquinone 4‐C = O vibration is characteristic of strong hydrogen bonding to the microenvironment, whereas the mode dominated by the 1‐C = O vibration indicates a weaker interaction. The asymmetric binding of the 1‐ and 4‐C = O groups to the protein might contribute to the factors governing different redox reactions of ubiquinone‐10 at the QA site as compared with its reactions at the QB site.

Time-resolved ESR and chemically induced dynamic electron polarisation of the primary reaction in a reaction center particle of Rhodopseudomonas sphaeroides wild type at low temperature.

Recently a very fast decaying ESR signal preceding the so-called signal I in plant photosynthesis has been observed [ 1 ] . The signal was emissive, which was taken to provide evidence for the occurrence of chemically induced dynamic electron polarisation (CIDEP). Such spin-polarisation effect may give insight into the events preceding the primary charge separation. Because the primary reactions in the bacterial photosystem are much better understood than those of the plant systems [2], we have investigated light-induced paramagnetism of bacterial reaction centers on a time scale of 20-300 ps. At a temperature of 100°K we have found a fast decaying, partly emissive, flash-induced ESR signal, with a complex timeand field-dependency, in particles obtained from Rhodopseudomonas sphaeroides wild type by treatment with sodium dodecyl sulphate (SDS). We have attempted to analyse the time-resolved spectra in the framework of current CIDEP theories. We can account for the spectral shape by assuming that the radical pair mechanism is operative, through which the ESR signals of the primary donor-acceptor pair become spinpolarised. This polarisation then decays in a time comparable to the spin-lattice relaxation time. Apparently, by monitoring the time evolution of the lightinduced ESR signal one can probe a spin-memory of the much faster events leading to the charge separation.

Heteronuclear 2D-correlations in a uniformly [13C, 15N] labeled membrane-protein complex at ultra-high magnetic fields.

One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C,15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the α-helical segments of the protein, mostly found in the membrane interior.

Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides.

The tyrosine-(M)210 of the reaction center of Rhodobacter sphaeroides 2.4.1 has been changed to a tryptophan using site-directed mutagenesis. The reaction center of this mutant has been characterized by low-temperature absorption and fluorescence spectroscopy, time-resolved sub-picosecond spectroscopy, and magnetic resonance spectroscopy. The charge separation process showed bi-exponential kinetics at room temperature, with a main time constant of 36 ps and an additional fast time constant of 5.1 ps. Temperature dependent fluorescence measurements predict that the lifetime of P* becomes 4–5 times slower at cryogenic temperatures. From EPR and absorbance-detected magnetic resonance (ADMR, LD-ADMR) we conclude that the dimeric structure of P is not significantly changed upon mutation. In contrast, the interaction of the accessory bacteriochlorophyll BA with its environment appears to be altered, possibly because of a change in its position.

Asymmetric binding of the primary acceptor quinone in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides R26, probed with Q-band (35 GHz) EPR spectroscopy

The reaction center (RC)‐bound primary acceptor quinone QA of the photosynthetic bacterium Rhodobacter sphaeroides R26 functions as a one‐electron gate. The radical anion Q•− A is proposed to have an asymmetric electron distribution, induced by the protein environment. We replace the native ubiquinone‐10 (UQ10) with specifically 13C‐labelled UQ10, and use Q‐band (35 GHz) EPR spectroscopy to investigate this phenomenon in closer detail. The direct observation of the 13C‐hyperfine splitting of the g z‐component of UQ10•− A in the RC and in frozen isopropanol shows that the electron spin distribution is symmetric in the isopropanol glass, and asymmetric in the RC. Our results allow qualitative assessment of the spin and charge distribution for Q•− A in the RC. The carbonyl oxygen of the semiquinone anion nearest to the S = 2 Fe2+‐ion and QB is shown to acquire the highest (negative) charge density.

