Bacterial Photosynthetic Apparatus Research Articles

Near-atomic cryo-EM structure of the light-harvesting complex LH2 from the sulfur purple bacterium Ectothiorhodospira haloalkaliphila.

Bacteria with the simplest system for solar energy absorption and conversion use various types of light-harvesting complexes for these purposes. Light-harvesting complex 2 (LH2), an important component of the bacterial photosynthetic apparatus, has been structurally well characterized among purple non-sulfur bacteria. In contrast, so far only one high-resolution LH2 structure from sulfur bacteria is known. Here, we report the near-atomic resolution cryoelectron microscopy (cryo-EM) structure of the LH2 complex from the purple sulfur bacterium Ectothiorhodospira haloalkaliphila, which allowed us to determine the predominant polypeptide composition of this complex and the identification of the most probable type of its carotenoid. Comparison of our structure with the only known LH2 complex from a sulfur bacterium revealed severe differences in the overall ring-like organization. Expanding the architectural universe of bacterial light-harvesting complexes, our results demonstrate that, as observed for non-sulfur bacteria, the LH2 complexes of sulfur bacteria may also exhibit various types of spatial organization.

Differential stability of bacterial photosynthetic apparatus of Rhodobacter alkalitolerans strain JA916T under alkaline and light environment.

In purple bacteria, photosynthesis is performed by densely packed pigment-protein complexes, including the light-harvesting complexes known as RC-LH1 and LH2, with carotenoids to assist in the functioning of photosynthesis. Most photosynthetic bacteria are exposed to various abiotic stresses such as light, temperature, alkalinity-acidity, and salinity. Rhodobacter (R.) alkalitolerans was discovered from the alkaline pond; here, we report the comparative study of the photosynthetic apparatus of R. alkalitolerans in various light intensities in relation to its high pH tolerance ability. With increased light intensity, the stability of photosystem complexes decreased in normal pH (npH pH 6.80 ± 0.05) conditions, whereas in high pH (hpH pH 8.60 ± 0.05), acclimation was observed to high light. The content of bacteriochlorophyll a, absorbance spectra, and circular dichroism data shows that the integrity of photosystem complexes is less affected in hpH compared with npH conditions. Large pore blue native polyacrylamide gel electrophoresis of photosystem protein complexes and sucrose density gradient of n-dodecyl β-D-maltoside solubilized intracytoplasmic membranes show that LH2 is more affected in npH than in hpH, whereas RC-LH1 monomer or dimer has shown interplay between monomer and dimer in hpH, although the dimer and monomer both increased in npH. Increased content and expression level of ATPase protein complex and subunit-"c" of ATPase, fast relaxation kinetics of p515, and relatively higher membrane lipid content in hpH along with less photooxidative stress and subsequently lesser superoxide dismutase activity exemplify photoprotection in hpH. Furthermore, the increased expression levels of antiporter NhaD in hpH signify its role in the maintenance of homeostatic balance in hpH.

Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity

In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.

Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity

In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.

Forces guiding assembly of light-harvesting complex 2 in native membranes

Interaction forces of membrane protein subunits are of importance in their structure, assembly, membrane insertion, and function. In biological membranes, and in the photosynthetic apparatus as a paradigm, membrane proteins fulfill their function by ensemble actions integrating a tight assembly of several proteins. In the bacterial photosynthetic apparatus light-harvesting complexes 2 (LH2) transfer light energy to neighboring tightly associated core complexes, constituted of light-harvesting complexes 1 (LH1) and reaction centers (RC). While the architecture of the photosynthetic unit has been described, the forces and energies assuring the structural and functional integrity of LH2, the assembly of LH2 complexes, and how LH2 interact with the other proteins in the supramolecular architecture are still unknown. Here we investigate the molecular forces of the bacterial LH2 within the native photosynthetic membrane using atomic force microscopy single-molecule imaging and force measurement in combination. The binding between LH2 subunits is fairly weak, of the order of k(B)T, indicating the importance of LH2 ring architecture. In contrast LH2 subunits are solid with a free energy difference of 90 k(B)T between folded and unfolded states. Subunit α-helices unfold either in one-step, α- and β-polypeptides unfold together, or sequentially. The unfolding force of transmembrane helices is approximately 150pN. In the two-step unfolding process, the β-polypeptide is stabilized by the molecular environment in the membrane. Hence, intermolecular forces influence the structural and functional integrity of LH2.

