Crystallization of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7750.

The B800-820 light-harvesting complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 121.8 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820 alpha 1, B800-820 alpha 2, B800-820 beta 1 and B800-820 beta 2). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000.     

Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050

Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.     

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.     

Fusion of proteoliposomes containing the B800-850 light-harvesting complex with membranes of the mutant strain NK3 of Rhodopseudomonas capsulata lacking B800-850

Intracytoplasmic membranes of the mutant strain NK3 of Rhodopseudomonas capsulata lacking the lightharvesting complex B800-850 were fused with proteoliposomes containing the B800-850 complex. Fluorescence emission spectroscopy at 77K showed that after fusion the fluorescence of the B850 bacteriochlorophyll disappeared nearly completely and the B870 fluorescence became prominent. This result and control experiments with proteoliposome-chromatophore mixture and with chromatophore and solubilized B800-850 complexes, respectively, indicate that in fused membranes a reorientation of membrane particles took place and excitons migrated from B850 to B870 bacteriochlorophyll.In fused proteoliposome-chromatophore vesicles a light-induced carotenoid band shift was observed, reflecting the building of an electrical membrane potential due to chargeseparation. Carotenoid band shift was not observed in separated proteoliposomes and NK3 chromatophores.It is concluded that by membrane fusion and lateral diffusion of membrane particles reaction center-light-harvesting B870 complexes came in functional contact with B800-850 antenna complexes.Abbreviations Bchl bacteriochlorophyll - LDAO lauryl dimethylamine oxide - RC reaction center Dedicated to Professor R. Clinton Fuller, Amherst, MA, USA, on the occasion of his 60th birthday in recognition of his work on photosynthetic bacteria and the cooperation between our laboratories     

Chemical cross-linking studies of the light-harvesting pigment-protein complex B800-850 of Rhodopseudomonas capsulata

The spatial relationship of the three polypeptides contained in the B800-850 light-harvesting complex of Rhodopseudomonas capsulata has been studied with chemical cross-linking of crude membrane preparations of the phototrophic negative mutant strain Y5. Samples were cross-linked with the cleavable reagent dithiobis (succinimidyl propionate) (1.1 nm chain length) and analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membranes labelled with 14C-amino acids were used to determine the compositional stoichiometry of cross-linked products. It was found that the two polypeptides with an apparent Mr of 8000 and 10 000, respectively, that are associated with the pigments bacteriochlorophyll a and carotenoid form homooligomers as well as heterooligomers. The data support the idea that these polypeptides are closely arranged in clusters probably containing at least four of each species. The third subunit with an Mr of 14 000, which is not associated with pigments, was found to be most susceptible to cross-linking and formed homooligomers but no heterooligomers with the other two subunits, and is thus likely to be loosely attached to these clusters. Comparative studies with the phototrophic positive wild type strain indicated that the results found with the phototrophic negative mutant strain Y5 reflect the organization of the B800-850 complex in the membrane of Rhodopseudomonas capsulata. Studies with the isolated B800-850 complex revealed that the sterical arrangement of the three constituent polypeptides in dodecyl dimethylamine-N-oxide containing solutions must be very similar to that in the membrane.     

Interactions stabilizing the structure of the core light-harvesting complex (LH1) of photosynthetic bacteria and its subunit (B820)

