Antenna organization in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum

Structural aspects of the core antenna in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum were studied by means of fluorescence emission and singlet-singlet annihilation measurements. In both species the number of bacteriochlorophylls of the core antenna between which energy transfer can occur corresponds to one core-reaction center complex only. From measurements of variable fluorescence we conclude that in C. tepidum excitation energy can be transferred back from the core antenna (B920) to the peripheral B800–850 complex in spite of the relatively large energy gap, and on basis of annihilation measurements a model of separate core-reaction center units accompanied by their own peripheral antenna is suggested. C. vinosum contains besides a core antenna, B890, two peripheral antennae, B800–820 and B800–850. Energy transfer was found to occur from the core to B800–850, but not to B800–820, and it was concluded that in C. vinosum each core-reaction center complex has its own complement of B800–850. The results reported here are compared to those obtained earlier with various strains and species of purple non-sulfur bacteria.Abbreviations BChl- bacteriochlorophyll - B800–820 and B800–850- antenna complexes with Q_y-band absorption maxima near 800 nm and 820 or 850 nm, respectively - B890 and B920- antenna complexes with Q_y-band absorption maxima near 890 and 920 nm, respectively - LH1- light harvesting 1 or core antenna - LH2- light harvesting 2 or peripheral antenna     

Purification and Characterization of the B808–866 Light-harvesting Complex from Green Filamentous Bacterium Chloroflexus aurantiacus

The integral membrane light-harvesting complex B808–866 from the thermophilic green filamentous bacterium Chloroflexus aurantiacus has been isolated and characterized. Reversed-phase HPLC analysis demonstrated that the number of bacteriochlorophyll (BChl) in the B808–866 antenna complex is 36 ± 2 per reaction center. The main carotenoid type is γ-carotene, and the molar ratio of BChl to carotenoid is 3:2. The steady-state absorption and fluorescence spectroscopy of the B808–866 complex are reminiscent of the well-studied LH2 peripheral antenna of purple bacteria, whereas the protein sequence and the circular dichroism spectrum of B808–866 is more similar to the LH1 inner core antenna. The efficiency of excitation transfer from carotenoid to BChl is about 25%. The above results combined with electron microscopy and dynamic light scattering analysis suggest that the B808–866 antenna is more like the LH1, whereas surrounds the reaction center but probably consists of 24 building blocks with a ring diameter of about 20 nm. The above results suggested that there are probably two reaction centers inside the ring of B808–866. The unique properties of this light-harvesting complex may provide insights on the protein–pigment interactions in bacterial photosynthesis.     

The effect of growth conditions on the light-harvesting apparatus in Rhodopseudomonas acidophila

The detailed effect on the light-harvesting apparatus of three different wild-type strains of Rhodopseudomonas acidophila in response to changes in both light-intensity and temperature have been investigated. In all three strains at high light-intensities (160 mol s m² and above) the only LH2 antenna complex synthesised is the B800–850 complex. In strains 7050 and 7750 as the light-intensity is lowered the B800–850 complex is gradually replaced by another type of LH2 the B800–820 complex. However, at no light-intensities studied is this changeover complete when the cells are grown at 30°C. If however, the light-intensity is lowered at temperatures below 25°C with strain 7750 there is a complete replacement of the B800–850 complex by the B800–820 complex. At all light-intensities and temperatures tested, strain 10050 only synthesised the B800–850 complex. Strain 7050 also responded to changes in light-intensity by altering its carotenoid composition. At high light-intensity the major carotenoids were rhodopin and rhodopin-glucoside, while at low light-intensities the major ones were rhodopinal and rhodopinal-glucoside. This change in carotenoid content started to occur at rather higher light-intensities than the switchover from B800–850 to B800–820.     

