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 $\text{p =}\text{(ΔA}{}_{\mathrm{<mtext>V</mtext>}}\text{? ΔA}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{(ΔA}{}_{\mathrm{<mtext>V</mtext>}}\text{+ ΔA}{}_{\mathrm{<mtext>H</mtext>}}$) is obtained when exciting and observing within the 870 nm band which is contradictory to the results of Mar and Gingras (Mar, T. and Gingras, 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. 76, 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 Q_x 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 centred at 805 nm. The Q_y transitions of the molecules constituting the bacteriochlorophyll dimer are nearly parallel (angle less than 25°).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 Q_y bacteriopheophytin transitions is 55° or 125°. Both Q_y transitions are nearly perpendicular to the 870 nm absorption band. Finally, the carotenoid molecules makes an angle greater than 70° with the 870 nm band and the other bacteriochlorophyll molecules.     

Absorption and fluorescence spectra and molecular organization of bacteriochlorophyll in reaction centers of Rhodopseudomonas spheroides

Second derivative spectroscopy, computer curve analysis and Stepanov's equation show that the absorbance and fluorescence spectra of primary electron donor in reaction center of Rhodopseudomonas sphaeroides are splitting each into two asymmetric Gaussian components. Their absorption maxima at -196 degrees are 880 and 896 nm and emission maxima-906 and 923 nm, respectively. The absorption spectrum of Bchl-800 splits in the near infrared region into two bands with maxima at 790 and 803 nm. These components are ascribed to an exciton coupling in the two dimers of bacteriochlorophyll in the reaction center. The Qy transition moments of the two bacteriochlorophyll molecules of primary electron donor make an angle of 110 degrees and the angle between two Qy transitions of the pigment in Bchl-800 dimer is 150 degrees. The distance between the centers of chromophores in the dimers is estimated to be 8-11 A.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor.

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550-650 and 700-950nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as deltaav/deltaah, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm , and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the "special pair" that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the "light-dark" difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550–650 and 700–950 nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as Δa_v/Δa_h, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810 nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm, and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the “special pair” that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the “light-dark” difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

Absorption and structure of an LM unit isolated with sodium dodecyl sulfate from reaction centers of Rhodopseudomonas sphaeroides

Low-temperature absorption, circular dichroism and resonance Raman spectra of the LM units isolated with sodium dodecyl sulfate from wild-type Rhodopseudomonas sphaeroides reaction centers (Agalidis, I. and Reiss-Husson, F. (1983) Biochim. Biophys. Acta 724, 340–351) are described in comparison with those of intact reaction centers. In LM unit, the Q_y absorption band of P-870 at 77 K shifted from 890 nm (in reaction center) to 870 nm and was broadened by about 30%. In contrast, the 800 nm bacteriochlorophyll absorption band including the 810 species remained unmodified. It was concluded that the 810 nm transition is not the higher excitonic component of P-870. The Q_x band of P-870 shifted from 602 nm (in reaction center) to 598 nm in LM, whereas the Q_x band of the other bacteriochlorophylls was the same in reaction center and LM and had two components at about 605 and 598 nm. The Q_xII band of bacteriopheophytin was upshifted to 538 nm and a slight blue shift of the Q_y band of bacteriopheophytin was observed. Resonance Raman spectra of spheroidene in LM showed that its native cis-conformation was preserved. Resonance Raman spectroscopy also demonstrated that in LM the molecular interactions assumed by the conjugated carbonyls of bacteriochlorophyll molecules were altered, but not those assumed by the bacteriopheophytins carbonyls. In particular at least one Keto group of bacteriochlorophyll free in reaction center, becomes intermolecularly bounded in LM (possibly with extraneous water). This group may belong to the primary donor molecules.     

