Isolation and characterization of light harvesting bacteriochlorophyll · protein complexes from Rhodopseudomonas capsulata

The isolation of two native light harvesting bacteriochlorophyl · protein complexes from Rhodopseudomonas capsulata is described. The light harvesting bacteriochlorophyll I (B 875) has been isolated from the blue-green mutant Ala⁺ lacking both carotenoids and light harvesting bacteriochlorophyll II. Light harvesting bacteriochlorophyll I is associated with a protein (light harvesting band 2) of 12 000 molecular weight.Light harvesting bacteriochlorophyll II complex has been isolated from the mutant Y5 lacking a reaction center and light harvesting bacteriochlorophyll I. Light harvesting bacteriochlorphyll II (B 800 + 850) together with carotenoids is associated with two polypeptides (light harvesting bands 3 and 4) having molecular weights of about 8000 and 10 000 (sodium dodecyl sulfate polyacrylamide gel electrophoresis). A third protein (light harvesting band 1) is in the purified light harvesting II fraction (mol. wt. approx. 14 000), but not associated with bacteriochlorophyll or carotenoids. The amino acid composition of the 3 antenna pigment II proteins is given. The polarity of these proteins was found to be 48%. From the amino acid composition the following molecular weights were calculated band 1: 17 350, band 3: 13 350 and band 4: 10 500.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

Identification of the carotenoid present in the B-800–850 antenna complex from Rhodopseudomonas capsulata as that which responds electrochromically to transmembrane electric fields

Mild proteolysis of Rhodopseudomonas capsulata chromatophores results in a parallel loss of the 800 nm bacteriochlorophyll absorption band and a blue shift in the carotenoid absorption bands associated with the B-800–850 light-harvesting complex. Both the light-induced and the salt-induced electrochromic carotenoid band shift disappear in parallel to the loss of the 800 nm bacteriochlorophyll absorption upon pronase treatment of chromatophores. During the time required for the loss of the 800 nm bacteriochlorophyll absorption and the loss of the electrochromic carotenoid band shift photochemistry is not inhibited and the ionic conductance of the membrane remains very low. We conclude that the carotenoid associated with the B-800–850 light-harvesting complex is the one that responds electrochromically to the transmembrane electric field. Analysis of the pigment content of Rps. capsulata chromatophores indicates that all of the carotenoid may be accounted for in the well defined pigment-protein complexes.     

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides

The pigment content of a B800–850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed.The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800–850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band.The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.     

The 7.5-A electron density and spectroscopic properties of a novel low-light B800 LH2 from Rhodopseudomonas palustris.

A novel low-light (LL) adapted light-harvesting complex II has been isolated from Rhodopseudomonas palustris. Previous work has identified a LL B800-850 complex with a heterogeneous peptide composition and reduced absorption at 850 nm. The work presented here shows the 850 nm absorption to be contamination from a high-light B800-850 complex and that the true LL light-harvesting complex II is a novel B800 complex composed of eight alpha beta(d) peptide pairs that exhibits unique absorption and circular dichroism near infrared spectra. Biochemical analysis shows there to be four bacteriochlorophyll molecules per alpha beta peptide rather than the usual three. The electron density of the complex at 7.5 A resolution shows it to be an octamer with exact 8-fold rotational symmetry. A number of bacteriochlorophyll geometries have been investigated by simulation of the circular dichroism and absorption spectra and compared, for consistency, with the electron density. Modeling of the spectra suggests that the B850 bacteriochlorophylls may be arranged in a radial direction rather than the usual tangential arrangement found in B800-850 complexes.     