Atomic force microscopy of the bacterial photosynthetic apparatus: plain pictures of an elaborate machinery

Photosynthesis both in the past and present provides the vast majority of the energy used on the planet. The purple photosynthetic bacteria are a group of organisms that are able to perform photosynthesis using a particularly simple system that has been much studied. The main molecular constituents required for photosynthesis in these organisms are a small number of transmembrane pigment-protein complexes. These are able to function together with a high quantum efficiency (about 95%) to convert light energy into chemical potential energy. While the structure of the various proteins have been solved for several years, direct studies of the supramolecular assembly of these complexes in native membranes needed maturity of the atomic force microscope (AFM). Here, we review the novel findings and the direct conclusions that could be drawn from high-resolution AFM analysis of photosynthetic membranes. These conclusions rely on the possibility that the AFM brings of obtaining molecular resolution images of large membrane areas and thereby bridging the resolution gap between atomic structures and cellular ultrastructure.

Biohybrid Photoelectrochemical Nanoengineered Interfaces

AbstractIncorporation of biophotonic components in artificial devices is an emerging trend in exploring biomimetic approaches for green technologies. In this study, highly efficient, nanoscaled light antenna structures from green photosynthetic bacteria, known as chlorosomes, comprised of bacteriochlorophyll-c pigment arrays that are stable in aqueous environments are studied in an electrochemical environment for their photoelectrogenic capacity. Biohybrid electrochemical cells containing chlorosomes coupled to the native bacterial photosynthetic apparatus have a higher dark charge storage density (at least 10-fold) than electrochemical cells with decoupled chlorosomes. Nevertheless, upon light stimulation, the charge storage density, also known as charge injection capacity, for both electrochemical systems increased the charge stored near the electrode. Decoupled chlorosome-based systems showed a light-intensity dose-dependent response reaching a maximum change of ˜300 nC/cm2at near sunlight intensities (˜80-100mW/cm2). Chronoamperometric studies under light stimulation conditions confirmed the photo-induced effect. Current studies are focused on optimization of the electrode/chlorosome interfacial properties across various heterogeneous interfaces. Successful implementation of harvesting photo-energy using the chlorosome or its derivatives may lead to substantial innovations in current biophotonic technologies, such as biofuel cells and retinal prosthetics.

Membrane insertion of Rhodopseudomonas acidophila light harvesting complex 2 investigated by high resolution AFM

Light harvesting complexes 2 (LH2) are the peripheral antenna proteins in the bacterial photosynthetic apparatus and are built of α/β-heterodimers containing three bacteriochlorophylls and two carotenoids each. Previously, we have found in 2D-crystals that the complexes could be inserted within the membrane with a tilt with respect to the membrane plane ( Rhodobacter sphaeroides) or without tilt ( Rubrivivax gelatinosus). To investigate whether the tilted insertion represents the native state or if it is due to specific 2D-crystal contacts, we have used atomic force microscopy to investigate LH2 from Rhodopseudomonas acidophila reconstituted at different lipid to protein ratios. High-resolution topographs could be acquired of two types of 2D-crystals or of densely packed membranes. Interestingly, in type 2 2D-crystals and in non-crystalline densely packed membranes, cylinders are integrated with their symmetry axis normal to the membrane plane, while in type 1 2D-crystals LH2 cylinders are integrated with a tilt of ∼4° with respect to the membrane plane. Therefore, we present strong evidence that the tilt of LH2 does not represent the native membrane state and is due to protein–protein contacts in specific 2D-crystals.

Watching the photosynthetic apparatus in native membranes.

Over the last 9 years, the structures of the various components of the bacterial photosynthetic apparatus or their homologues have been determined by x-ray crystallography to at least 4.8-A resolution. Despite this wealth of structural information on the individual proteins, there remains an urgent need to examine the architecture of the photosynthetic apparatus in intact photosynthetic membranes. Information on the arrangement of the different complexes in a native system will help us to understand the processes that ensure the remarkably high quantum efficiency of the system. In this work we report images obtained with an atomic force microscope of native photosynthetic membranes from the bacterium Rhodospirillum photometricum. Several proteins can be seen and identified at molecular resolution, allowing the analysis and modeling of the lateral organization of multiple components of the photosynthetic apparatus within a native membrane. Analysis of the distribution of the complexes shows that their arrangement is far from random, with significant clustering both of antenna complexes and core complexes. The functional significance of the observed distribution is discussed.

High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2.