The role of chromophore coupling in tuning the spectral properties of peripheral light-harvesting protein of purple bacteria

The publication of a structure for the peripheral light-harvesting complex of a purple photosynthetic bacterium (McDermott et al. (1995), Nature 374: 517–521) provides a framework within which we can begin to understand various functional aspects of these complexes, in particular the relationship between the structure and the red-shift of the bacteriochlorophyll Q_y transition. In this article we describe calculations of some of the spectral properties expected for an array of chromophores with the observed geometry. We report the stability of the calculated absorption spectrum to minor structural alterations, and deduce that the observed red shift of the 850 nm Q_y transition in the B800–850 antenna complexes is about equally attributable to chromophore-chromophore and chromophore-protein interactions, while chromophore-chromophore interactions predominate in generating the red-shift of the 820 nm Q_y transition in B800–820 type peripheral liggt-harvesting complexes. Finally we suggest that the red shift in the absorbance of the monomeric Bchl a found in antenna complexes to 800 nm, from 770 nm as observed in most solvents, is largely attributable to a hydrogen bond with the 2-acetyl group of this chromophore.     

Studies on the light-harvesting complexes from the thermotolerant purple bacterium Rhodopseudomonas cryptolactis

The antenna complexes from Rps. cryptolactis have been isolated and purified. Rps. cryptolactis contains two types of variable antenna complex, B800-850 and B800-820 as well as the core B875 antenna complex. The variable antenna complexes contain more than two types of antenna apoprotein, and have a Bchla:carotenoid ratio of 2:1. They can both be crystallised, but the B800-820 complex is the easiest with which to get relatively large single 3-D crystals (up to 0.5 mm in each dimension).     

A Fluorescence Detected Magnetic Resonance Investigation of the Carotenoid Triplet States Associated with Photosystem II of Isolated Spinach Thylakoid Membranes

The carotenoid triplet populations associated with the fluorescence emission chlorophyll forms of Photosystem II have been investigated in isolated spinach thylakoid membranes by means of fluorescence detected magnetic resonance in zero field (FDMR). The spectra collected in the 680–690 nm emission range, have been fitted by a global analysis procedure. At least five different carotenoid triplet states coupled to the terminal emitting chlorophyll forms of PS II, peaking at 682 nm, 687 nm and 692 nm, have been characterised. The triplets associated with the outer antenna emission forms, at 682 nm, have zero field splitting parameters |D| = 0.0385 cm⁻¹, |E| = 0.00367 cm⁻¹; |D| = 0.0404 cm⁻¹, |E| = 0.00379 cm⁻¹ and |D| = 0.0386 cm⁻¹, |E| = 0.00406 cm⁻¹ which are very similar to those previously reported for the xanthophylls of the isolated LHC II complex. Therefore the FDMR spectra recorded in this work provide insights into the organisation of the LHC II complex in the unperturbed environment represented by thylakoid membranes. The additional carotenoid triplet populations, detected by monitoring the chlorophyll emission at 687 and 692 nm, are assigned to carotenoids bound to inner antenna complexes and hence attributed to β-carotene molecules.     

Singlet-singlet annihilation at low temperatures in the antenna of purple bacteria

Chromatophores of the purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter (Rhodopseudomonas) sphaeroides were excited by means of 35-ps flashes at 532 nm of varying intensities, both at room temperature and at 4 K. With increasing exciting energy densities the integrated yield of fluorescence produced by these flashes was found to decrease considerably due to singlet-singlet annihilation. An analysis of the results showed that in R. rubrum the number of connected antenna molecules between which energy transfer is possible decreases from about 1000 to about 150 when the temperature is lowered from 298 to 4 K. In Rb. sphaeroides the B875 light-harvesting complex appears to contain about 100 connected bacteriochlorophyll (BChl) 875 molecules at 4 K, while the B800–850 complex contains about 45 BChl 850 molecules. The data are explained by a model for the antenna of Rb. sphaeroides in which units of B875, containing about four reaction centres, are separated by an array of B800–850 units that surrounds B875. By applying a random walk model we found that in both species the rate of energy transfer between neighbouring antenna molecules decreased about 10-fold upon lowering the temperature. The rate of energy transfer from antenna molecules to either open or closed reaction centres decreased only 3- to 4-fold in R. rubrum and remained approximately constant in Rb. sphaeroides upon cooling. A blue shift of the emission spectra at 4 K of both species was observed when the excitation energy density was increased to a level where singlet-singlet annihilation plays a significant role. This observation appears to support the notion that an additional long-wave pigment exists in the antenna of these bacteria.     