Pigment-pigment interactions and secondary structure of reconstituted algal chlorophyll a/b-binding light-harvesting complexes of Chlorella fusca with different pigment compositions and pigment-protein stoichiometries

Earlier we have shown by in vitro reconstitution experiments that the pigment composition of the chlorophyll alb-binding light-harvesting complex of the green alga Chlorella fusca could be altered in a relatively broad range (Meyer and Wilhelm 1993). In this study we used these reconstituted complexes of different pigment loading to analyze the excitonic interactions between the pigment molecules and the secondary structure by means of circular dichroism spectra in the visible and the far UV spectral regions, respectively. We found that, in contrast to the expectations, the pigment composition and pigment content hardly affected the circular dichroism spectra in the visible spectral region. Reconstituted complexes, independent of their pigment composition, exhibited the most characteristic circular dichroism bands of the native light-harvesting complex, even if one polypeptide bound only 3 chlorophyll a, 3 chlorophyll b and 1–2 xanthophyll molecules. Full restoration of the protein secondary structure, however, could not be achieved. The -helix content depended significantly on the pigment composition as well as on the pigment-protein ratio of the reconstituted complexes. Further binding of pigments resulted in restoration of the minor excitonic circular dichroism bands, the amplitudes of which depended on the pigment content of the reconstituted complexes. These data suggest that in the reconstitution of light-harvesting complexes a central cluster of pigment molecules plays an important role. Further binding of pigments to the peripheral binding sites appeared also to stabilize the protein secondary structure of the reconstituted complexes.Abbreviations CD- circular dichroism - LHC- chlorophyll a/b light-harvesting complex(es) - LHC II- light-harvesting complex(es) of Photosystem II of higher plants - LHCP- light-harvesting Chl a/b-binding protein(s) - PP- polypeptide(s)     

Energy transfer in purple bacterial photosynthetic units from cells grown in various light intensities

Three photosynthetic membranes, called intra-cytoplasmic membranes (ICMs), from wild-type and the ?pucBA_abce mutant of the purple phototrophic bacterium Rps. palustris were investigated using optical spectroscopy. The ICMs contain identical light-harvesting complex 1–reaction centers (LH1–RC) but have various spectral forms of light-harvesting complex 2 (LH2). Spectroscopic studies involving steady-state absorption, fluorescence, and femtosecond time-resolved absorption at room temperature and at 77 K focused on inter-protein excitation energy transfer. The studies investigated how energy transfer is affected by altered spectral features of the LH2 complexes as those develop under growth at different light conditions. The study shows that LH1 → LH2 excitation energy transfer is strongly affected if the LH2 complex alters its spectroscopic signature. The LH1 → LH2 excitation energy transfer rate modeled with the Förster mechanism and kinetic simulations of transient absorption of the ICMs demonstrated that the transfer rate will be 2–3 times larger for ICMs accumulating LH2 complexes with the classical B800–850 spectral signature (grown in high light) compared to the ICMs from the same strain grown in low light. For the ICMs from the ?pucBA_abce mutant, in which the B850 band of the LH2 complex is blue-shifted and almost degenerate with the B800 band, the LH1 → LH2 excitation energy transfer was not observed nor predicted by calculations.     

Energy transfer in the B800-850-carotenoid light-harvesting complex of various mutants of Rhodopseudomonas sphaeroides and of Rhodopseudomonas capsulata

Emission and absorption spectra in the temperature range 4–300 K have been obtained for bacteriochlorophyll light-harvesting complexes (B800–850 complexes) from several mutants of Rhodopseudomonas sphaeroides and a nonphotosynthetic mutant of Rhodopseudomonas capsulata. The energy-transfer properties of these complexes were remarkably similar despite differences in carotenoid composition. Between 300 and 200 K the excitation densities in B800 and B850 are in thermal equilibrium, indicating rapid energy transfer from B800 to B850 and vice versa. The temperature dependence of the ratio of the B800 and B850 emission yields allows the determination of the ratio of the number of B800 and B850 molecules in the complex which is close to 0.5. Below 200 K thermal equilibrium no longer exists. At 4–100 K the B800 emission yield increases with decreasing temperature and becomes dependent on the wavelength of excitation. From the B800 emission yield at 4 K the B800–850 dipole-dipole distance was calculated to be equal to or smaller than 21 Å for all B800–850 complexes. Excitation spectra for B800 and B850 emission show that the overall energy-transfer efficiencies from carotenoid and B800 to B850 are greater than 90% at all temperatures. At 4 K the carotenoid transfers its excitation energy preferentially to B850. Experiments with chromatophores indicated that the energy-transfer properties of the B800–850 complexes were not modified by the isolation procedures.     