Orientation of reaction center and antenna chromophores in the photosynthetic membrane of rhodopseudomonas viridis

Whole cells of Rhodopseudomonas viridis were oriented in a magnetic field. The degree of orientation of the cells was determined by using a photoselection technique. In order to deduce the orientation of the antennae and chromophores of the reaction centers with respect to the membrane plane, we performed linear dichroism measurements of absolute spectra and light induced difference spectra linked to states P⁺I and PI^? on oriented cells. These measurements lead to the following conclusions:The antennae bacteriochlorophyll molecular plane is nearly perpendicular to the membrane. The Q_y and Q_x transitions moments of these molecules make respectively angles of 20 and 70°ith the membrane plane. The antenna carotenoid molecules make an angle of 45°ith the membrane.The primary electron donor possesses two transition moments centered respectively at 970 and 850 nm. The 970 nm transition moment is parallel to the membrane plane, the 850 nm transition is tilted out of the plane. Upon photooxidation of this primary electron donor, a monomer-like absorption band appears at 805 nm. Its transition makes an angle smaller than 25° with the membrane. The photooxidation of the dimer also induces an absorption band shift for the two other bacteriochlorophyll molecules of the reaction center. The absorption band shifts of the two bacteriochlorophyll molecules occur in opposite direction.One bacteriopheophytin molecule is photoreduced in state PI^?. This photoreduction induces an absorption band shift for only one bacteriochlorophyll molecule. Finally, the geometry of the dimeric primary donor seems to be affected by the presence of a negative charge in the reaction center.     

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.     

Stark effect in wild-type and heterodimer-containing reaction centers from Rhodobacter capsulatus

The effect of an external electric field on the optical absorption spectra of wild-type Rhodobacter capsulatus and two Rb. capsulatus reaction centers that have been genetically modified through site-directed mutagenesis (HisM200----LeuM200 and HisM200----PheM200) was measured at 77 K. The two genetically modified reaction centers replace histidine M200, the axial ligand to the M-side bacteriochlorophyll of the special pair, with either leucine or phenylalanine. These substitutions result in the replacement of the M-side bacteriochlorophyll with bacteriopheophytin, forming a bacteriochlorophyll-bacteriopheophytin heterodimer. The magnitude of the change in dipole moment from the ground to excited state (delta mu app) and the angle delta between the Qy transition moment and the direction of delta mu app were measured for the special pair absorption band for all three reaction centers. The values for delta mu app and delta obtained for wild-type Rb. capsulatus (delta mu app = 6.7 +/- 1.0 D, delta = 38 +/- 3 degrees) were the same within experimental error as those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. The values for delta mu app and delta obtained for the red-most Stark band of both heterodimers were the same, but delta mu was substantially different from that of wild-type reaction centers (HisM200----LeuM200, delta mu app greater than or equal to 14.1 D and delta = 33 +/- 3 degrees; HisM200----PheM200, delta mu app greater than or equal to 15.7 D and delta = 31 +/- 4 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriochlorophyll a-types in chromatophore and subchromatophore preparations from Rhodopseudomonas sphaeroides.

Comparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850 nm absorption band had a positive correlation with the 850 nm and 870 nm CD bands. The 800 nm and 870 nm absorption bands seemed not to correlate with any CD bands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870 nm absorption band, but not to that of the 800 nm and 850 nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870 nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850 nm absorption band originates from exciton-coupling of bacteriochlorophyll a.     

Polarized fluorescence and absorption of macroscopically aligned Light Harvesting Complex II.

Polarized absorption and fluorescence measurements have been performed at 77 K on isotropic and anisotropic preparations of trimeric Light Harvesting Complex II (LHC-II) from spinach. The results enable a decomposition of the absorption spectrum into components parallel and perpendicular to the trimeric plane. For the first time, it is shown quantitatively that the strong absorption band around 676 nm is polarized essentially parallel to the plane of the trimer, i.e., the average angle between the corresponding transition dipole moments and this plane is at most 12 degrees. The different absorption bands for LHC-II should not be considered as corresponding to individual pigments but to collective excitations of different pigments. Nevertheless, the average angle between the Qy transition dipole moments of all chlorophyll a pigments in LHC-II and the trimeric plane could be determined and was found to be 17.5 degrees +/- 2.5 degrees. For the chlorophyll b pigments, this angle is significantly larger (close to 35 degrees). At 77 K, most of the fluorescence stems from a weak band above 676 nm and the corresponding transition dipole moments are oriented further out of plane than the dipole moments corresponding to the 676-nm band. The results are shown to be of crucial significance for understanding the relation between the LHC-II structure and its spectroscopy.     