Reconstitution experiments with a chemically synthesized core light-harvesting (LH1) beta-polypeptide analogue having 3-methylhistidine instead of histidine in the position that normally donates the coordinating ligand to bacteriochlorophyll (Bchl) have provided the experimental data needed to assign to B820 one of the two possible alphabeta.2Bchl pairs that are observed in the crystal structure of LH2 from Phaeospirillum (formerly Rhodospirillum) molischianum, the one with rings III and V of Bchl overlapping. Consistent with the assigned structure, experimental evidence is provided to show that significant stabilizing interactions for both the subunit complex (B820) and LH1 occur between the N-terminal regions of the alpha- and beta-polypeptides. On the basis of the results with the chemically synthesized polypeptides used in this study, along with earlier results with protease-modified polypeptides, mutants, and chemically synthesized polypeptides, the importance of a stretch of 9-13 amino acids at the N-terminal end of the alpha- and beta-polypeptides is underscored. A progressive loss of interaction with the LH1 beta-polypeptide was found as the first three N-terminal amino acids of the LH1 alpha-polypeptide were removed. The absence of the N-terminal formylmethionine (fMet), or conversion of the sulfur in this fMet to the sulfoxide, resulted in a decrease in LH1 formation. In addition to the removal of fMet, removal of the next two amino acids also resulted in a decrease in K(assoc) for B820 formation and nearly eliminated the ability to form LH1. It is suggested that the first three amino acids (fMetTrpArg) of the LH1 alpha-polypeptide of Rhodospirillum rubrum form a cluster that is most likely involved in close interaction with the side chain of His -18 (see Figure 1 for numbering of amino acids) of the beta-polypeptide. The results provide evidence that the folding motif of the alpha- and beta-polypeptides in the N-terminal region observed in crystal structures of LH2 is also present in LH1 and contributes significantly to stabilizing the complex.     

Fluorescence spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila strain 10050

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The frequency and size of these fluorescence peak movements were found to increase linearly with excitation intensity. Using the modified Redfield theory, changes in the realization of the static disorder accounted for the observed changes in spectral shape and intensity. Long lifetimes of the quasi-stable states suggest large free energy barriers between the different realizations.     

The hopanoids of the purple non-sulfur bacteria Rhodopseudomonas palustris and Rhodopseudomonas acidophila and the absolute configuration of bacteriohopanetetrol

Five complex hopanoids have been detected in the purple non-sulfur bacterium Rhodopseudomonas acidophila. Next to the polyfunctionalized methylcyclopentane bacteriohopanetetrol ether already isolated from Methylobacterium organophilum, 35-carbamoylbacteriohopane-32,33,34-triol, 34,35-dicarbamoylbacteriohopane-32,33-diol and two nucleoside analogues, (22R)-30-(5'-adenosyl)hopane and (22S)-30-(5'-adenosyl)hopane were isolated and identified by spectroscopic and chemical methods. In Rhodopseudomonas palustris, however, only 35-amino-bacteriohopane-32,33,34-triol was detected. Chemical correlation between adenosylhopane and bacteriohopanetetrol, as well as comparison of derivatives obtained from bacterial and synthetic hopanoids, permitted the determination of the configurations of all asymmetric centres of the side-chain of bacteriohopanetetrol as 22R, 32R, 33R and 34S. According to the stereochemistry, this side-chain could be a D-ribose derivative linked through its C-5 carbon atom to the hopane skeleton.     

The complete amino-acid sequence of the large bacteriochlorophyll-binding polypeptide from light-harvesting complex II (B800-850) of Rhodopseudomonas capsulata

The large bacteriochlorophyll-a-binding polypeptide of the light-harvesting complex II (B800-850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16-35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C-terminal part is also hydrophobic while the N-terminal part consists of hydrophilic amino acids. The polarity of the total amino acids was determined to be 28.3%.     

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 light-harvesting core-complex and the B820-subunit from Rhodopseudomonas marina. Part I. Purification and characterisation.

The BChla-containing B880-complex (core-complex) of Rhodopseudomonas marina (Rhodospirillaceae) was isolated with a new purification method. The isolation of the B880-complex was performed by solubilisation of the photosynthetic membranes with the detergent LDAO and subsequent fractionated ammonium-sulfate precipitation with about 50% recovery. The B880-complex retained its original spectral properties as revealed with absorption, fluorescence and circular dichroism spectroscopy. Furthermore, we dissociated the B880-complex with the detergent n-octyl-beta-glucoside (OG) and purified the developed subcomplex by the method of Miller et al. [1], which showed an absorption maximum at 820 nm (B820). The alpha- to beta-polypeptide ratio and the alpha- or beta-polypeptide to BChla ratio, respectively, were estimated to be 1:1 in both complexes. The molecular weights of the B880 and the B820-complexes, determined by gel filtration chromatography, were 181 and 32 kDa, respectively. Thus, it appears that the B880-complex of Rp. marina consists of 24 polypeptides and the B820-complex of four polypeptides. Six B820-complexes or possible subunits could form the B880-complex. On the basis of these data we propose a model for the structure of BChla containing core-complexes.     

