Pigment-protein complexes from Rhodopseudomonas palustris: isolation, characterization, and reconstitution into liposomes.

We have employed detergent solubilization and sucrose density gradient centrifugation to obtain pigment-protein complexes from Rhodopseudomonas palustris. Two types of detergent buffers were used, containing either octyl-beta-glucopyranoside (OG) plus sodium dodecyl sulfate (SDS) or OG alone. The fractions thus obtained were analyzed spectrophotometrically and by polyacrylamide gel electrophoresis to determine their pigment and protein composition. OG-SDS solubilization yields four fractions. The least dense of these fractions (OG-SDS a and b) are nonspecific mixtures of peptides and pigments. The next fraction, OG-SDS c, is an accessory light-harvesting complex, LHII, called B800-850. The largest particle, OG-SDS d, is a combination of reaction center (RC) and primary light-harvesting complex (LHI), B880. Solubilization using OG alone yields one fraction, a single large complex consisting of RC, LHI, and LHII. We have inserted the two large OG-SDS complexes and the OG complex into phospholipid liposomes to determine the size of such complexes in freeze-fractured membranes. On the basis of morphological, biochemical, and available biophysical data, we propose the following models for pigment-protein complexes in R. palustris membranes: 5-nm particles as free RC or LHI tetramers, 7.5-nm particles as LHI or LHII octamers (or both); 10-nm particles as RC-LHI core complexes (1 RC plus 12 LHI) or large LHII oligomers (or both), and large particles of 12.5 and 15 nm and LHII associated with the RC-LHI core complex.     

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.     

Turnover of the B870-α pigment-binding protein in a mutant of Rhodopseudomonas capsulata which is defective in assembling reaction center and B870 into membranes

Abstract The photosynthetically negative mutant strain Y142 of Rhodopseudomonas capsulata , which synthesizes bacteriochlorophyll (Bchl), carotenoids and the light-harvesting (LH) complex B800–850, but no reaction center and LH complex B870, is capable of synthesizing the Bchl-binding polypeptide (α, 12 kDa) of B870. In contrast to the high stability of the polypeptides of the B800–850 complex, the 12 kDa polypeptide was rapidly degraded after synthesis and insertion into the membrane.     

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.     

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.     

Effect of the in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer in light-harvesting complex from Rhodobacter sphaeroides 601

The oxidation of bacteriochlorophylls (BChls) in peripheral light-harvesting complexes (LH2) from Rhodobacter sphaeroides was investigated by spectroelectrochemistry of absorption, fluorescence emission, and femtosecond (fs) pump-probe, with the aim obtaining information about the effect of in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer within LH2. The experimental results revealed that: (a) the generation of the BChl radical cation in both B800 and B850 rings dramatically induced bleaching of the characteristic absorption in the NIR region and quenching of the fluorescence emission from the B850 ring for the electrochemical oxidized LH2; (b) the BChl-B850 radical cation might act as an additional channel to compete with the unoxidized BChl-B850 molecules for rapidly releasing the excitation energy, however the B800-B850 energy transfer rate remained almost unchanged during the oxidation process.     

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 B800-850 complex and its dependence on the spectral composition of the light is discussed.     

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).     

Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll alpha-protein complexes.

Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. The material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll alpha and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.     

Comparative studies of two membrane fractions isolated from chemotrophically and phototrophically grown cells of Rhodopseudomonas capsulata.

Light and heavy membrane fractions have been isolated by equilibrium sucrose density centrifugation from Rhodopseudomonas capsulata 938 GCM grown aerobically in the dark (chemotrophically) and anaerobically in the light (phototrophically). The densities of the light and heavy fractions from phototrophic cells were 1.1004 to 1.1006 and 1.1478, respectively, and the densities of the light and heavy fractions from chemotrophic cells were 1.0957 to 1.0958 and 1.1315, respectively. Both fractions were active in photochemical and respiratory functions and in electron transport-coupled phosphorylation. The light membrane fraction isolated from chemotrophic cells contained the reaction center and the light-harvesting pigment-protein complex B 870, but not the variable light-harvesting complex B 800-850. A small amount of the complex B 800-850 was present in the light fraction isolated from phototrophically grown cells, but it was not energetically coupled to the photosynthetic apparatus. From inhibitor studies, difference spectroscopy, and measurement of enzyme activities it was tentatively concluded that the light membrane fraction contains only the reduced nicotinamide adenine dinucleotide-oxidizing electron transport chain having a KCN-insensitive, low-potential cytochrome c oxidase, whereas the heavy fraction contains additionally the succinate dehydrogenase and a high-potential cytochrome b terminal oxidase sensitive to KCN. The light membrane fraction was more labile than the heavy fraction in terms of phosphorylating activity.     

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.     

Oxido-reduction of B800-850 and B880 holochromes isolated from three species of photosynthetic bacteria as studied by electron-paramagnetic resonance and optical spectroscopy

Certain redox properties of bacteriochlorophyll alpha were used to probe the structure of several light-harvesting pigment-protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800-850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll alpha could be oxidized by ferricyanide as witnessed by the bleaching of their near-infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800-850 and the B880 holochromes is that oxidation of the latter gives rise to a g = 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37 mT to 0.48 mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800-850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800-nm and 850-nm bands. This indicates that, contrary to the B880 holochromes, the B800-850 holochromes may not form a homogeneous class.     