The complete amino acid sequence of the antenna polypeptide B806-866-β from the cytoplasmic membrane of the green bacterium Chloroflexus auranliacus

The bacteriochlorophyll a-binding polypeptide B806–866-β was extracted from membranes of the green thermophilic bacterium Chloroflexus aurantiacus with chloroform/methanol/ammonium acetate. Purification of the antenna polypeptide (6.3 kDa) was achieved by chromatography on Sephadex LH-60, Whatman DE-32 and by FPLC. The complete amino acid sequence (53 amino acid residues) was determined. The B806–866-β polypeptide is sequence homologous to the antenna β-polypeptides of purple bacteria (27–40%) and exhibits the characteristic three domain structure of the B870, B800–850 and B800–820 antenna complexes. The two typical His residues, conserved in all antenna β-polypeptides of purple bacteria, were found: His-24 lies within the N-terminal hydrophilic domain and His-42 within the central hydrophobic domain. This polypeptide together with the previously described α-polypeptide form the basic structural unit of the B806–866 antenna complex from C. aurantiacus.     

Effects of Carotenoid Inhibition on the Photosynthetic RC–LH1 Complex in Purple Sulphur Bacterium Thiorhodospira sibirica

Core complexes (LH1–RC) were isolated using preparative gel electrophoresis from photosynthetic membranes of the purple bacterium, Thiorhodospira sibirica, grown in the absence or presence of the carotenoid biosynthesis inhibitor, diphenylamine. The biosynthesis of carotenoids is affected by diphenylamine both quantitavely and qualitatively: after inhibition, the level of carotenoids in core complexes reaches only 10% of the normal content, as analyzed by HPLC and absorption spectroscopy. The normally grown bacterium biosynthesizes spirilloxanthin, rhodopin, anhydrorhodovibrin and lycopene, whereas after inhibition only neurosporene, ζ-carotene and their derivatives are found in the complexes. There is no concomitant accumulation of appreciable amounts of colorless carotenoid precursors. Interestingly, the main absorption band of the core light harvesting complex isolated from carotenoid-inhibited cells, shows a red shift to 889 nm, instead of a blue shift observed in many carotenoid-deficient species of purple photosynthetic bacteria. The stability of isolated core complexes against n-octyl-β-D-glucopyranoside clearly depends on the presence of carotenoids. Subcomplexes resulting from the detergent treatment, were characterized by non-denaturating gel electrophoresis combined with in situ absorption spectroscopy. Core complexes with the native carotenoid complement dissociate into three subcomplexes: (a) LH1 complexes partially depleted of carotenoids, with an unusual spectrum in the NIR region (λ_max = 791, 818, 847 and 875 nm), (b) reaction centers associated with fragments of LH1, (c) small amounts of a carotenoidless B820 subcomplex. The core complex from the carotenoid-deficient bacterium is much less stable and yields only the two sub-complexes (b) and (c). We conclude that carotenoids contribute critically to stability and interactions of the core complexes with detergents.     

The quenching of tryptophan fluorescence by protonated and unprotonated imidazole

The fluorescence life-time of N-acetyl-tryptophan-amide (NATA) was measured by multifrequency phase fluorometry, in the presence of increasing concentrations of imidazole. Two pH values were tested, pH 4.5 where imidazole is fully protonated and pH 9.0 where it is fully unprotonated. At both pH values, the inverse life-time increases in a non-linear way with the imidazole concentration, showing that imidazole is not a high efficiency collisional quencher. The data can be analysed in terms of the formation of a complex with a reduced fluorescence life-time. The rate constants for association (at 25°C) are around 5 (±0.2) × 10⁹ M^–1 s^–1 and are thus diffusion controlled. The association equilibrium constant is strongly pH dependent and is much higher than the expected value of 0.4 M^–1 for a collisional complex. The intrinsic fluorescence life-time of the complex is 1.56 (±0.02) ns at pH 9.0 and 1.82 (±0.03) ns at pH 4.5, as compared to 2.37 (±0.03) ns for free NATA at pH 9.0 and 2.83 (±0.05) at pH 4.5 (all atI = 0.34). This means that at both pH values the fluorescence life-time of NATA in the complex is reduced to 61 (±0.5)% of its value in the free state. Despite this, the protonated form of imidazole is a better quencher at low concentrations, owing to a longer residence-time of the complex. At high viscosity the association equilibration is too slow and the system is described by two life-times. The quenching effect ofHis-18 on the fluorescence of the proximalTrp-94 of barnase (Locwenthal et al. 1991, Willaert et al. 1991) is discussed in terms of these findings.     

