Magnetophotoselection of the triplet state of reaction centers from Rhodopseudomonas sphaeroides R-26.

Reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, give rise to large triplet state EPR signals upon illumination at low temperature (11 K). Utilizing monochromatic polarized light to generate the EPR spectra (magnetophotoselection) we have shown that the intensities of the observed triplet signals are strongly dependent upon the wavelength and polarization direction of the excitation. These data can be used to calculate the orientations of the excited transition moments with respect to each other and with respect to the triplet state principal magnetic axes system. Our quantitative approach is to follow the procedure outlined in a previous publication (Frank, H.A., Friesner, R., Nairn, J.A., Dismukes, G.C. and Sauer, K. (1979) Biochim. Biophys. Acta 547, 484-501) where computer simulations of the observed triplet state spectra were employed. The results presented in the present work indicate that the transition moment at 870 nm which is associated with the bacteriochlorophyll 'special pair' lies almost entirely along one of the principal magnetic axes of the triplet state. Aso, the 870 nm transition moment makes an angle of approx. 60 degrees with the 546 nm transition moment which is associated with a bacteriopheophytin. This latter result is in agreement with previous photoselection studies on the same bacterial species (Vermeglio, A., Breton, J., Paillotin, G. and Cogdell, R. (1978) Biochim. Biophys. Acta 501, 514-530).     

EPR-spectroscopy of reduced oxyferrous-P450cam.

X-irradiation of the ternary complex of P450:substrate:O2 at 77 K produces a reduced intermediate by electron addition to the Fe:O2 complex which can be studied by EPR-spectroscopy. The EPR spectrum of the new species exhibits rhombic symmetry with g-factors of 2.27, 2.17 and 1.95, respectively. Increasing the temperature of the sample to 190 K results in loss of intensity of the intermediate signals. X-irradiation of oxymyo- and oxyhemoglobin produces similar EPR signals indicating that the added electron is resident on the Fe:O2 compleX (Kappl, R., et al. (1985) Biochim. Biophys. Acta 870, 20-30).     

The orientations of reaction center transition moments in the chromatophore membrane of Rhodopseudomonas sphareroides, bases on new linear dichroism and photoselection measurements.

Chromatophore membranes from Rhodopseudomonas sphaeroides were oriented by drying suspensions on the surfaces of glass slides, Polarized spectra of light-induced absorption changes were obtained between 500 and 1000 nm. As observed earlier, these spectra showed negative bands, reflecting photooxidation of the bacteriochlorophyll 'special pair' in the reaction centers, centered near 870, 810, 630 and 600 nm. These bands have been designated BY1, BY2, BX1 and BX2, respectively, corresponding to two QY transitions and two QX transitions of the dimeric special pair. We found the BY1 and BX1 transition moments to be parallel (within 20 degrees) to the plane of the membrane, whereas the BX2 moment makes an angle of 55--63 degrees with the plane. Using the photoselection technique we found that the angle between the BY1 and BX1 transition moments is 30 degrees, while that between BY1 and BX2 is 75 degrees. The BX1 and BX2 moments were found to be orthogonal, consistent with the prediction of molecular exciton theory for a dimer. By combining these data, we have calculated the orientations of the transition moments of the bacteriochlorophyll dimer in spherical polar coordinates, with the pole of the coordinate system normal to the plane of the membrane. The orientations of the QY and QX transition moments of the two bacteriopheophytin molecules in the reaction center were also computed in this coordinate system by transforming the data reported by Clayton, C.N., Rafferty, R.K. and Vermeglio, A. ((1979) Biochim. Biophys. Acta 545, 58--68). We have derived the transformation equations for two polar coordinate systems: in one, the pole is an axis of symmetry as defined by the orientations of purified reaction centers in stretched gelatin films (Rafferty, C.N. and Clayton, R.K. (1979) Biochim. Biophys. Acta 545, 106--121). In the other, the pole is normal to the plane of the chromatophore membrane. These two polar axes are approximately orthogonal.     