The electronic structure of the primary electron donor of reaction centers of purple bacteria at atomic resolution as observed by photo-CIDNP 13C NMR

Composed of the two bacteriochlorophyll cofactors, PL and PM, the special pair functions as the primary electron donor in bacterial reaction centers of purple bacteria of Rhodobacter sphaeroides. Under light absorption, an electron is transferred to a bacteriopheophytin and a radical pair is produced. The occurrence of the radical pair is linked to the production of enhanced nuclear polarization called photochemically induced dynamic nuclear polarization (photo-CIDNP). This effect can be used to study the electronic structure of the special pair at atomic resolution by detection of the strongly enhanced nuclear polarization with laser-flash photo-CIDNP magic-angle spinning NMR on the carotenoid-less mutant R26. In the electronic ground state, PL is strongly disturbed, carrying a slightly negative charge. In the radical cation state, the ratio of total electron spin densities between PL and PM is 2:1, although it is 2.5:1 for the pyrrole carbons, 2.2:1 for all porphyrinic carbons, and 4:1 for the pyrrole nitrogen. It is shown that the symmetry break between the electronic structures in the electronic ground state and in the radical cation state is an intrinsic property of the special pair supermolecule, which is particularly attributable to a modification of the structure of PL. The significant difference in electron density distribution between the ground and radical cation states is explained by an electric polarization effect of the nearby histidine.

15N photochemically induced dynamic nuclear polarization magic-angle spinning NMR analysis of the electron donor of photosystem II

In natural photosynthesis, the two photosystems that operate in series to drive electron transport from water to carbon dioxide are quite similar in structure and function, but operate at widely different potentials. In both systems photochemistry begins by photo-oxidation of a chlorophyll a, but that in photosystem II (PS2) has a 0.7 eV higher midpoint potential than that in photosystem I (PS1), so their electronic structures must be very different. Using reaction centers from 15N-labeled spinach, these electronic structures are compared by their photochemically induced dynamic nuclear polarization (photo-CIDNP) in magic-angle spinning (MAS) NMR measurements. The results show that the electron spin distribution in PS1, apart from its known delocalization over 2 chlorophyll molecules, reveals no marked disturbance, whereas the pattern of electron spin density distribution in PS2 is inverted in the oxidized radical state. A model for the donor of PS2 is presented explaining the inversion of electron spin density based on a tilt of the axial histidine toward pyrrole ring IV causing π-π overlap of both aromatic systems.

FTIR spectroscopy shows weak symmetric hydrogen bonding of the QB carbonyl groups in Rhodobacter sphaeroides R26 reaction centres

The absorption frequencies of the C = O and C = C (neutral state) and of the C̲⋯O (semiquinone state) stretching vibrations of QB have been assigned by FTIR spectroscopy, using native and site‐specifically 1‐, 2‐, 3‐ and 4‐13C‐labelled ubiquinone‐10 (UQ10) reconstituted at the QB binding site of Rhodobacter sphaeroides R26 reaction centres. Besides the main C = O band at 1641 cm−1, two smaller bands are observed at 1664 and 1651 cm−1. The smaller bands at 1664 and 1651 cm−1 agree in frequencies with the 1‐ and 4‐C = O vibrations of unbound UQ10, showing that a minor fraction is loosely and symmetrically bound to the protein. The larger band at 1641 cm−1 indicates symmetric H‐bonding of the 1‐ and 4‐C = O groups for the layer fraction of UQ10 but much weaker interaction as for the 4‐C = O group of QA The FTIR experiments show that different C = O protein interactions contribute to the factors determining the different functions of UQ10 at the QA and the QB binding sites.

Determination of the number of detergent molecules associated with the reaction center protein isolated from the photosynthetic bacterium Rhodopseudomonas viridis : effects of the amphiphilic molecule 1,2,3-heptanetriol

Detergent‐free reaction center (RC) proteins from the photosynthetic bacterium Rhodopseudomonas viridis were obtained using Bio‐Beads SM‐2. With these RCs, the amount of detergent molecules associated with the protein was measured by determining the detergent concentration at which re‐solubilization occurred as a function of the RC concentration. For N,N dimethyl dodecylamine‐N‐oxide (LDAO), Triton X‐100 and β‐octylglucoside 260 ± 30,105 ± 10 and 360 ± 100 detergent molecules were necessary to dissolve the protein, respectively. With this technique we have studied the effect of the amphiphilic molecule 1,2,3‐heptanetriol, which is essential in the crystallization process of these RCs. Addition of 5% 1,2,3‐heptanetriol reduces the value for LDAO to 120 ± 20 LDAO/RC, supporting the notion that crystallization of the RCs is promoted by increasing the number of protein‐protein contacts.