The light-harvesting core-complex and the B820-subunit from Rhodopseudomonas marina. Part II. Electron microscopic characterisation.

Electron micrographs of photosynthetic membranes of the BChla-containing bacterium Rp. marina showed a quasi-crystalline structure. The photoreceptor units are arranged in a hexagonal lattice with a reaction center to reaction center distance of 102 +/- 3 A. Purified B880-complex was concentrated up to an OD880 of 60 which induced the formation of large protein vesicles. The protein complexes within these vesicles were highly ordered and showed a hexagonal lattice with the same center to center distance of 102 +/- 3 A as was observed in the native membranes. Image processing of the micrographs revealed a ring-like structure of the B880-complex at 26 A resolution and suggests that the B880-complex consists of 5 or 6 subunits. For the first time it can be shown that an isolated core-complex is in a stable, ring-like structure even without the reaction center which is supposed to be located in the middle of the B880-ring. The data indicate that the isolated B880-complex exhibits the same structure as in the native membrane.     

Selective release, removal, and reconstitution of bacteriochlorophyll a molecules into the B800 sites of LH2 complexes from Rhodopseudomonas acidophila 10050.

A method is described which allows the selective release and removal of the Bchla-B800 molecules from the LH2 complex of Rhodopseudomonas acidophila 10050. This procedure also allows reconstitution of approximately 80% of the empty binding sites with native Bchla. As shown by circular dichroism spectroscopy, the overall structures of the B850-only and reconstituted complexes are not affected by the pigment-exchange procedure. The pigments reconstituted into the B800 sites can also efficiently transfer excitation energy to the Bchla-B850 molecules.     

Reconstitution of carotenoids into the light-harvesting complex B800-850 of Chromatium minutissimum

Chromatophores and peripheral light-harvesting complexes B800–850 with a trace of carotenoids were isolated from Chromatium minutissimum cells in which carotenoid biosynthesis was inhibited by diphenylamine. Three methods previously used for the reconstitution of carotenoids into either the light-harvesting (LH1) type complexes or reaction centers (RC) of carotenoidless mutants were examined for the possibility of carotenoid reconstitution into the carotenoid depleted chromatophores. All these methods were found to be unsuitable because carotenoid depleted complex B800–850 from Chr. minutissimum is characterized by high lability. We have developed a novel method maintaining the native structure of the complexes and allowing reconstitution of up to 80% of the carotenoids as compared to the control. The reconstituted complex has a similar CD spectrum in the carotenoid region as the control, and its structure restores its stability. These data give direct proof for the structural role of carotenoids in bacterial photosynthesis.     

The structure and thermal motion of the B800-850 LH2 complex from Rps.acidophila at 2.0A resolution and 100K: new structural features and functionally relevant motions

The structure at 100K of integral membrane light-harvesting complex II (LH2) from Rhodopseudomonas acidophila strain 10050 has been refined to 2.0A resolution. The electron density has been significantly improved, compared to the 2.5A resolution map, by high resolution data, cryo-cooling and translation, libration, screw (TLS) refinement. The electron density reveals a second carotenoid molecule, the last five C-terminal residues of the alpha-chain and a carboxy modified alpha-Met1 which forms the ligand of the B800 bacteriochlorophyll. TLS refinement has enabled the characterisation of displacements between molecules in the complex. B850 bacteriochlorophyll molecules are arranged in a ring of 18 pigments composed of nine approximate dimers. These pigments are strongly coupled and at their equilibrium positions the excited state dipole interaction energies, within and between dimers, are approximately 370cm(-1) and 280cm(-1), respectively. This difference in coupling energy is similar in magnitude to changes in interaction energies arising from the pigment displacements described by TLS tensors. The displacements appear to be non-random in nature and appear to be designed to optimise the modulation of pigment energy interactions. This is the first time that LH2 pigment displacements have been quantified experimentally. The calculated energy changes indicate that there may be significant contributions to inter-pigment energy interactions from molecular displacements and these may be of importance to photosynthetic energy transfer.     