Absorption studies of primary reactions in Photosystem I. Yield and rate of formation of the P-700 triplet state

The quantum yield of P-700 triplet state formation has been found from flash-induced absorption studies in the microsecond time range to be 0.45 and 0.35 at 294 K and 6–20 K, respectively, in CP1-SDS particles which lack the secondary acceptors. From these quantum yield measurements, yields of formation of the P-700 triplet state from the primary biradical (P-700⁺–A⁻₀) were calculated to be around 0.6 at both temperatures, whereas double-laser flash experiments allowed us to derive upper limits for this yield (0.84 at 294 K and 0.79 at 20 K). These values agree with the high values that have been previously calculated from an earlier absorption study (Sétif, P., Hervo, G. and Mathis, P. (1981) Biochim. Biophys. Acta 638, 257–267) but appear significantly higher than the yield calculated from EPR experiments (5–10%) (Gast, P., Swarthoff, T., Ebskamp, F.C.R. and Hoff, A.J. (1983) Biochim. Biophys. Acta 722, 163–175). Possible explanations for this discrepancy are discussed. From absorption studies in the submicrosecond time range as well as from double-laser flash experiments, the lifetime of the biradical (P-700⁺–A⁻₀), from which the P-700 triplet state is formed by recombination, has been measured to increase from 30–50 ns at room temperature up to 120–130 ns between 10 and 110K.     

The triplet state of the primary donor of the photosynthetic bacterium Rhodopseudomonas viridis

The absorbance-detected magnetic-resonance technique has been applied to the study of the triplet state of the primary donor in chromatophores of the photosynthetic bacterium Rps. viridis. The results confirm the triplet-minus-singlet absorbance-difference spectrum and its interpretation as previously obtained for isolated reaction centers (Den Blanken, H.J. and Hoff, A.J. (1982) Biochim. Biophys. Acta 681, 365–374). Our present results affirm that the primary donor is a bacteriochlorophyll b dimer, and that there is no blue exciton band at 850 nm. We show that the reaction centers are not identical, but have a small heterogeneity in their properties. In chromatophores and sometimes in isolated reaction centers a shoulder is observed in the long-wavelength absorbance-difference band of the primary donor. This shoulder is possibly caused by charge transfer interaction of the donor with an adjacent chromophore (Vermeglio, A. and Paillotin. G. (1982) Biochim. Biophys. Acta 681, 32–40; Maslov, V.G., Klevanik, A.V., Ismailov, M.A. and Shuvalov, V.A. (1983) Doklady Akad. Nauk. SSSR 269, 1217–1221) or it reflects a slight heterogeneity in the reaction-center geometry, which cannot be removed with the selection offered by the magnetic resonance technique. The zero-field triplet-ESR spectrum and the sublevel decay rates of the triplet state of the primary donor are presented, as detected in whole cells at the antenna fluorescence, and in chromatophores and isolated reaction centers at the absorbance-difference band at 838 nm. We do not observe the expected reversal of the sign of the ESR transitions monitored with the two techniques. A tentative explanation is given in terms of energy transfer from unrelaxed excited states of the antenna pigments to the reaction center.     

Picosecond photodichroism studies on reaction centers from the green photosynthetic bacterium Chloroflexus aurantiacus

Picosecond photodichroism (photoselection) measurements have been carried out on reaction centers from the facultative green photosynthetic bacterium Chloroflexus aurantiacus using weak 30 ps flashes in the long-wavelength band of the primary electron donor, P. Absorption changes due to the chemical and photochemical oxidation of P and the reduction of quinone also have been examined. Our results on Chloroflexus suggest that the Q_y transition-dipoles of the bacteriopheophytin molecules participating in, or affected by, the primary reactions are oriented essentially perpendicular to the 865 nm transition dipole of P. This is in agreement with previous work on reaction centers from purple bacteria, such as Rhodopseudomonas sphaeroides. The data also suggest that the 812 nm ground-state transition is oriented at an angle of 45–65° with respect to the 865 nm transition. The new band that appears near 800 nm upon oxidation of P is polarized mainly parallel to the 865 nm band. These relative polarizations of the absorption bands are in very good agreement with the results of recent linear dichroism studies (Vasmel, H., Meiburg, R.F., Kramer, H.J.M., De Vos, L.J. and Amesz, J. (1983) Biochim. Biophys. Acta 724, 333–339). Possible origins for the absorption changes and the photodichroism spectra are discussed. The data are consistent with either a monomeric or dimeric structure of P-865.     

