M. Plato

Unidirectionality of charge separation in reaction centers of photosynthetic bacteria

Abstract 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 novel high-field/high-frequency EPR and ENDOR spectrometer operating at 3 mm wavelength

A high-field/high-frequency EPR and ENDOR system operating at 3 mm wavelength is described. The probe-head designs of two different resonator types, i.e. an open Fabry-Perot resonator and a cylindrical TE011 cavity, are presented in detail. The advantages and limitations of high-field/high-frequency EPR and ENDOR spectroscopy are demonstrated for selected examples. The performance data of the spectrometer suggest that it will be very useful for broad applications in physics, chemistry and biology.

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.

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.

General TRIPLE resonance on free radicals in solution. Determination of relative signs of isotropic hyperfine coupling constants

For the first time a general type of electron–nuclear–nuclear TRIPLE resonance has been performed on radicals in liquid solution. In this method two inequivalent nuclei are irradiated simultaneously at their respective NMR frequencies, the resonance being detected by an enhancement of a saturated ESR line. This general TRIPLE experiment, the analog of which is known already in the solid state (DOUBLE ENDOR), gives direct information about relative signs of hyperfine coupling constants. Several important aspects of the experimental setup are described. The experimental results are discussed on the basis of a simple relaxation model.

Molecular orbital study of polarity and hydrogen bonding effects on the g and hyperfine tensors of site directed NO spin labelled bacteriorhodopsin

Semiempirical molecular orbital methods (PM3, INDO, ZINDO/S) have been used to calculate the effects of local electric fields and of hydrogen bonding on the g and hyperfine tensors of a nitroxide spin label model system. The results yield a linear correlation between the two principal tensor components g xx and A N zz at label sites of varying polarity. Hydrogen bonding with a single water molecule produces a constant shift of Δg xx ≅ −4 × 10−4. These theoretical results are used to interpret recent high field (3.4 T, 95 GHz) electron paramagnetic resonance investigations on site-directed spin labelled bacteriorhodopsin. This protein reveals a close correlation between proticity and polarity at the various label sites. The slope of the g xx versus A N zz dependence is affected strongly by polarity induced structural strains of the spin label.

Molecular orbital investigation of dimer formations of bacteriochlorophyll a. Model configurations for the primary donor of photosynthesis

Abstract 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.

On the role of tryptophan as a superexchange mediator for quinone reduction in photosynthetic reaction centers

The role of tryptophan (W) in the quinone reduction in the reaction centers of R. viridis and Rb. sphaeroides is studied by a quantum chemical calculation of the electronic coupling matrix elements within the bacteriopheophytin‐W‐quinone subsystem, which is based on the X‐ray structural data. The results favor the superexchange mechanism via W over the direct coupling. Uncertainties in the available X‐ray coordinates and structural relaxation accompanying the formation of the P+H− state result in large changes in the relative contributions of the electronic couplings due to electron and hole superexchange. Aromatic amino acid residues may serve as essential functional components in electron transfer.

ESR, ENDOR and TRIPLE resonance studies of the primary donor radical cation P960+• in the photosynthetic bacterium Rhodopseudomonas viridis

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.

Observation of deuterium quadrupole splittings of aromatic free radicals in liquid crystals by ENDOR and TRIPLE resonance

First measurements of deuterium quadrupole coupling constants of a polyatomic doublet state radical are reported. The quadrupole couplings were determined by ENDOR and electron–nuclear–nuclear TRIPLE resonance in liquid crystals. For the partially deuterated perinaphthenyl radical the out‐of‐plane component of the deuterium quadrupole coupling was measured to be e2q33Q/h =−(94±3) kHz for both molecular sites. Within experimental error these values are equal to those measured earlier for deuterated alternant hydrocarbons in the diamagnetic singlet ground state.