Nuclear wavepacket motion producing a reversible charge separation in bacterial reaction centers

The excitation of bacterial reaction centers (RCs) at 870 nm by 30 fs pulses induces the nuclear wavepacket motions on the potential energy surface of the primary electron donor excited state P*, which lead to the fs oscillations in stimulated emission from P* [M.H. Vos, M.R. Jones, C.N. Hunter, J. Breton, J.-C. Lambry and J.-L. Martin (1994) Biochemistry 33, 6750-6757] and in Qy absorption band of the primary electron acceptor, bacteriochlorophyll monomer B(A) [A.M. Streltsov, S.I.E. Vulto, A.Y. Shkuropatov, A.J. Hoff, T.J. Aartsma and V.A. Shuvalov (1998) J. Phys. Chem. B 102, 7293-7298] with a set of fundamental frequencies in the range of 10-300 cm(-1). We have found that in pheophytin-modified RCs, the fs oscillations with frequency around 130 cm(-1) observed in the P*-stimulated emission as well as in the B(A) absorption band at 800 nm are accompanied by remarkable and reversible formation of the 1020 nm absorption band which is characteristic of the radical anion band of bacteriochlorophyll monomer B(A)-. These results are discussed in terms of a reversible electron transfer between P* and B(A) induced by a motion of the wavepacket near the intersection of potential energy surfaces of P* and P+B(A)-, when a maximal value of the Franck-Condon factor is created.     

A study on the heterogeneity of the light-harvesting complex II from Ectothiorhodospira sp. after acid/chaotropic treatment

The light-harvesting complex II of the purple bacteria has two strong near infrared electronic absorption bands, around 800 (B800) and 850 (B850) nm, arising from the Qy transitions of bacteriochlorophyll a. It was previously reported that under some specific acid/chaotropic conditions the B850 bacteriochlorophylls of the light-harvesting complex II of Ectothiorhodospira sp. are strongly reorganised. Part of these pigments absorbs at 843 nm while another set absorbs around 858 nm. The current work should investigate whether a mix of two different complexes could generate the 843- and 858-nm bands. Acid/chaotropic conditions inducing the reorganisation of B850 were reproduced on a sample bound to an ionic-exchange column. The chromatographic pattern was found strongly homogeneous. The findings indicate that the heterogeneity of the reorganised B850 results from two forms of differently structured bacteriochlorophylls bound to the same polypeptide backbone.     

The orientations of transition moments in reaction centers of Rhodopseudomonas sphaeroides, computed from data of linear dichroism and photoselection measurements.

Linear dichroism measurements of reaction centers of Rhodopseudomonas sphaeroides in stretched gelatin films have yielded angles that various optical transition moments make with an axis of symmetry in the reaction center. Photoselection experiments have yielded angles that certain transition moments make with each other. We have combined these data so as to compute the orientations of the Qx and Qy transition moments of the two molecules of bacteriopheophytin and of the bacteriochlorophyll special pair (photochemical electron donor) in the reaction center. Orientations are expressed in spherical polar coordinates with the symmetry axis as the pole. We have also computed additional angles between pairs of transition moments. In this treatment we have assumed that the bacteriopheophytins are independent monomers with little or no exciton coupling.     

Properties of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films.: Linear dichroism and low temperature spectra

Methods of preparing dried gelatin films containing purified reaction centers of Rhodopseudomonas sphaeroides are described. The spectral properties of reaction centers in solution are essentially maintained in dried gelatin films. These films are uniform and have excellent optical properties, showing little particulate scattering at temperatures down to about 4K. Film contraction on cooling to 90K is less than 1% in linear dimension. Linear dichroism spectra are reported for films at room and low temperature. Reaction centers show a moderate amount of linear dichroism in unstretched gelatin films; the magnitude of the linear dichroism becomes much greater when the films are stretched. In stretched films, linear dichroic ratios ($\text{A}{}_{\mathrm{\parallel }}\text{A}{}_{\mathrm{\perp }}$; absorbance measured with electric vector parallel and perpendicular to stretching direction) between 1.7 and 2.2 were obtained for the 860 nm absorption band of the bacteriochlorophyll component that undergoes primary photooxidation. The relative polarizations of light-induced absorption changes of reaction centers in stretched films are similar to those reported by Vermeglio and Clayton ((1976) Biochim. Biophys. Acta 449, 500–515) and support their hypothesis that absorbance decreases, maximal near 860 and 810 nm, and an increase near 790 nm are associated with the respective disappearance and appearance of discrete bands characteristic of the reduced and oxidized bacteriochlorophyll dimer. This interpretation is also supported by the polarization of the absolute absorption spectrum near 810 and 860 nm. An absorption band near 540 nm, ascribed to the Q_x transitions of two molecules of bacteriopheophytin in the reaction center, is split at low temperatures into two bands having similar polarizations. This splitting is probably not due to exciton coupling of the two molecules, since excition theory predicts different polarizations.     