Formation of Bacteriochlorophyll Form B820 in Light Harvesting 2 Complexes from Purple Sulfur Bacteria Treated with Dioxane

Treatment of some sulfur bacteria (Allochromatium minutissimum, Thiorhodospira sibirica, and Ectothiorhodospira halovacuolata WN22) with dioxane results in formation of the bacteriochlorophyll form B820 in the light harvesting complex LH2. This form characterized by absorption maximum at 820 nm has the same absorption spectrum as B820 subcomplex from LH1 complex. Appearance of the B820 form was accompanied by a sharp decrease in absorption in the carotenoid region. This phenomenon observed in all LH2 complexes investigated may be attributed to formation of colorless carotenoid aggregates. This is very similar to the previously reported dissociation of the LH1 complex with carotenoids into B820 subcomplexes. Although the B820 form corresponded the bacteriochlorophyll dimer, its circular dichroism spectrum showed that pigment molecules in this dimer exhibit different interaction than those in the B820 subcomplex. The dioxane treatment of LH2 complexes isolated from Rhodopseudomonas palustris bacteria grown under normal or low intensity illumination did not result in formation of such dimers. It is suggested that bacteriochlorophyll B820 formation is related to unique structure of LH2 complexes from the sulfur bacteria.     

The effect of norflurazon on protein composition and chlorophyll organization in pigment–protein complex of Photosystem II

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides in the 29.5–21 kDa region. The Chl a forms at 668, 676, and 690 nm that belong to LHC and antenna part of PS I disappear completely after treatment. The intensity of the Chl b form at 648 nm is sharply decreased in treated seedlings grown under 30 or 100 lx light intensity. The bands of carotenoid absorption at 421, 448 (Chl a), 452, 480, 492, 496 (β-carotene), and 508 nm also disappear. The band shift from 740 to 720 nm and decrease in its intensity relative to the 687 nm emission peak in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chla form at 710–712 nm.     

Trapping of an assembly intermediate of photosynthetic LH1 antenna beyond B820 subunit. Significance for the assembly of photosynthetic LH1 antenna

Most photosynthetic LH1 antennae undergo dissociation into B820 subunits, suggesting their universal character as structural modules. However, dissociation into subunits seems to occur reversibly only in the absence of carotenoids and the subunits were never found to bind carotenoids. The interactions of carotenoids with B820 have been studied in a newly developed reconstitution assay of the LH1 antenna from Rhodospirillum rubrum (Fiedor, L., Akahane, J., and Koyama, Y. (2004) Biochemistry 43, 16487-16496). These model studies show that B820 subunits strongly interact with carotenoids and spontaneously form stable LH1-like complexes with substoichiometric carotenoid content. This is the first experimental evidence that B820 may occur as a short-lived intermediate in the assembly of LH1 in vivo. The resulting complex of B820 subunits with carotenoid, termed iB873, is homogeneous, according to ion exchange chromatography and reproducible pigment composition. The iB873-bound carotenoid is as efficient in energy transfer to bacteriochlorophyll as the one in native antenna. To our knowledge, iB873 is the first complex binding functional carotenoid, with the spectral and biochemical properties intermediate between that of B820 and the fully assembled LH1.     

Formation of a subunit form of the core light-harvesting complex from sulfur purple bacteria <Emphasis Type="Italic">Ectothiorhodospira haloalkaliphila</Emphasis> with different carotenoid composition

B820 subunits from a purple sulfur bacterium Ectothiorhodospira haloalkaliphila strain ATCC 51935^T were obtained by treatment of carotenoid free LH1-RC complexes of this bacterium with ß-octylglucopyranoside (ß-OG). The same complexes with 100% carotenoid content were unable to dißsociate to B820 subunits, but disintegrated to monomeric bacteriochlorophyll (BChl) regardless of their carotenoid composition. The degree of dissociation of the LH1-RC complexes with an intermediate content of carotenoids (the B820 formation) was directly dependent on the quantity of carotenoids in the samples. The resulting B820 subunits did not contain carotenoids. B820 subunits easily aggregated to form a complex with an absorption peak at 880 nm at decreased ß-OG concentration. Analysis of the spectra of the LH1-RC complexes isolated from the cells with different levels of carotenogenesis inhibition led to the conclusion of the heterogeneity of the samples with a predominance of them in (a) the fraction with 100% of carotenoids and (b) the fraction of carotenoid-free complexes.     