The orientations of reaction center transition moments in the chromatophore membrane of Rhodopseudomonas sphaeroides, based on new linear dichroism and photoselection measurements

Chromatophore membranes from Rhodopseudomonas sphaeroides were oriented by drying suspensions on the surfaces of glass slides. Polarized spectra of light-induced absorption changes were obtained between 500 and 1000 nm. As observed earlier, these spectra showed negative bands, reflecting photooxidation of the bacteriochlorophyll ‘special pair’ in the reaction centers, centered near 870, 810, 630 and 600 nm. These bands have been designated BY₁, BY₂, BX₁ and BX₂, respectively, corresponding to two Q_y transitions and two Q_x transitions of the dimeric special pair. We found the BY₁ and BX₁ transition moments to be parallel (within 20°) to the plane of the membrane, whereas the BX₂ moment makes an angle of 55–63° with the plane.Using the photoselection technique we found that the angle between the BY₁ and BX₁ transition moments is 30°, while that between BY₁ and BX₂ is 75°. The BX₁ and BX₂ moments were found to be orthogonal, consistent with the prediction of molecular exciton theory for a dimer.By combining these data, we have calculated the orientations of the transition moments of the bacteriochlorophyll dimer in spherical polar coordinates, with the pole of the coordinate system normal to the plane of the membrane. The orientations of the Q_y and Q_x transition moments of the two bacteriopheophytin molecules in the reaction center were also computed in this coordinate system by transforming the data reported by Clayton, C.N., Rafferty, R.K. and Vermeglio, A. ((1979) Biochim. Biophys. Acta 545, 58–68). We have derived the transformation equations for two polar coordinate systems: in one, the pole is an axis of symmetry as defined by the orientations of purified reaction centers in stretched gelatin films (Rafferty, C.N. and Clayton, R.K. (1979) Biochim. Biophys. Acta 545, 106–121). In the other, the pole is normal to the plane of the chromatophore membrane. These two polar axes are approximately orthogonal.     

Study of the long-wavelength fluorescence band at 920 nm of isolated reaction centers of the photosynthetic bacterium Rhodopseudomonas spaeroides R-26 with fluorescence-detected magnetic resonance in zero field

The triplet state of isolated reaction centers of Rhodopseudomonas sphaeroides R-26 has been studied by fluorescence-detected electron spin resonance in zero magnetic field (FDMR) at 4.2 K. The sign of the FDMR resonance monitored at the long-wavelength fluorescence band is positive (fluorescence increase); this confirms the earlier interpretation (Hoff, A.J. and Gorter de Vries, H. (1978) Biochim. Biophys. Acta 503, 94–106) that the negative sign of the FDMR resonance of the reaction center triplet state in whole bacterial cells is caused by resonant transfer of the singlet excitations from the antenna pigments to the trap. By monitoring the FDMR response as a function of the wavelength of fluorescence, we have recorded microwave-induced fluorescence spectra. In addition to the positive microwave-induced fluorescence band peaking at 935 nm, at 905 nm a negative band was found. The resonant microwave frequencies for these two bands, i.e., the values of the zero-field splitting parameters |D| and |E| of the triplet state being monitored, were different, those of the 905 nm microwave-induced fluorescence band being identical to the resonant microwave frequencies measured with absorption-detected zero-field resonance (Den Blanken, H.J., Van der Zwet, G.P. and Hoff, A.J. (1982) Chem. Phys. Lett. 85, 335–338), a technique that monitors the bulk properties of the sample. From this result and its negative sign, we tentatively attribute the 905 nm microwave-induced fluorescence band to a small (possibly less than 1%) fraction of antenna bacteriochlorophylls that are in close contact with the trap. The positive 935 nm microwave-induced fluorescence band with resonant microwave frequencies deviating from the bulk material is ascribed to a minority of primary donor bacteriochlorophyll dimers, which have a higher than normal fluorescence yield because of a somewhat slower charge-separation reaction. Is it likely that practically all long-wavelength fluorescence of isolated reaction centers stems from such impaired reaction centers.     

