Membrane adhesion in photosynthetic bacterial membranes. Light harvesting complex I (LHI) appears to be the main adhesion factor

We have reconstituted pigment-protein complexes isolated from Rhodopseudomonas palustris photosynthetic membranes into phospholipid liposomes. The various complexes were tested for their ability to promote adhesion of the liposome membrane in the presence and absence of Mg²⁺ ions. Samples containing a reaction center (RC)/light-harvesting I (LHI) complex appeared to stack in a manner resembling control thylakoids in 2 and 5 mM Mg²⁺. We also tested for the effects of Mg²⁺ on detergent extractablity of pigment-protein complexes from intact membranes. Mg²⁺ sharply reduced the amount of LHI solubilized from membranes, while having little effect on the extractability of the light harvesting II complex (LHII) and the RC. Based on these results we suggest that LHI is the principal adhesion factor of R. palustris thylakoids.Abbreviations LHC light harvesting complex - OG octyl glucoside - RC reaction center This paper is dedicated to Professor G. Drews on the occasion of his 60th birthday     

Pleiotropic effects of localized Rhodobacter capsulatus puf operon deletions on production of light-absorbing pigment-protein complexes.

The polycistronic puf operon of Rhodobacter capsulatus encodes protein components for the photosynthetic reaction center and one of the two antenna complexes involved in the capture of light energy. We report here that deletions within specific puf genes alter the synthesis and/or assembly in the photosynthetic membranes of pigment-protein complexes not affected genetically by the deletion. The pufX gene is required for normal ratios of antenna complexes, and its deletion results in an increase of membrane-bound light-harvesting I (LHI) complex-specific proteins. Expression of pufQ in strains deleted for the genes encoding the LHI and the photosynthetic reaction center (RC) yields a novel A868 peak that has not been associated with any of the pigment-protein complexes described previously. While deletions in the RC-coding region resulted in decreased LHI absorbance, no quantitative alteration in membrane-bound LHI protein was observed, suggesting that an intact RC complex is required for correct assembly of LHI in the membrane.     

褐藻裙带菜中PSII复合物及其亚复合物的分离和特性研究

用不连续梯度蔗糖密度超离心,从经TritonX-100增溶的褐藻裙带菜类囊体膜中分离到3种色素蛋白复合物条带,分别是捕光复合物、具有光氧化活性的PSII复合物颗粒(区带II)以及PSI(区带III)。PSII颗粒经毛地黄皂苷增溶后,再次超离心分离得到3条PSII的亚复合物条带。吸收和荧光激发谱显示其中的区带II-1为墨角藻黄素-Chla/c-蛋白复合物,区带II-2为Chla/c-蛋白复合物,两者都只含20kDa多肽;而鲜绿色的区带II-3为不含捕光复合物的活性PSII核心。     

Studies on the expression of the pufX polypeptide and its requirement for photoheterotrophic growth in Rhodobacter sphaeroides.

The puf operon in Rhodobacter sphaeroides contains the genes for the light-harvesting antenna complex I (LHI), the reaction centre (RC) L and M subunits and an additional small open reading frame identified as pufX. It has been demonstrated before that a photosynthetically incompetent pufLMX deletion strain was not complemented by a plasmid-borne truncated puf operon version lacking only pufX, although expression of the pufL and pufM gene products was restored. We demonstrate here that the functional reinsertion of only the pufX open reading frame into the same construct is sufficient and necessary for complementation of the non-photosynthetic phenotype. We also demonstrate that the observed lack of photoheterotrophic growth in the absence of pufX is not the result of decreased light-harvesting ability, but rather the result of an impairment in light-driven cyclic electron transfer. Western blots using polyclonal antibodies against a synthetic peptide corresponding to a portion of the DNA-derived pufX amino acid sequence showed that the pufX open reading frame is expressed and that the gene product has an M(r) of 8-10,000 on SDS gels; a value close to the predicted mass of 9 kDa. The pufX polypeptide was localized to the intracytoplasmic membrane fraction and appeared to co-purify with the RC-LHI complex. It is suggested that the pufX polypeptide is associated with the RC-LHI complex and that it may play a critical role in facilitating the interaction between this complex and other components required for light-driven cyclic electron transfer.     