Light-harvesting complexes 2 (LH2) are the accessory antenna proteins in the bacterial photosynthetic apparatus and are built up of alphabeta-heterodimers containing three bacteriochlorophylls and one carotenoid each. We have used atomic force microscopy (AFM) to investigate reconstituted LH2 from Rubrivivax gelatinosus, which has a C-terminal hydrophobic extension of 21 amino acids on the alpha-subunit. High-resolution topographs revealed a nonameric organization of the regularly packed cylindrical complexes incorporated into the membrane in both orientations. Native LH2 showed one surface which protruded by approximately 6 A and one that protruded by approximately 14 A from the membrane. Topographs of samples reconstituted with thermolysin-digested LH2 revealed a height reduction of the strongly protruding surface to approximately 9 A, and a change of its surface appearance. These results suggested that the alpha-subunit of R.gelatinosus comprises a single transmembrane helix and an extrinsic C-terminus, and allowed the periplasmic surface to be assigned. Occasionally, large rings ( approximately 120 A diameter) surrounded by LH2 rings were observed. Their diameter and appearance suggest the large rings to be LH1 complexes.

Light-harvesting mechanisms in purple photosynthetic bacteria

The processes by which photosynthetic bacteria capture light and transfer the energy to the reaction centre continue to be studied using an array of methodologies, both physical and biological. With the publication this year of the crystal structure of the LH2 complex from Rhodopseudomonas acidophila and the projection structure of the LH1 complex from Rhodospirillum rubrum, structural models now exist for all the components in the bacterial photosynthetic apparatus.

23S rRNA processing in Rhodobacter capsulatus is not involved in the oxygen-regulated formation of the bacterial photosynthetic apparatus

23S rRNA processing in Rhodobacter capsulatus is not involved in the oxygen-regulated formation of the bacterial photosynthetic apparatus

23S rRNA processing in Rhodobacter capsulatus is not involved in the oxygen-regulated formation of the bacterial photosynthetic apparatus

The chemical mutant strain Fm65 differs from wild-type strains of Rhodobacter capsulatus in several respects: (a) Fm65 does not form photosynthetic complexes because it does not synthesize bacteriochlorophyll; (b) the level of puf mRNA encoding pigment binding proteins increases little after reduction of oxygen; (c) Fm65 does not process 23S rRNA into 14S and 16S rRNA species, and as a consequence of altered rRNA processing, has lower growth rates. Here we show that transfer of wild-type bchBN and bchF genes into Fm65 restores normal oxygen regulation of the formation of photosynthetic complexes, but not rRNA processing. We conclude that the processing of 23S rRNA to 16S and 14S rRNA species in wild-type strains is not involved in the regulation of the photosynthetic apparatus formation. Abnormal oxygen regulation of puf transcription in strain Fm65 could be attributed to the defect in bacteriochlorophyll synthesis, suggesting that porphyrins are directly or indirectly involved in oxygen regulation of the synthesis of pigment binding proteins.

Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides.

Carotenoids have two major functions in bacterial photosynthesis, photoprotection and accessory light harvesting. The genes encoding many carotenoid biosynthetic pathways have now been mapped and cloned in several different species, and the availability of cloned genes which encode the biosynthesis of carotenoids not found in the photosynthetic genus Rhodobacter opens up the possibility of introducing a wider range of foreign carotenoids into the bacterial photosynthetic apparatus than would normally be available by producing mutants of the native biosynthetic pathway. For example, the crt genes from Erwinia herbicola, a gram-negative nonphotosynthetic bacterium which produces carotenoids in the sequence of phytoene, lycopene, beta-carotene, beta-cryptoxanthin, zeaxanthin, and zeaxanthin glucosides, are clustered within a 12.8-kb region and have been mapped and partially sequenced. In this paper, part of the E. herbicola crt cluster has been excised and expressed in various crt strains of Rhodobacter sphaeroides. This has produced light-harvesting complexes with a novel carotenoid composition, in which the foreign carotenoids such as beta-carotene function successfully in light harvesting. The outcome of the combination of the crt genes in R. sphaeroides with those from E. herbicola has, in some cases, resulted in an interesting rerouting of the expected biosynthetic sequence, which has also provided insights into how the various enzymes of the carotenoid biosynthetic pathway might interact. Clearly this approach has considerable potential for studies on the control and organization of carotenoid biosynthesis, as well as providing novel pigment-protein complexes for functional studies.

The use of non-denaturing Deriphat-polyacrylamide gel electrophoresis to fractionate pigment-protein complexes of purple bacteria

The suitability of Deriphat-polyacrylamide gel electrophoresis as a method for separating purple bacterial pigment-protein complexes has been tested. When appropriate non-denaturing detergents are used to solubilize chromatophores, this method provides a rapid, easy and microscale procedure for analyzing the composition of the bacterial photosynthetic apparatus with minimal disruption of individual pigment-proteins. Its usefulness is further illustrated by employing it to test for suitable detergents with which to solubilize purple bacterial chromatophores, and as an assay to study variation in the composition of the photosynthetic unit of bacterial cultures grown under different conditions.