Model studies to low-temperature optical transitions of photosynthetic reaction centers. II. Rhodobacter sphaeroides and Chloroflexus aurantiacus

The analysis of optical spectra for Rhodopseudomonas viridis from part I (Knapp, E.W., Scherer, P.O.J. and Fischer, S.F. (1986) Biochim. Biophys. Acta 852, 295–305) is extended to two other structurally similar reaction centers with different prosthetic groups (Rhodobacter sphaeroides and Chloroflexus aurantiacus). Assuming that the structure of the different reaction centers is similar, the interactions between the six prosthetic groups are calculated using the structure data from Rps. viridis. The absorbance, linear dichroism (LD), circular dichroism (CD), triplet-minus-singlet absorption-detected magnetic resonance (T — S ADMR) and its linear dichroism are simulated by an exciton model. The results point to partial delocalization of the special pair triplet state and its excitations.     

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.     

Spatial relationship between cytochrome a and a3

We have studied the spatial relationship between cytochromes a and a3 by the enhancement of the spin relaxation of cytochrome a3-NO EPR signals by the paramagnetic a heme at 15 K. An Fe-Fe distance of 12-19A is estimated from the absence of dipolar broadening and from the observation of spin relaxation enhancement in the a3-NO complex. When this result is combined with resonance x-ray diffraction data reported by Blasie et al. (Blasie, J. K., Pachence, J. M., Tavormina, A., Dutton, P. L., Stamatoff, J., Eisenberger, P., and Brown, G. (1982) Biochim. Biophys. Acta 679, 188-197) and the contribution from the exchange interaction is considered, we can limit the iron-iron distance to 12-16 A and estimate the angle between the Fe-Fe vector and mitochondrial membrane normal as 30-60 degrees. We also consider the possible effects of CuA on cytochrome a3-NO.     

Structures of photolyzed carboxymyoglobin

The structures of photoactivated carboxymyoglobin (Mb*CO) at temperatures to 10 K have been investigated by Fourier transform infrared (FT-IR) spectroscopy, visible spectroscopy, and near-infrared spectroscopy. Two energy states for *CO are observed by FT-IR, which are altered in frequency by 94% and 88% of the difference from the ground-state heme CO toward free CO gas [Alben, J. O., Beece, D., Bowne, S. F., Doster, W., Eisenstein, L. Frauenfelder, H., Good, D., McDonald, J. D., Marden, M. C., Moh, P. P., Reinisch, L., Reynolds, A. H., Shyamsundar, E., & Yue, K. T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3744-3748]. Ground-state MbCO shows no absorption in the near-infrared from 700 to 1200 nm. Conversely, Mb*CO shows an absorption near 766 nm, similar to that of ferrous myoglobin (deoxy-Mb) at 758 nm. These data are compared with M?ssbauer isomer shifts and quadrupole splitting [Spartalian, K., Lang, G., & Yonetani, T. (1976) Biochim. Biophys. Acta 428, 281-290] and magnetic susceptibility measurements [Roder, H., Berendzen, J., Bowne, S. F., Frauenfelder, H., Sauke, T. B., Shyamsunder, E., & Weissman, M. B. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2359-2363], which clearly indicate that the iron in both Mb*CO and deoxy-Mb is in the high-spin Fe(II) state, as does the heme transition in the Soret [Iizuka, T., Yamamoto, H., Kotani, M., & Yonetani, T. (1974) Biochim. Biophys. Acta 371, 126-139]. Thus the electronic structure of iron in Mb*CO is nearly identical with that of deoxy-Mb, and *CO is only slightly perturbed from the free gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation and linear dichroism of Mastigocladus laminosus phycocyanin trimer and Nostoc sp. phycocyanin dodecamer in stretched poly(vinyl alcohol) films