Probing the structure of the core light-harvesting complex (LH1) of Rhodopseudomonas viridis by dissociation and reconstitution methodology

A subunit complex was formed from the core light-harvesting complex (LH1) of bacteriochlorophyll(BChl)-b-containing Rhodopseudomonas viridis. The addition of octyl glucoside to a carotenoid-depleted Rps. viridis membrane preparation resulted in a subunit complex absorbing at 895 nm, which could be quantitatively dissociated to free BChl b and then reassociated to the subunit. When carotenoid was added back, the subunit could be reassociated to LH1 with a 25% yield. Additionally, the Rps. viridis - and -polypeptides were isolated, purified, and then reconstituted with BChl b. They formed a subunit absorbing near 895 nm, similar to the subunit formed by titration of the carotenoid depleted membrane, but did not form an LH1-type complex at 1015 nm. The same results were obtained with the -polypeptide alone and BChl b. Isolated polypeptides were also tested for their interaction with BChl a. They formed subunit and LH1-type complexes similar to those formed using polypeptides isolated from BChl-a-containing bacteria but displayed 6–10 nm smaller red shifts in their long-wavelength absorption maxima. Thus, the larger red shift of BChl-b-containing Rps. viridis is not attributable solely to the protein structure. The -polypeptide of Rps. viridis differed from the other -polypeptides tested in that it could form an LH1-type complex with BChl a in the absence of the - and -polypeptides. It apparently contains the necessary information required to assemble into an LH1-type complex. When the -polypeptide was tested in reconstitution with BChl a and BChl b with the - and -polypeptides, it had no effect; its role remains undetermined.Abbreviations B820 the subunit form of the core light-harvesting complex in BChl-a-containing bacteria which has an absorption maximum at or near 820 nm - B875 the core light-harvesting complex of Rhodobacter sphaeroides which has an absorption maximum at 875 nm - B881 the core light-harvesting complex of wild-type Rhodospirillum rubrum which has an absorption maximum at 881 nm - B895 the subunit form of the core light-harvesting complex in Rps. viridis which has an absorption maximum near 888–895 nm - B1015 the core light-harvesting complex of Rps. viridis which has an absorption maximum at 1015 nm - CD circular dichroism - LH1 the core light-harvesting complex - OG n-octyl -d-glucopyranoside     

Rhodopseudomonas acidophila strain 10050 contains photosynthetic LH2 antenna complexes that are not enriched with phosphatidylglycerol,and the phospholipids have a fatty acyl composition that is unusual for purple non-sulfur bacteria

The phospholipid composition of Rhodopseudomonas acidophila strain 10050 grown aerobically or anaerobically in the light was determined. The major phospholipids present in the aerobic cells were phosphatidylethanolamine (PE; 54%), phosphatidylglycerol (PG; 24%) and cardiolipin (diphosphatidylglycerol, DPG) (14%), together with phosphatidylcholine (PC; 5%). On moving the cells to anaerobic photosynthetic growth in the light PE remained the major phospholipid (37-49%), but there was a major change in the proportion of PC, which increased to 31-33%, and corresponding reductions in the contents of PG to 11-16% and DPG to 4-5%. The fatty acid composition of the phospholipids was unusual, compared with other purple non-sulfur photosynthetic bacteria, in that it contained 16:0 (29%), 17:1 (20%) and 19:1 (9%) plus several mainly unsaturated 2-OH fatty acids (9% total) as major components, when grown aerobically in the dark. In contrast when grown photosynthetically under anaerobic conditions there was <2% 17:1 or 19:1 present, while the amounts of 16:1 and 18:1 increased, and 16:0 decreased. The phospholipid composition of the purified light-harvesting complex 2 (LH2) complex was PE (43%), PC (42%) and DPG (15%). Unexpectedly, there was no PG associated with the purified LH2. These findings contrast with previous studies on several other photosynthetic bacteria, which had shown an increase in PG upon photosynthetic growth [Biochem. J. 181 (1979) 339]. The prior hypothesis that phosphatidylglycerol has some specific role to play in the function of light-harvesting complexes cannot be true for Rps. acidophila. It is suggested that specific integral membrane proteins may strongly influence the phospholipid content of the host membranes into which they are inserted.     

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.