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.     

Direct observation of sub-picosecond equilibration of excitation energy in the light-harvesting antenna of Rhodospirillum rubrum.

Excitation energy transfer in the light-harvesting antenna of Rhodospirillum rubrum was studied at room temperature using sub-picosecond transient absorption measurements. Upon excitation of Rs. rubrum membranes with a 200 fs, 600 nm laser flash in the Qx transition of the bacteriochlorophyll-a (BChl-a) absorption, the induced transient absorption changes in the Qy region were monitored. In Rs. rubrum membranes the observed delta OD spectrum exhibits ground state bleaching, excited state absorption and stimulated emission. Fast Qx --> Qy relaxation occurs in approximately 100-200 fs as reflected by the building up of stimulated emission. An important observation is that the zero-crossing of the transient difference absorption (delta OD) spectrum exhibits a dynamic redshift from 863 to 875 nm that can be described with by a single exponential with 325 fs time constant. The shape of the transient difference spectrum observed in a purified subunit of the core light-harvesting antenna, B820, consisting of only a single interacting pair of BChl-as, is similar to the spectrum observed in Rs. rubrum membranes and clearly different from the spectrum of BChl-a in a protein/detergent mixture. In the B820 and monomeric BChl-a preparations the 100-200 fs Qx --> Qy relaxation is still observed, but the dynamic redshift of the delta OD spectrum is absent. The spectral kinetics observed in the Rs. rubrum membranes are interpreted in terms of the dynamics of excitation equilibration among the antenna subunits that constitute the inhomogeneously broadened antenna. A simulation of this process using a set of reasonable physical parameters is consistent with an average hopping time in the core light harvesting of 220-270 fs, resulting in an average single-site excitation lifetime of 50-70 fs. The observed rate of this equilibration process is in reasonable agreement with earlier estimations for the hopping time from more indirect measurements. The implications of the findings for the process of excitation trapping by reaction centers will be discussed.     

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assembly of peripheral light-harvesting complexes in purple sulfur bacteria <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> ATCC 17899

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodopin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphenylamine (DPA) concentrations not exceeding 53 μM, while at illumination intensity decreased to 500 lx they could grow at 71 μM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoene synthetase (resulting in a decrease in the total carotenoid content) and of phytoene desaturase, which results in formation of neurosporene hydroxy derivatives and ζ-carotene. In the cells grown at 500 lx, small amounts of spheroidene and OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was ~15% of the control (without inhibition) for the B800-850 and ~20% of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with carotenoids due to disintegration of some carotenoidless complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.     

Spectroscopic characterization of the light-harvesting complex of Rhodospirillum rubrum and its structural subunit

The spectroscopic properties of the light-harvesting complex of Rhodospirillum rubrum, B873, and a detergent-isolated subunit form, B820, are presented. Absorption and circular dichroism spectra suggest excitonically interacting bacteriochlorophyll alpha (BChl alpha) molecules give B820 its unique spectroscopic properties. Resonance Raman results indicate that BCHl alpha is 5-coordinate in both B820 and B873 but that the interactions with the BChl C2 acetyl in B820 and B873 are different. The reactivity of BChl alpha in B820 in light and oxygen, or NaBH4, suggests that it is exposed to detergent and the aqueous environment. Excited-state lifetimes of the completely dissociated 777-nm-absorbing form [1.98 ns in 4.5% octyl glucoside (OG)], the intermediate subunit B820 (0.72 ns in 0.8% OG), and the in vivo like reassociated B873 (0.39 ns in 0.3% OG) were measured by single-photon counting. The fluorescence decays were exponential when emission was detected at wavelengths longer than 864 nm. An in vivo like B873 complex, as judged by its spectroscopic properties, can be formed from B820 without the presence of a reaction center.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30–35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808–866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Q_y transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Q_y transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808–866 complex of C. aurantiacus and the B800–850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 was derived.Abbreviations BChl bacteriochlorophyll - BPheo bacteriopheophytin - CD circular dichroism - LD linear dichroism - Tris Tris(hydroxymethyl)aminomethane