The linear dichroism (LD) spectra of the C-phycocyanin (C-PC) trimer disks oriented in poly(vinyl alcohol) films (PVA) at room temperature and at 95 K were determined. Utilizing the known atomic coordinates of the chromophores (Schirmer, T., Bode, W. and Huber, R. (1987) J. Mol. Biol. 196, 677-695) and theoretical estimates of the orientations of the transition dipole moments relative to the molecular framework, the LD spectra were simulated using the pairwise exciton interaction model of Sauer and Scheer (Biochim. Biophys. Acta 936 (1988) 157-170); in this model, the alpha 84 and beta 84 transition moments are coupled by an exciton mechanism, while the beta 155 chromophore remains uncoupled. Linear dichroism spectra calculated using this exciton model, as well as an uncoupled chromophore (molecular) model, were compared with experimental LD spectra. Satisfactory qualitative agreement can be obtained in both the exciton and molecular models using somewhat different relative values of the theoretically estimated magnitudes of the beta 155 oscillator strength. Because the relative contributions of each of the chromophores (and thus exciton components) to the overall absorption of the C-PC trimer are not known exactly, it is difficult to differentiate successfully between the molecular and exciton models at this time. The linear dichroism spectra of PC dodecamers derived from phycobilisomes of Nostoc sp. oriented in stretched PVA films closely resemble those of the C-PC trimers from Mastigocladus laminosus, suggesting that the phycocyanin chromophores are oriented in a similar manner in both cases, and that neither linker polypeptides nor the state of aggregation have a significant influence on these orientations and linear dichroism spectra. The LD spectra of oriented phycocyanins in stretched PVA films at low temperatures (95 K) appear to be of similar quality and magnitude as the LD spectra of single C-PC crystals (Schirmer, T. and Vincent, M.G. (1987) Biochim. Biophys. Acta 893, 379-385).     

EPR spectroscopy of soybean lipoxygenase-1. Description and quantification of the high-spin fe(III) signals

The interaction of soybean lipoxygenase-1 with 13-Ls-hydroperoxy-9-cis,11-trans-octadecadienoic acid, the product of the enzymic dioxygenation of linoleic acid, results in the formation of either a yellow or a purple coloured enzyme form depending on the amount of product used. The composition of the high-spin Fe(III) signals in the EPR spectra of both enzyme forms has been studied and the amount of EPR-visible iron determined by integration and simulation. Sets of g values of the species building up the high-spin Fe(III) signal around g 6 are derived from both third-order perturbation calculation and exact numerical diagonalization of the spin Hamiltonian describing the system. The results of these calculations are generally applicable to systems having S = 5/2. The iron in the native, colourless enzyme is almost EPR-nondetectable. The yellow form of the enzyme shows a complex EPR signal around g 6 which consists of contributions of at least three species with different ligand symmetry. The signal corresponds to approx. 75% of the total iron content. The g 6 signal of the purple Fe(III) enzyme corresponds roughly to the same amount of iron but the ratio between the different species is not the same as in the yellow enzyme. This enzyme form also shows an additional g 4.3 signal with a large amplitude but a relatively low integrated intensity (approx. 10% of the total iron content). The results are consistent with the suggested mechanism of the catalytic function of iron in lipoxygenase which was based on qualitative EPR results (De Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J., Wever, R. and van Gelder, B.F. (1975) Biochim. Biophys. Acta 377, 71--79).     

Magnetic field-induced increase of the yield of (bacterio)chlorophyll emission of some photosynthetic bacteria and of Chlorella vulgaris.

In photosynthetic bacteria, in which the iron-ubiquinone complex X is prereduced, a magnetic field induces an increase of the emmission yield, which is correlated with the decrease in reaction center triplet yield reported previously (Hoff, A.J., Rademaker, H., van Grondelle, R. and Duysens, L.N.M. (1977) Biochim. Biophys. Acta 460, 547--554). Our results support the hypothesis that under these conditions charge recombination of the oxidized primary donor and the reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll. In Chlorella vulgaris and spinach chloroplasts, at 120 K, the magnetic field has an effect similar to that found in bacteria, which suggests that an intermediary electron acceptor between P-680 and Q is present in Photosystem II also.     

Pulse ENDOR and density functional theory on the peridinin triplet state involved in the photo-protective mechanism in the peridinin-chlorophyll a-protein from Amphidinium carterae

The photoexcited triplet state of the carotenoid peridinin in the Peridinin-chlorophyll a-protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the alpha- and beta-protons of the peridinin conjugated chain. Assignments of beta-protons belonging to two different methyl groups, with aiso=7.0 MHz and aiso=10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole pi-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186-195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll-peridinin pair, is functioning as a bridge in the triplet-triplet energy transfer between the two pigments.     