Energy selection is not correlated in the Qx and Qy bands of a Mg-porphyrin embedded in a protein

The Qx-Qy splitting observed in the fluorescence excitation spectra of Mg-mesoporphyrin-IX substituted horseradish peroxidase (MgMP-HRP) and of its complex with naphthohydroxamic acid (NHA) was studied by spectral hole burning techniques. The width of a hole directly burnt in the Qy band and that of a satellite hole indirectly produced in Qy as a result of hole burning in Qx was compared. We also studied the dependence of the satellite hole in the Qy band on the burning frequency used in the Qx band. Both the directly and indirectly burnt holes were very broad in the (higher energy) Qy band. The width of the satellite hole in the Qy band was equal to the entire width of the inhomogeneously broadened band, independently from the position of hole burning in Qx. This is indicative of a clear lack of correlation between the electronic transition energies of the Qx and Qy bands. A photoproduct was produced by laser irradiation of the MgMP-HRP/NHA complex and was identified as a species with lowered Q-splitting. Conversion of the photoproduct could be achieved by thermal activation measured in temperature-cycling experiments, with a characteristic temperature of 25 K. We attribute the phototransformation to a conformational change of MgMP.     

The type, amount, location, and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides.

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7',8',-tetrahydro-psi,psi-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1, 2, 7', 8'-tetrahydro-psi,psi-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm (Qx) transition of the bacteriochlorophyll complex.     

Spectroscopy and structure of bacteriochlorophyll dimers. I. Structural consequences of nonconservative circular dichroism spectra.

The origin of the nonconservative nature of the circular dichroism (CD) spectrum of bacteriochlorophyll dimers is investigated. It is shown that coupling between the Qy and Qx transitions can, under rather restricting circumstances, lead to an asymmetrical CD spectrum: only for a limited set of relative orientations of the monomers within the dimer is the spectrum found to be asymmetrical. The relation between intensity and asymmetry of the CD spectrum is elucidated. The results are applied to the B820 subunit of the LH1 antenna system and subsequently to the antenna system LH1 itself. Differences in the geometry of the BChls in LH1 versus the LH2 structure are discussed.     

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.     

Exciton interactions in dimers of bacteriochlorophyll and related molecules

Formalisms are developed for calculating the absorption wavelengths, dipole strengths and rotational strengths for dimers of bacteriochlorophyll and related molecules. The expressions explicitly consider the mixing of bacteriochlorophyll's four main excited states (Q_y, Q_x, B_x and B_y) in the ground and excited states of the dimer. This mixing must be considered in order to account for the hyperchromism and nonconservative circular dichroism found experimentally in oligomers of bacteriochlorophyll and bacteriopheophytin. The spectroscopic properties of the eight absorption bands of a bacteriopheophytin dimer are calculated as functions of the geometry of the dimer. The importance of the mixing of nondegenerate excited states, and of the mixing of doubly-excited states into the dimer's ground state, is evaluated by comparisons with calculations in which these phenomena are neglected. Structures for bacteriopheophytin dimers are found for which most of the calculated spectroscopic properties are consistent with the properties seen experimentally. Possible explanations are considered for the remaining discrepancies between the calculated and observed properties.     

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.

Two independent sets of measurements using a variety of methods show that a sample with A_{800 nm} = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.

Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.

These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: _{802 nm} = 288 (± 14) mM⁻¹ · cm⁻¹ and _{865 nm} = 128 (± 6) mM⁻¹ · cm⁻¹. For reversible bleaching of the 865-nm band, Δ^{red - ox}_865nm = 112 (± 6) mM⁻¹ · cm⁻¹ (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.