Possible role of the highly conserved amino acids Trp-8 and Pro-13 in the N-terminal segment of the pigment-binding polypeptide LHI alpha of Rhodobacter capsulatus

Trp-8 and Pro-13 of the Rhodobacter capsulatus light-harvesting (LH) I alpha polypeptide are highly conserved among LHI and LHII alpha proteins of several species of the Rhodospirillaceae. Exchange of Trp-8 and Pro-13 to other amino acyl residues similar in structure and/or hydrophobicity indicates that Trp-8 is involved in the insertion of the LHI alpha polypeptide into the intracytoplasmic membrane (ICM). Pro-13, however, seems not to participate in the integration process of the LHI alpha protein but seems to be important for stable insertion of the LHI beta partner protein in the ICM.     

The light-harvesting complex II (B800-850) of Rhodobacter sulfidophilus: Characterization and formation under different growth conditions

Abstract The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800–850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 μg Bchl per mg cell protein when grown at high light intensity (20 000 lux) to 60 μg Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographie analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of a and one type of β polypeptide in the purified complex, of which the N-terminal sequences have been determined.     

Light-harvesting pigment-protein complexes of Rhodopseudomonas sphaeroides forma sp. denitrificans

The composition of the light-harvesting system of Rhodopseudomonas sphaeroides forma sp. denitrificans was investigated. When chromatophores were solubilized by sodium dodecyl sulfate (SDS) at 0 degrees C and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), at least two B800-B850 pigment-protein complexes, three B870 pigment-protein complexes, a reaction center (RC) complex and two pigmented bands which contained B800, B850, and B870 were resolved. In the re-electrophoresis, the B870 pigment-protein complexes gave rise to a series of multiple pigmented bands. All of these multiple pigment-protein complexes showed almost the same polypeptide composition and absorption spectrum characteristic of the B870 complex. The apparent molecular weights of these B870 complexes showed a regular interval of about 7,000 indicating that these complexes were oligomers of a subunit. It was also found that a predominant B800-B850 pigment-protein complex could be degraded into a small complex via some intermediates. These results indicate that essentially two kinds of pigment-protein complexes construct the light-harvesting system of this bacteria and, upon treatment with SDS, these complexes are degraded into many classes of subunit aggregates showing a complicated profile of pigmented bands on the gel. Pigmented bands which contained both of B800-B850 and B870 complexes were considered to arise from occasional co-migration of distinct B800-B850 and B870 pigment-protein complexes.     

Heterologous synthesis and assembly of functional LHII antenna complexes from <Emphasis Type="Italic">Rhodovulum sulfidophilum</Emphasis> in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> mutant

The light harvesting complexes, including LHII and LHI, are the important components of photosynthetic apparatus. Rhodovulum (Rdv.) sulfidophilum and Rhodobacter (R.) sphaeroides belong to two genera of photosynthetic bacteria, and they are very different in some physiological characteristics and light harvesting complexes structure. The LHII structural genes (pucBsAs) from Rdv. sulfidophilum and the LHI structural genes (pufBA) from R. sphaeroides were amplified, and cloned into an expression vector controlled by puc promoter from R. sphaeroides, which was then introduced into LHI and LHII-minus R. sphaeroides mutants; the transconjugant strains synthesized heterologous LHII and native LHI complexes, which played normal roles in R. sphaeroides. The Rdv. sulfidophilum LHII complex from pucBsAs had near-infrared absorption bands at ~801–853 nm in R. sphaeroides, and was able to transfer energy efficiently to the native LHI complex. The results show that the pucBsAs genes from Rdv. sulfidophilum could be expressed in R. sphaeroides, and the functional foreign LHII and native LHI were assembled into the membrane of R. sphaeroides.     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