X-ray diffraction demonstrates that phosphatidyldiacylglycerol and phosphatidylcholesterol are not lamellar above the main transition temperature

X-ray diffraction was used to investigate the lattice structure of aqueous dispersions of two phosphatidyldiacylglycerols and of a phosphatidylcholesterol above and below the chain melting transition temperature. Previously, Noggle et al. (Biochim. Biophys. Acta (1982) 691, 240-248) had investigated these lipids and had concluded on the basis of electron microscopy that the lipids were in a lamellar state above the transition temperature. However, they found the 31P-NMR signals were not characteristic of lamellar phases. It was, therefore, concluded that these lipids were yielding unexpected 31P-NMR spectra. The present X-ray results demonstrate that, in fact, the lipids are not in a lamellar state above the transition temperature and that the 31P-NMR and X-ray data are not necessarily in disagreement. Characteristics of the phases both above and below the chain melt temperature are discussed.     

The orientation of the primary donor in bacterial photosynthesis.

The triplet state EPR spectra of magnetically aligned whole cells of Rhodopseudomonas viridis and Rhodopseudomonas palustris display a marked dependence on the orientation of the static EPR field with respect to the alignment field direction. This observation implies that the primary donor species on which the triplets are localized are ordered within the membranes. We have developed a theoretical model for the system to enable calculation of the orientation of the magnetic axes of the primary donor species with respect to the membranes in which they reside. The triplet state spectra are generated by an ensemble of partially ordered magnetic systems and a computer simulation of the experimental results. The triplet orientation is very similar for the two organisms studied, where one axis lies predominantly in the plane of the membrane and the other two axes have approximately equal projections onto the normal to the membrane.     

Orientation of plgments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. IV. The 100 K linear dichroism spectra of thylakoids from wild-type and chlorophyll b-less barley thylakoids

Using a polyacrylamide gel squeezing technique, linear dichroism spectra of thylakoids from wild-type and chlorophyll-b less barley have been obtained at 100 K. The calculated difference linear dichroism spectra, based on normalization at 690–695 nm, are identical to those of the light-harvesting complex (LHC) isolated by Triton solubilization. This observation is in agreement with previous conclusions (Tapie, P., Haworth, P., Hervo, G. and Breton, J. (1982) Biochim. Biophys. Acta 682, 339–344) regarding: (i) scattering artifacts are absent in linear dichroism spectra determined using polyacrylamide gels, (ii) the in vivo orientation of LHC pigments is maintained in the isolated complex and (iii) the largest dimension(s) of the isolated LHC is (are), in vivo, parallel to the plane of the photosynthetic membrane.     

High-resolution optical absorption-difference spectra of the triplet state of the primary donor in isolated reaction centers of the photosynthetic bacteria Rhodopseudomonas sphaeroides R-26 and Rhodopseudomonas viridis measured with optically detected magnetic resonance at 1.2 K

We have recorded triplet optical absorption-difference spectra of the reaction center triplet state of isolated reaction centers from Rhodopseudomonas sphaeroides R-26 and Rps. viridis with optical absorption-detected electron spin resonance in zero magnetic field (ADMR) at 1.2 K. This technique is one to two orders of magnitude more sensitive than conventional flash absorption spectroscopy, and consequently allows a much higher spectral resolution. Besides the relatively broad bleachings and appearances found previously (see, e.g., Shuvalov V.A. and Parson W.W. (1981) Biochim. Biophys. Acta 638, 50–59) we have found strong, sharp oscillations in the wavelength regions 790–830 nm (Rps. sphaeroides) and 810–890 nm (Rps. viridis). For Rps. viridis these features are resolved into two band shifts (a blue shift at about 830 nm and a red shift at about 855 nm) and a strong, narrow absorption band at 838 nm. For Rps. sphaeroides R-26 the features are resolved into a red shift at about 810 nm and a strong absorption band at 807 nm. We conclude that the appearance of the absorption bands at 807 and 838 nm, respectively, is due to monomeric bacteriochlorophyll. Apparently, the exciton interaction between the pigments constituting the primary donor is much weaker in the triplet state than in the singlet state, and at low temperature the triplet is localized on one of the bacteriochlorophylls on an optical time scale. The fact that for Rps. sphaeroides the strong band shift and the monomeric band found at 1.2 K are absent at 293 K and very weak at 77 K indicates that these features are strongly temperature dependent. It seems, therefore, premature to ascribe the temperature dependence between 293 and 77 K of the intensity of the triplet absorption-difference spectrum at 810 nm (solely) to a delocalization of the triplet state on one of the accessory bacteriochlorophyll pigments.