Theory of protein-induced lateral phase separation in lipid membranes

An account is given of the current status of theoretical modeling of the phase equilibria in lipid membranes with intrinsic proteins. Special attention is paid to the description of lateral phase separation, which is important for membrane function since it may lead to biologically differentiated regions. We discuss in particular the mattress-model approach by Mouritsen and Bloom, who take matching between protein and lipid hydrophobic thicknesses as a determining factor for the phase behavior. The model has been developed in the framework of phenomenological thermodynamic solution theory. The predictions of the theory are compared to a variety of experimental measurements, including those of membrane recombinants of the protein content of the reaction center and antenna protein of the bacterial photosynthetic apparatus as well as the erythrocyte band 3 protein. The physical effects of lipid-protein interactions are contrasted to those of lipid-cholesterol interactions. The concept of hydrophobic matching is then used as a basis for discussing a possible relationship between membrane thickness and physiological function.

A Genetic System for Rapidly Assessing Herbicides That Compete for the Quinone Binding Site of Photosynthetic Reaction Centers

To facilitate genetic analysis and manipulation of the bacterial photosynthetic apparatus, a new plasmid vector bearing the reaction center and light harvesting structural genes (puf operon) of Rhodobacter capsulatus was constructed. This plasmid (pU2922) can be conjugated from E. coli strains (tra+) to Rb. capsulatus with high frequency and shows improved fidelity and stability over Co1E1 derivatives. The puf genes may be expressed in trans from pU2922 in Rb. capsulatus deletion backgrounds to assay the effects of directed mutations or to isolate plasmid–borne spontaneous mutations. We have used this genetic system to characterize a total of 27 mutations at five sequence positions in the secondary quinone–binding domain of the reaction center, where six different herbicides assayed in this study act as competitive inhibitors. The broad spectrum of herbicide response ranging from increased resistance to increased sensitivity that is observed in these engineered bacterial strains may provide a sensitive indicator of the interactions of a given herbicide with the binding site. This collection should prove useful for screening new derivatives for their herbicidal activity and for understanding the chemistry of herbicide binding.

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.

Quantitative analysis of membrane distortions induced by mismatch of protein and lipid hydrophobic thickness

The phase transitional behaviour of bilayers of the phospholipid l-α-ditridecanoylphos-phatidylcholine is studied as a function of protein content for the reaction center (RC) and an antenna protein (LHCP) of the bacterial photosynthetic apparatus. As membrane and protein are structurally well characterized the experimental results can be quantitatively compared with those of calculations based upon elastic models within the Landaude Gennes-theory. Agreement between theory and experiment demonstrates that dominant elastic forces result from a mismatch of hydrophobic regions of membrane and protein. The data also indicate that RC are present in a monomeric form and LHCP in a highly aggregated form. In addition, the latter protein responds to changes in the lipid environment.

Fluorescence quantum yields, lifetimes, and critical distances for energy transfer for chlorophyll ? and its pheophytin in solutions

Fluorescence properties of bacteriochlorophyll are essential for understanding its two most important functions in the bacterial photosynthetic apparatus: transformation of the light energy into the energy of separated charges in the reaction centers and transfer of the excitation energy [i, 2]. In both cases the fluorescence competes either with the electron transfer process or with the electronic energy transfer. Data on fluorescence parameters of bacteriochlorophyll pigments are rather sparse. It has been demonstrated [3] that the fluorescence lifetime data on BChl a are erroneous in many cases and describe a product of BChl a degradation. Data on the BChl a quantum yield (Q) are even poorer. The values of Q are estimated [3] for a number of solvents from the relation Q = T/Tnat, where Tnat is the natural fluorescence lifetime determined from the So § $I absorption band. A shortcoming of such approach is the necessity of knowing an exact value of the molar extinction coefficient in the band maximum as well as the spectral limits of the band. There is also an unresolved problemof other electronic transitions or some impurity bands within the limits of the So § $I transition for BChl a and Chl a. Nevertheless, the variations in T and Q in different solvents are attributed to changes in transition probability and the possibility of internal conversion is discussed [3] without any strong experimental support. Another possible approach is to measure Q directly. The aim of this work is to measure fluorescence quantum spectra, determine the fluorescencequantum yields in a number of solvents, andto measure the fluorescence lifetimes under the same conditions. The experimentally determined Q and T values can be used for determining Tnat without knowing the extinction coefficients and without making any arbitrary assumptions and complicated calculations which introduce additional errors. The determination of the fluorescence quantum spectra for BChl a and BPh a has become a basis for calculating a number of parameters which characterize inductive resonance interactions in the pigments: the overlap integrals between the absorption and fluorescence spectra ~nd the critical distances Ro for the energy transfer. A comparison of the critical distances for energy transfer for BChl a and Chl a has been also conducted.