Jacques Breton

The Photosynthetic Bacterial Reaction Center II

Alexander Ogrodnik, Thomas Langenbacher, Ulrich Eberl, Martin Yolk and Maria E. Michel-Beyerle Institut fUr Physikalische und Theoretische Chemie Technische Universitiit Miinchen, Lichtenbergstr.4, D-8046 Garching (Germany) Electric field effects on reaction centers from Rb. sphaeroides have been detected in ps transient absorption at 90K. Upon application of a field of 7·IOSY /cm the bleaching of the bacteriopheophytin at the A-branch (HA, 545nm) monitored after 30ps mirrors a reduction of the Quantum yield of P+HA formation by 0:11% (P being the special pair). A similar field induced reduction of the Quantum yield is observed when probing the bleaching of P at 870nm. However, this effect evolves with a time constant of o:900ps. We conclude that the loss channel employs an intermediate state with this lifetime before repopulating the ground state. Field induced formation of P+Hii (B-branch) was excluded from measurements in the Qx transition of HB at 533nm. Measurements in the blue and red wings of the 800nm Qy band of the two bacteriochlorophylls (BA and BB) are compatible with the state P+Bii being the long-lived intermediate involved in the loss of quantum yield.

Analysis of picosecond laser induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach

A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law.

The Lhca antenna complexes of higher plants photosystem I.

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while beta-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.

Orientation of the chromophores in the reaction center of Rhodopseudomonas viridis. Comparison of low-temperature linear dichroism spectra with a model derived from X-ray crystallography☆

Abstract Linear dichroism (LD) and absorption (A) spectra of reaction centers from Rhodopseudomonas viridis included in the native chromatophores or reconstituted in planar aggregates have been recorded at 10 K. The samples were oriented in squeezed polyacrylamide gels and the primary donor P was in the reduced or (chemically) oxidized state. The LD spectra of reaction centers in these two states are in favor of a dimeric model of P in which excitonic coupling between the two non-parallel QY transitions leads to a main transition at 990 nm (parallel to the membrane plane) and another one of smaller oscillator strength at 850 nm (tilted at approx. 60° out of the membrane plane). These assignments are in close agreement with the ones proposed in a previous LD study at room temperature (Paillotin, G., Vermeglio, A. and Breton, J. (1979) Biochim. Biophys. Acta 545, 249–264). The main QX excitonic component of P has a broad absorption peaking at 620 nm and it corresponds to dipoles exhibiting the same orientation as those responsible for the 850 nm transition. On the basis of the present LD study and of CD data of chemically oxidized-minus-reduced reaction centers, we proposed that the minor QX excitonic component of P is oriented close to the membrane plane and absorbs around 660 nm. The two monomeric bacteriochlorophylls exhibit a positive LD for both their QY transitions (unresolved at 834 nm) and their QX transitions (resolved at 600 and 607 nm), indicating that the planes of these molecules are only slightly tilted out of the membrane plane. The two bacteriopheophytins exhibit strong negative LD with identical LD/A values for their QY transitions (resolved at 790 and 805 nm) and small positive LD for their QX transitions (resolved at 534 and 544 nm), demonstrating that these two molecules are strongly tilted out of the membrane plane with each of the QY transitions tilted at approx. 50° out of that plane. A comparison of these LD data with the structural model derived from X-ray crystallography (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) clearly suggests that a good agreement exists between the results of the two techniques under the following conditions: (i) the C-2 symmetry axis of the reaction center runs along the membrane normal; (ii) excitonic coupling is present only in the primary donor special pair; and (iii) the direction of the optical transitions of the monomeric bacteriochlorophylls and of the bacteriopheophytins is not significantly perturbed by the interactions among the pigments. In addition, a carotenoid is detected in the isolated reaction center with an orientation rather perpendicular to the C-2 symmetry axis. Finally, a comparison of these data with similar ones obtained on the bacteriochlorophyll a-containing reaction center of Rhodopseudomonas sphaeroides 241 points towards a geometrical arrangement of the chromophores which is indistinguishable from the one observed in the reaction center of Rps. viridis.

Orientation of pigments and structural proteins in the photosynthetic membrane of spinach chloroplasts: a linear dichroism study.