The isolation and partial characterisation of the light-harvesting pigment-protein complement of Rhodopseudomonas acidophila

The photosynthetic membranes of two strains of Rhodopseudomonas acidophila (7750 and 7050) have been resolved into their constituent light-harvesting pigment-protein complexes. Four different types of antenna complexes (B880, B800–830 and two types of B800–850) have been isolated and partially purified. In each case the light-harvesting pigments (bacteriochlorophyll a and carotenoids) are bound to rather low molecular weight polypeptides (in the 5000–9000 region).     

Mutants of Sweetclover (Melilotus alba) Lacking Chlorophyll b: Studies on Pigment-Protein Complexes and Thylakoid Protein Phosphorylation

Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorption spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid membranes lacking the three Chl b-containing pigment-protein complexes (AB-1, AB-2, and AB-3) while still containing A-1 and A-2, Chl a complexes derived from photosystems I and II, respectively. Complete solubilization and denaturation of the thylakoid proteins from this mutant revealed very little apoprotein from the Chl b-containing light-harvesting complexes, the major thylakoid proteins in normal plants. The normal and mutant sweetclover plants had active thylakoid protein kinase activities and numerous polypeptides were labeled following incubation with [γ-³²P]ATP. With the U374 mutant, however, there was very little detectable label co-migrating with the light-harvesting complex apoproteins on polyacrylamide gels. The Chl b-deficient chlorina-f2 mutant of barley (Hordeum vulgare) also had an active protein kinase activity capable of phosphorylating numerous polypeptides, including ones migrating with the same mobility as the light-harvesting complex apoproteins. These results indicate that the sweetclover mutants may be useful systems for studies on the function and organization of Chl b in thylakoid membranes of higher plants.     

Energy transfer in an LH4-like light harvesting complex from the aerobic purple photosynthetic bacterium Roseobacter denitrificans

A peripheral light-harvesting complex from the aerobic purple bacterium Roseobacter (R.) denitrificans was purified and its photophysical properties characterized. The complex contains two types of pigments, bacteriochlorophyll (BChl) a and the carotenoid (Car) spheroidenone and possesses unique spectroscopic properties. It appears to lack the B850 bacteriochlorophyll a Q(y) band that is typical for similar light-harvesting complex 2 antennas. Circular dichroism and low temperature steady-state absorption spectroscopy revealed that the B850 band is present but is shifted significantly to shorter wavelengths and overlaps with the B800 band at room temperature. Such a spectral signature classifies this protein as a member of the light-harvesting complex 4 class of peripheral light-harvesting complexes, along with the previously known light-harvesting complex 4 from Rhodopseudomonas palustris. The influence of the spectral change on the light-harvesting ability was studied using steady-state absorption, fluorescence, circular dichroism, femtosecond and microsecond time-resolved absorption and time-resolved fluorescence spectroscopies. The results were compared to the properties of the similar (in pigment composition) light-harvesting complex 2 from aerobically grown Rhodobacter sphaeroides and are understood within the context of shared similarities and differences and the putative influence of the pigments on the protein structure and its properties.     

Characterization of light-harvesting mutants of Rhodopseudomonas sphaeroides. I. Measurement of the efficiency of energy transfer from light-harvesting complexes to the reaction center

Light-harvesting mutants of Rhodopseudomonas sphaeroides lacking either the B800-850 complex or the B875 complex have been characterized by their absorption spectra in the visible and near-infrared region, and by their ability to transfer energy from the light-harvesting complexes to the reaction center. A new method of measuring the relative efficiency of energy transfer from the light-harvesting complexes to the reaction center is described. The B875- mutant had absorption maxima in the near-infrared at 800 and 849 nm with no evidence of an 875-nm shoulder. The efficiency of energy transfer from the light-harvesting complexes to the reaction center in the B875- mutant was 24% of the value measured for the wild-type strain and the B800-850- mutant. Yet, despite the fact that the efficiency of energy transfer for the B800-850- mutant and the wild-type strain were the same, there was a large difference in their photosynthetic unit size. These results are discussed in the context of a model in which light energy captured by the B800-850 complexes is transferred through the B875 complexes to the reaction center.     

Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting the B800-850 complex and its dependence on the spectral composition of the light is discussed.     

Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Q_y bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of ?0.2 was observed upon excitation in the Q_x BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Q_y transition dipoles essentially parallel and their Q_x transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.     

Polypeptides and bacteriochlorophyll organization in the light-harvesting complex B850 of Rhodobacter sphaeroides R-26.1

The light-harvesting complex (LHC) B850 from Rhodobacter sphaeroides was dissociated into several fragments by treatment with sodium dodecyl sulfate. The molecular weight of each fragment was determined by using transverse polyacrylamide gel electrophoresis under nondenaturing conditions and gel filtration techniques. Four B850 LHCs were observed, having molecular weights of 60,000, 72,000-75,000, 105,000, and 125,000-145,000, and two small bacteriochlorophyll (Bchl)-polypeptide complexes having molecular weights of 6000-8000 and 12,000-14,000. Each of the B850 complexes contains ca. one Bchl a for each 6.5-kDa protein. The optical absorption and circular dichroism of the B850 LHCs recorded directly from the gels are similar to those measured previously for a 22-24-kDa B850 LHCs by Sauer and Austin [(1978) Biochemistry 17, 2011-2019]. These data, combined with studies of other groups, indicate that the smallest LHC in LH1 and LH2 is a Bchl-polypeptide tetramer. Each tetramer contains two Bchl dimers that probably have the structure of P-860, the primary electron donor in Rhodobacter sphaeroides, and two alpha-beta-polypeptide pairs. Interactions among the paired Bchls shift their individual Qy transitions from 780-800 to 850-860 nm, and interactions among two such pairs induce the circular dichroism signal of the LHCs. Three Bchl-polypeptide tetramers probably form a dodecamer having C3 symmetry, and six such dodecamers organize into a large hexagon that can accommodate one or two reaction center complexes.     

Fluorescence properties of the light-harvesting bacteriochlorophyll protein from Rhodopseudomonas sphaeroides R-26

The light-harvesting bacteriochlorophyll-protein (BChl-protein) from Rhodopseudomonas sphaeroides, R-26 mutant, exhibits a strong optical absorption peak near 850 nm (Q_y band) and a weaker peak at 590 nm (Q_x band). This pigment-protein appears to contain two BChl molecules per subunit, and previous circular dichroism studies indicated the presence of excitonic interactions between the BChl molecules. The complex exhibits a fluorescence maximum near 870 nm at room temperature. Excitation in the Q_y region results in polarization p values that vary only from +0.12 at 820 nm to +0.14 near 900 nm. These values are appreciably smaller than that for monomeric BChl in viscous solvents (p > 0.4). By contrast, using Q_x excitation the p value is ?0.25 for the BChl-protein complex, which is close to that observed for the BChl monomer. For the BChl-protein these polarization values do not change greatly at a temperature of 90 K; however, the Stokes' shift of the fluorescence emission increases significantly over that at room temperature.     

Contents to volume 178

UV CD and IR spectra of the water-soluble bacteriochlorophyll-protein antenna isolated from Prosthecochloris aestuarii indicate that about 50% of the protein is in a β-sheet conformation while for the dominant antenna complexes isolated from bacteria (B800-850) and from green plants (LHC), the α-helix (45%) is more abundant than the β-sheet (~ 10%) conformation. Furthermore, IR dichroism studies show that the α-helical segments of a large variety of intrinsic membrane Chl-protein complexes (antenna and reaction centers) are tilted on the average at 30–35° away from the membrane normal. The observation that in these complexes the Chl planes are also tilted at about the same angle suggests that the transmembrane orientation of the α-helices determines the positioning of the Chl molecules in photosynthetic membranes.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: III. A mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca⁺²-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca⁺²-binding site. This site was reconstructed from the fourth potential Ca⁺²-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca⁺²-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca²⁺ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin. For communication II, see [1].     

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