Light harvesting in photosystem II

Water oxidation in photosynthesis takes place in photosystem II (PSII). This photosystem is built around a reaction center (RC) where sunlight-induced charge separation occurs. This RC consists of various polypeptides that bind only a few chromophores or pigments, next to several other cofactors. It can handle far more photons than the ones absorbed by its own pigments and therefore, additional excitations are provided by the surrounding light-harvesting complexes or antennae. The RC is located in the PSII core that also contains the inner light-harvesting complexes CP43 and CP47, harboring 13 and 16 chlorophyll pigments, respectively. The core is surrounded by outer light-harvesting complexes (Lhcs), together forming the so-called supercomplexes, at least in plants. These PSII supercomplexes are complemented by some “extra” Lhcs, but their exact location in the thylakoid membrane is unknown. The whole system consists of many subunits and appears to be modular, i.e., both its composition and organization depend on environmental conditions, especially on the quality and intensity of the light. In this review, we will provide a short overview of the relation between the structure and organization of pigment-protein complexes in PSII, ranging from individual complexes to entire membranes and experimental and theoretical results on excitation energy transfer and charge separation. It will become clear that time-resolved fluorescence data can provide invaluable information about the organization and functioning of thylakoid membranes. At the end, an overview will be given of unanswered questions that should be addressed in the near future.     

Isolation and characterization of a subunit form of the light-harvesting complex of Rhodospirillum rubrum

A new method is described for the isolation of subunits of the light-harvesting complex from Rhodospirillum rubrum (wild type and the G-9 mutant) in yields that approach 100%. The procedure involved treating membrane vesicles with ethylenediaminetetraacetic acid-Triton X-100 to remove components other than the light-harvesting complex and reaction center. In the preparation from wild-type cells, a benzene extraction was then employed to remove carotenoid and ubiquinone. The next step involved a careful addition of the detergent n-octyl beta-D-glucopyranoside, which resulted in a quantitative shift of the long-wavelength absorbance maximum from 873 to 820 nm. This latter complex was then separated from reaction centers by gel filtration on Sephadex G-100. The pigment-protein complex, now absorbing at 820 nm, contained two polypeptides of about 6-kilodalton molecular mass (referred to as alpha and beta) in a 1:1 ratio and two molecules of bacteriochlorophyll (BChl) for each alpha beta pair. This complex is much smaller in size than the original complex absorbing at 873 nm but probably is an associated form such as alpha 2 beta 2 X 4BChl or alpha 3 beta 3 X 6BChl. The 820-nm form could be completely shifted back to a form once again having a longer wavelength lambda max near 873 nm by decreasing the octyl glucoside concentration. Thus, the complex absorbing at 820 nm appears to be a subunit form of the original 873-nm complex.     

Structure of a bacterial photosynthetic membrane. Isolation, polypeptide composition, and selective proteolysis

A procedure for the isolation of highly purified bacterial photosynthetic membranes from Rhodopseudomonas viridis is described. The purity of the final membrane fraction has been confirmed by electron microscopy. Seven major polypeptide bands are associated with the photosynthetic membranes, and all seven are resistant to solubilization in Triton X-100 detergent. Two pigmented bands with apparent molecular weights of 44K and 41K are thought to be cytochromes. The three polypeptides with apparent molecular weights of 38K, 32K, and 28K have been reported in reaction center preparations of other laboratories. Two low-molecular-weight (16K and 11K) bands bind bacteriochlorophyll b and may represent light-harvesting bacteriochlorophyll-protein complexes. The structures that were isolated seem to represent complete photosynthetic membranes, consisting of reaction center, electron transport, and light-harvesting components, all arranged in the regular lattice characteristic of viridis. Selective proteolysis of these membranes indicates that all membrane components are accessible to digestion by trypsin and pronase, except for the light-harvesting complexes.     

Effect of modification of histidine residues on organization of the pigment--protein complexes of chromatium minutissimum

The effect of diethyl pyrocarbonate on chromatophores and isolated pigment--protein complexes of Chromatium minutissimum was studied. It is shown that modification of histidine residues results in the destruction of the core antenna LHI (B880) and in a spectral shift from 850 to 830 nm in the peripheral antenna LHII (B800-850). In the purple sulfur bacterium Chromatium minutissimum the pigment--protein complexes B800-B850 (peripheral antenna, LHII) and B880 (core antenna, LHI) collect and transmit the absorbed light energy to the reaction centers. The composition of pigments and proteins as well as primary structure of the majority of polypeptides in both types of complexes from various photosynthetic bacteria have been determined.     

Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803: localization of the CupA subunit of NDH-1

The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2).     

The marginal regions of thylakoid membranes: A partial characterization by polyoxyethylene sorbitan monolaurate (Tween 20) solubilization of spinach thylakoids

The detergent Tween-20 solubilized preferentially portions of the marginal regions of Spinacea oleracea L. thylakoid membranes and, thus, opened the inside of the grana to the external media. Differential centrifugation. following Tween-20 solubilization. enabled separate fractions of grana and stromal-exposed membranes to be isolated. Analysis of Tween-20 solubilized material, after pelleting all membrane material by centrifugation at 100 000 g, revealed polypeptides associated with the coupling factor (CF₁) particles, cytochrome b₆/f and photosystem II complexes, suggesting that the marginal membranes contain these proteins. Concomitantly, the 100 000 g pellet was depleted in cytochrome b₆/f and P700, determined spectroscopically, Thus. our results reveal the margin to be a distinct membrane region, which does not contain the light-harvesting centers of photosystem II (LHC II). The implication of these results, in terms of the energetic interaction of components of granal and stromalexposed membrane regions, is discussed.     

Self-assembling peptide detergents stabilize isolated photosystem I on a dry surface for an extended time

We used a class of designed peptide detergents to stabilize photosystem I (PS-I) upon extended drying under N2 on a gold-coated-Ni-NTA glass surface. PS-I is a chlorophyll-containing membrane protein complex that is the primary reducer of ferredoxin and the electron acceptor of plastocyanin. We isolated the complex from the thylakoids of spinach chloroplasts using a chemical detergent. The chlorophyll molecules associated with the PS-I complex provide an intrinsic steady-state emission spectrum between 650 and 800 nm at -196.15 degrees C that reflects the organization of the pigment-protein interactions. In the absence of detergents, a large blue shift of the fluorescence maxima from approximately 735 nm to approximately 685 nm indicates a disruption in light-harvesting subunit organization, thus revealing chlorophyll-protein interactions. The commonly used membrane protein-stabilizing detergents, N-dodecyl-beta-D-maltoside and N-octyl-beta-D-glucoside, only partially stabilized the approximately 735-nm complex with approximately 685-nm spectroscopic shift. However, prior to drying, addition of the peptide detergent acetyl-AAAAAAK at increasing concentration significantly stabilized the PS-I complex. Moreover, in the presence of acetyl-AAAAAAK, the PS-I complex is stable in a dried form at room temperature for at least 3 wk. Another peptide detergent, acetyl-VVVVVVD, also stabilized the complex but to a lesser extent. These observations suggest that the peptide detergents may effectively stabilize membrane proteins in the solid-state. These designed peptide detergents may facilitate the study of diverse types of membrane proteins.     

Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes

Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It utilizes light for photochemical energy conversion, and is heavily involved in the regulation of the energy flow. We investigated the structural organization of photosystem II and its associated light-harvesting antenna by electron microscopy, multivariate statistical analysis, and classification procedures on partially solubilized photosystem II membranes from spinach. Observation by electron microscopy shortly after a mild disruption of freshly prepared membranes with the detergent n-dodecyl-alpha,D-maltoside revealed the presence of several large supramolecular complexes. In addition to the previously reported supercomplexes [Boekema, E. J., van Roon, H., and Dekker, J. P. (1998) FEBS Lett. 424, 95-99], we observed complexes with the major trimeric chlorophyll a/b protein (LHCII) in a third, L-type of binding position (C2S2M0-2L1-2), and two different types of megacomplexes, both identified as dimeric associations of supercomplexes with LHCII in two types of binding sites (C4S4M2-4). We conclude that the association of photosystem II and its associated light-harvesting antenna is intrinsically heterogeneous, and that the minor CP26 and CP24 proteins play a crucial role in the supramolecular organization of the complete photosystem. We suggest that different types of organization form the structural basis for photosystem II to specifically react to changing light and stress conditions, by providing different routes of excitation energy transfer.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.