Abstract 1. The rotation of the plane of polarization, when monochromatic light traverses an anisotropic sample, is proportional to the linear dichroism ( ΔA ) of this sample. A sensitive technique of measurement of this rotation (sensitivity corresponding to 2·10 −5 absorbance unit for ΔA ) has been applied to mechanically or magnetically oriented chloroplasts (800-185 nm). The contribution of artifacts encountered in linear dichroism measurements (textural dichroism, selective polarized scattering, selective polarized reflection) is discussed and is shown to be negligible. 2. A high degree of orientation of the pigments with respect to the normal to the plane of the lamellae has been detected. By comparing linear dichroism and absorption data, this orientation is analyzed in terms of the value of the angle ϕ between the normal and the directions of the transition dipole moments. 3. The directions of y polarized transitions of Ca-680 and longer wavelength forms of chlorophyll a lie close to, or in the lamellar plane ( ϕ >60–65°), Ca-670 is less oriented or oriented with ϕ slightly >55°. 4. The negative dichroism in the Soret band of chlorophyll a implies that the directions of x polarized transitions are tilted out of the membrane plane ( ϕ⋍48° ) 5. Chlorophyll b molecules are oriented in a similar way. 6. Carotenoid molecules lie nearly parallel to the lamellar plane. 7. For structural proteins in intact thylakoid membranes an orientation of tryptophan residues is detected and the 220-185-nm signals are tentatively assigned to an orientation of α-helical regions parallel to the membrane plane.

The neoxanthin binding site of the major light harvesting complex (LHCII) from higher plants

The localisation of the xanthophyll neoxanthin within the structure of the major light harvesting complex (LHCII) of higher plants has been investigated by site‐directed mutagenesis and spectroscopic methods. Mutation analysis performed on pigment binding sites in different helix domains leads to selective loss of neoxanthin for mutations on helix C thus localising this pigment between the helix C and helix A/B domains. Recombinant proteins binding two lutein molecules per polypeptide but lacking neoxanthin have been used in order to determine the contribution of neoxanthin to the absorption and linear dichroism spectra. The data were used to derive the orientation of the neoxanthin transition moment, lying in the polyene chain, which was thus determined to form an angle of 57±1.5° with respect to the normal to the membrane plane where the protein is inserted. On the basis of these results we propose a model for the localisation of the carotenoid site in the LHCII structure which is still unresolved.

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes.

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680+ Phe-) and charge stabilization (formation of P-680+ QA-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced QA was about 30% of that measured with oxidized QA. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.

A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site‐directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA‐His676 and PsaB‐His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls.

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides: A photoselection study

The relative orientation of the pigments of reaction centers from Rhodopseudomonas sphaeroides has been studied by the photoselection technique. A high value (+0.45) of p=(delta AV--delta AH)/(delta AV + delta AH) is obtained when exciting and observing within the 870 nm band which is contradictory to the results of Mar and Gingras (Mar, T. and Gringras, G. (1976) Biochim. Biophys. Acta 440, 609-621) and Shuvalov et al. (Shuvalov, V.A., Asadov, A.A. and Krakhmaleva, I.N. (1977) FEBS Lett. 16, 240-245). It is shown that the low values of p obtained by both groups were erroneous due to excitation conditions. Analysis of the polarization of light-induced changes when exciting with polarized light in single transitions (spheroiden band and bacteriopheophytin Qx bands) enable us to propose a possible arrangement of the pigments within the reaction center. It is concluded that the 870 nm band corresponds to a single transition and is one of the two bands of the primary electron donor (P-870). The second band of the bacteriochlorophyll dimer is centered at 805 nm. The Qx transitions of the molecules constituting the bacteriochlorophyll dimer are nearly parallel (angle less than 25 degrees). The two bacteriopheophytin molecules present slightly different absorption spectra in the near infra-red. Both bacteriopheophytin absorption bands are subject to a small shift under illumination. The angle between the Qy bacteriopheophytin transitions is 55 degrees or 125 degrees. Both Qy transitions are nearly perpendicular to the 870 nm absorption band. Finally, the carotenoid molecules makes an angle greater than 70 degrees with the 870 nm band and the other bacteriochlorophyll molecules.

A protein conformational change associated with the photoreduction of the primary and secondary quinones in the bacterial reaction center

A comparison is made between the PQA → P+Q− A and PQAQB → P+QAQ− B transitions in Rps. viridis and Rb. sphaeroides reaction centers (RCs) by the use of light‐induced Fourier transform infrared (FTIR) difference spectroscopy. In Rb. sphaeroides RCs, we identify a signal at 1650 cm−1 which is present in the P+QA‐minus‐PQA spectrum and not in the P+QAQ− B‐minus‐PQAQB spectrum. In contrast, this signal is present in both P+Q− A‐minus‐PQ− A and P+QAQ− B‐minus‐PQAQB spectra of Rps. viridis RCs. These data are interpreted in terms of a conformational change of the protein backbone near QA (possible at the peptide C≡O of a conserved alanine residue in the QA pocket) and of the different bonding interactions of QB with the protein in the RC of the two species.