Isolation and characterization of bacteriochlorophyll-protein complexes from an aerobic bacterium,Pseudomonas radiora

Bacteriochlorophyll(Bchl)-protein complexes were isolated from a strictly aerobic and facultative methylotrophic bacterium Pseudomonas radiora strain MD-1. They were identified as the reaction center (RC)-B870 and the B870 complexes on the basis of their absorption spectra, light induced spectral changes and polypeptide compositions. The RC-B870 complex of this bacterium showed similar properties to those of typical purple photosynthetic bacteria, and contained c-type cytochrome which was oxidized upon illumination.Abbreviations Bchl bacteriochlorophyll - RC reaction center - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. This material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll a and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.     

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.     

Changes in the content of pigment-protein complexes in Rhodobacter sphaeroides forma sp. denitrificans grown under photosynthetic and photo-denitrifying conditions

Changes in the relative content of pigment-protein complexes, RC-B880 and B800-850, were studied in membranes of Rhodobacter sphaeroides forma sp. denitrificans cultured under various anaerobic conditions. The content of each pigment-protein complex was determined by the decomposition of the absorption spectra of membranes in the near-infrared region into the spectra of RC-B880 and B800-850. The standard spectrum of each complex in the membranes was obtained using two absorption spectra of membranes with different ratios of the complexes by eliminating the spectrum of first one than the other complex. Spectra composed from the two standard spectra were in good agreement with original membrane spectra after subtraction of the contribution of scattering in various membrane samples. Bacteriochlorophyll (BChl) content in the membrane was dependent on the light intensity during growth. The relation between the total BChl content in the membrane and BChl content in the RC-B880 and B800-850 complex was linear above 15 nmol BChl per mg membrane protein, regardless of the culturel conditions, photosynthetic or photo-denitrifying. The linear relationship reached a point where all BChl molecules were contained in RC-B880 at 13 nmol BChl per mg membrane protein. This means that only RC-B880 would be synthesized below the threshold, and above the threshold additional BChl was distributed between RC-B880 and B800-850 in a constant ratio (1:5.7). The results suggest that the syntheses of B800-850 and RC-B880 are not regulated independently.     

固氮红细菌(Rhodobacter azotoforman)色素蛋白复合体的分离纯化与特性

【目的】为揭示不产氧光合细菌产氢菌株色素蛋白复合体(PPC)色素组成和含量与光合放氢的关系奠定基础。【方法】以PPC特征光谱为检测指标,采用硫酸铵分级分离、DEAE-纤维素层析、吸收光谱和SDS-PAGE等方法进行了固氮红细菌(Rhodobacter azotoformans,R.azotoformans)R7产氢菌株PPC的分离纯化、纯度分析和鉴定;采用表面增强激光解吸电离离子飞行时间质谱、HPLC-MS和荧光光谱法对其中一种PPC进行了组成分析和能量传递活性测定。【结果】从R7菌株获得了3种纯化的PPC,1种为反应中心与中心捕光色素蛋白复合体(RC-LH1),2种为外周捕光色素蛋白复合体(LH2),其中一种LH2的吸收光谱具有异常的423nm强吸收峰,其蛋白的两种亚基的分子量分别为5356.8Da和5697.8Da,类胡萝卜素属球形烯系,分子量为562Da,激发光能够从类胡萝卜素向细菌叶绿素以及细菌叶绿素向细菌叶绿素传递。【结论】固氮红细菌产氢菌株含有2种不同光谱特性的LH2,其中一种具有新光谱特性。     

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.     

The crystallographic structure of the B800-820 LH3 light-harvesting complex from the purple bacteria Rhodopseudomonas acidophila strain 7050

The B800-820, or LH3, complex is a spectroscopic variant of the B800-850 LH2 peripheral light-harvesting complex. LH3 is synthesized by some species and strains of purple bacteria when growing under what are generally classed as "stressed" conditions, such as low intensity illumination and/or low temperature (<30 degrees C). The apoproteins in these complexes modify the absorption properties of the chromophores to ensure that the photosynthetic process is highly efficient. The crystal structure of the B800-820 light-harvesting complex, an integral membrane pigment-protein complex, from the purple bacteria Rhodopseudomonas (Rps.) acidophila strain 7050 has been determined to a resolution of 3.0 A by molecular replacement. The overall structure of the LH3 complex is analogous to that of the LH2 complex from Rps. acidophila strain 10050. LH3 has a nonameric quaternary structure where two concentric cylinders of alpha-helices enclose the pigment molecules bacteriochlorophyll a and carotenoid. The observed spectroscopic differences between LH2 and LH3 can be attributed to differences in the primary structure of the apoproteins. There are changes in hydrogen bonding patterns between the coupled Bchla molecules and the protein that have an effect on the conformation of the C3-acetyl groups of the B820 molecules. The structure of LH3 shows the important role that the protein plays in modulating the characteristics of the light-harvesting system and indicates the mechanisms by which the absorption properties of the complex are altered to produce a more efficient light-harvesting component.     

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.