Low-temperature Linear Dichroism Research Articles

Orientation and excitonic interactions of the Fenna—Matthews—Olson bacteriochlorophyll a protein in membranes of the green sulfur bacterium Chlorobium tepidum

Linear and circular dichroism spectra of isolated bacteriochlorophyll a proteins (FMO proteins) and membrane vesicles containing FMO protein from the green sulfur bacterium Chlorobium tepidum were measured at room temperature and 77 K. The orientation of membranes and isolated FMO protein was obtained by gel squeezing. Linear dichroism (LD) data indicate that isolated FMO protein and membrane vesicles associated with the FMO protein are oriented in a similar way in a squeezed polyacrylamide gel. Both samples show a characteristic negative LD band around 814 nm with flanking positive bands at 802 and 824 nm ascribed to the Qy excitonic transitions of BChl a of the FMO protein. This confirms that the C3 symmetry axis of the trimer is perpendicular to the membrane plane, which is supported by the model of the disc-like structure of FMO protein trimers of Cb. tepidum [Li Yi-Fen, Zhou W, Blankenship RE, and Allen JP (1997) J Mol Biol 272: 456–471]. The LD data are consistent with either BChl 3 or 6, but not 7 as the principal contributor to the low temperature band at 825 nm. The low temperature linear and circular dichroism spectra of FMO protein trimers from Chlorobium tepidum show significant differences from the low temperature LD and CD spectra of FMO protein trimers from Prosthecochloris aestuarii. The data are interpreted in terms of somewhat different pigment-protein and pigment-pigment interactions in the two complexes.

Probing native-like orientation of pigments in modified reaction centers from Rhodobacter sphaeroides R26 by linear dichroism

Site-specific pigment modifications are useful to investigate structure-function relationships in photosynthesis. In reaction centers bearing modified (bacterio)pheophytins, changed electron transfer kinetics have been related to the changed redox potentials of the pigments introduced (Huber, H. et al. (1995) Chemical Physics, Special Issue, vol 197 (Hochstrasser, R.M. and Hofacker, G.L. eds.) pp. 297–305; [1]). In order to analyze potentially interfering structural changes induced in these reaction centers by the exchange procedure, in particular mispositioning or misorientation of the pigments, low-temperature linear dichroism spectra have been measured for reaction centers from Rhodobacter sphaeroides containing modified bacteriopheophytins and bacteriochlorophylls at the sites H AB and B A,B, respectively. They show that all modified pigments are oriented similar to the native ones, and that they do not affect significantly the linear dichroism of the monomeric bacteriochlorophylls and bacteriopheophytins or of the primary donor.

Orientation of Photosystem-I pigments: low temperature linear dichroism spectroscopy of a highly-enriched P700 particle isolated from spinach

The linear dichroism of Photosystem I particles containing 10 chlorophylls per P700 has been investigated at 10 K. The particles were oriented by uniaxial squeezing of polyacrylamide gels. The oxidation state of P700 was altered either by incubation of the gels with redox mediators or by low temperature illumination. The QY transitions of the primary electron donor P700, of the remaining unoxidized chlorophyll in P700(+) and of a chlorophyll molecule absorbing at 686 nm, which presumably corresponds to the primary electron acceptor A0, are all preferentially oriented perpendicular to the gel squeezing direction. The QY transition of the chlorophyll forms absorbing at 670 and 675 nm appear tilted at 40 ± 5° from this orientation axis. This orientation of the various chlorophylls is compared to that previously reported for more native Photosystem I particles.

Orientation of the pigments in Photosystem II: A low-temperature linear dichroism and polarized fluorescence emission study of chlorophyll-protein complexes isolated from Chlamydomonas reinhardtii

The orientation of the various absorbing and fluorescing dipoles in Photosystem II have been investigated by linearly polarized light spectroscopy at 5 K, performed on macroscopically oriented PS II complexes derived from Chlamydomonas reinhardtii. Linear dichroism and absorption spectra show that the Q Y transitions of the chlorophyll molecules are mostly tilted at less than 35° from the plane of largest cross-section of the particle (which in vivo coincides with the plane of the thylakoid membrane). The chlorophyll forms absorbing at 676 and 683 nm are oriented closer to the membrane than the forms absorbing at 665 and 670 nm which are tilted at approximately 35° from the plane. A dip observed around 680 nm in the LD/absorption spectra indicates a component tilted at a larger angle away from the membrane plane than the 676 nm- and 683 nm-absorbing species. A component weakly absorbing around 693 nm and exhibiting a negative LD (tilt larger than 35°) is clearly resolved. The amplitude of the LD at 693 nm relative to that observed at the maximum (676 nm) varies from sample to sample. In the blue spectral region, two populations of carotenoids are observed; one absorbs around 460 and 490 nm, while the other absorbs around 510 nm. They are oriented out of and near to the thylakoid plane, respectively. Comparison of polarized absorption and fluorescence spectra from the same oriented samples allows the assignment of the 695 nm fluorescence emission to the dipoles responsible for the LD signal at 693 nm.

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

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 Q Y 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., Verméglio, A. and Breton, J. (1979) Biochim. Biophys. Acta 545, 249–264). The main Q X 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 Q X 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 Q Y transitions (unresolved at 834 nm) and their Q X 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 Q Y transitions (resolved at 790 and 805 nm) and small positive LD for their Q X transitions (resolved at 534 and 544 nm), demonstrating that these two molecules are strongly tilted out of the membrane plane with each of the Q Y 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 photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I 110, PS I 65, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I 65 (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F 735 and also as the Q Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl- b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.

Orientation of the pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. III. A quantitative comparison of the low-temperature linear dichroism spectra of thylakoids and isolated pigment-protein complexes

The low-temperature linear dichroism spectrum of thylakoids oriented in polyacrylamide gel can be adequately described by a linear combination of the corresponding spectra of particles of light-harvesting complex, Photosystem I and Photosystem II, isolated by Triton X-100 extraction. The main conclusions which can be derived from this observation are: (1) The in vivo orientation of the pigments within each of the three complexes is not significantly affected by the extraction and purification procedures. (2) The various photosynthetic pigments are oriented roughly to the same extent in each of the three main biochemical constituents of the thylakoid. (3) All the complexes investigated behave like ellipsoids, the largest dimensions of which are lying in the plane of the photosynthetic membrane.

Linear dichroism of microalgae, developing thylakoids and isolated pigment-protein complexes in stretched poly (vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields. Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b (648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl a b ratio of approx. 6 and the LD spectrum was positive with a maximum at 690–694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack Chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.

Electronic spectra of dithieno analogues of phenanthrene

The magnetic circular dichroism (MCD) spectra of some dithieno analogues of phenanthrene: benzo [2,1-b;3,4-b′] dithiophene (I) benzo [1,2-b;4,3-b′] dithiophene (II), benzo [1,2-c;3,4-c′] dithiophene (III) and benzo [1,2-b;3,4-b′] dithiophene (IV) are reported. For I–III the spectra corresponding to two different transition moment directions could be obtained from low-temperature linear dichroism spectra. The results compare well with theoretical energies, oscillator strengths, moment directions and MCD B-terms which were obtained from semi-empirical quantum mechanical calculations in the π-electron approximation.