Study of the structure of the photosynthetic reaction center of the green thermophilic bacteria Chloroflexus aurantiacus

Analysis of the Chloroflexus aurantiacus reaction centre (RC) using both protein and recombinant DNA techniques resulted in determination of its polypeptide composition and the primary structures of its two subunits. A model of the polypeptide chains' folding in the membrane is suggested based on: i) homology between L- and M-subunits of Chloroflexus aurantiacus RC and their counterparts in purple bacteria; ii) comparison of their hydropathy plots, and iii) data on the tertiary structures of purple bacteria RCs. The role of a number of functionally important amino acid residues in the RC electron transport activity is discussed. Limited proteolysis of the RC under non-denaturing conditions was used to determine the contribution of the N-terminal regions to its thermal stability.     

Structural and spectroscopic properties of a reaction center complex from the chlorosome-lacking filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii

The photochemical reaction center (RC) complex of Roseiflexus castenholzii, which belongs to the filamentous anoxygenic phototrophic bacteria (green filamentous bacteria) but lacks chlorosomes, was isolated and characterized. The genes coding for the subunits of the RC and the light-harvesting proteins were also cloned and sequenced. The RC complex was composed of L, M, and cytochrome subunits. The cytochrome subunit showed a molecular mass of approximately 35 kDa, contained hemes c, and functioned as the electron donor to the photo-oxidized special pair of bacteriochlorophylls in the RC. The RC complex appeared to contain three molecules of bacteriochlorophyll and three molecules of bacteriopheophytin, as in the RC preparation from Chloroflexus aurantiacus. Phylogenetic trees based on the deduced amino acid sequences of the RC subunits suggested that R. castenholzii had diverged from C. aurantiacus very early after the divergence of filamentous anoxygenic phototrophic bacteria from purple bacteria. Although R. castenholzii is phylogenetically related to C. aurantiacus, the arrangement of its puf genes, which code for the light-harvesting proteins and the RC subunits, was different from that in C. aurantiacus and similar to that in purple bacteria. The genes are found in the order pufB, -A, -L, -M, and -C, with the pufL and pufM genes forming one continuous open reading frame. Since the photosynthetic apparatus and genes of R. castenholzii have intermediate characteristics between those of purple bacteria and C. aurantiacus, it is likely that they retain many features of the common ancestor of purple bacteria and filamentous anoxygenic phototrophic bacteria.     

Excitation energy transfer and trapping dynamics in the core complex of the filamentous photosynthetic bacterium Roseiflexus castenholzii

The light-harvesting core complex of the thermophilic filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii is intrinsic to the cytoplasmic membrane and intimately bound to the reaction center (RC). Using ultrafast transient absorption and time-resolved fluorescence spectroscopy with selective excitation, energy transfer, and trapping dynamics in the core complex have been investigated at room temperature in both open and closed RCs. Results presented in this report revealed that the excited energy transfer from the BChl 800 to the BChl 880 band of the antenna takes about 2?ps independent of the trapping by the RC. The time constants for excitation quenching in the core antenna BChl 880 by open and closed RCs were found to be 60 and 210?ps, respectively. Assuming that the light harvesting complex is generally similar to LH1 of purple bacteria, the possible structural and functional aspects of this unique antenna complex are discussed. The results show that the core complex of Roseiflexus castenholzii contains characteristics of both purple bacteria and Chloroflexus aurantiacus.     

SANS investigation of the photosynthetic machinery of Chloroflexus aurantiacus

Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.     

Supramolecular organization of photosynthetic membrane proteins in the chlorosome-containing bacterium Chloroflexus aurantiacus

The arrangement of core antenna complexes (B808-866-RC) in the cytoplasmic membrane of filamentous phototrophic bacterium Chloroflexus aurantiacus was studied by electron microscopy in cultures from different light conditions. A typical nearest-neighbor center-to-center distance of ~18 nm was found, implying less protein crowding compared to membranes of purple bacteria. A mean RC:chlorosome ratio of 11 was estimated for the occupancy of the membrane directly underneath each chlorosome, based on analysis of chlorosome dimensions and core complex distribution. Also presented are results of single-particle analysis of core complexes embedded in the native membrane.     

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone-10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+QA-) formation. Since the C. aurantiacus RC lacks H-subunit, this subunit was not an obligatory component for electrogenic QB protonation.     

Preliminary Analysis of Lipids and Fatty Acids of Green Bacteria and Chloroflexus aurantiacus

The complex lipids and fatty acids of the seven type species of green bacteria and three strains of Chloroflexus aurantiacus were analyzed. The green bacteria contained lipids that behaved as cardiolipin and phosphatidylglycerol on thin-layer chromatography. They did not contain phosphatidylethanolamine or phosphatidylserine. Similarly, Chloroflexus contained lipids that behaved as phosphatidylglycerol and phosphatidylinositol on thin-layer chromatography and did not contain phosphatidylethanolamine or phosphatidylserine. The green bacteria contained glycolipids I and II of Constantopoulos and Bloch (monogalactosyldiglyceride and a galactose- and rhamnose-containing diglyceride). Chloroflexus exhibited galactose-containing glycolipids that behaved identically with the mono- and digalactosyldiglycerides of spinach on thin-layer chromatography, and each contained galactose as well as at least one other sugar. The fatty acids of both groups of bacteria consisted entirely of saturated and monounsaturated fatty acids. In the green bacteria, myristic, palmitic, and hexadecenoic acids predominated. In Chloroflexus, palmitic, stearic, and oleic acids predominated. The positions of the double bonds in the monounsaturated fatty acids of Chloroflexus indicated synthesis by the anaerobic pathway. The lipid analyses suggest a close relationship between the green bacteria and Chloroflexus and further suggest that these groups of photosynthetic bacteria are more closely related to the blue-green algae than are the purple bacteria.     

The primary structure of cytochrome c-554 from the green photosynthetic bacterium Chloroflexus aurantiacus.

The complete nucleotide sequence of the cytochrome c-554 gene from the green photosynthetic bacterium Chloroflexus aurantiacus has been determined. The derived amino acid sequence showed that the cytochrome precursor protein consists of 414 residues and contains 4-Cys-X-X-Cys-His- heme binding motifs. The only regions of the cytochrome c-554 sequence that were found to be significantly similar to the sequences of cytochromes from other organisms were the heme binding sites. The highest similarity was found with the heme binding segments in the four-heme reaction center cytochrome subunit from the purple photosynthetic bacterium Rhodopseudomonas viridis. The importance of this similarity for the evolutionary relationship between Chloroflexus and the purple bacteria is discussed.     

The complete amino acid sequence of a bacteriochlorophyll a binding polypeptide isolated from the cytoplasmic membrane of the green photosynthetic bacterium Chloroflexus aurantiacus

A polypeptide soluble in organic solvents was isolated from whole membrane fractions of the green thermophilic bacterium Chloroflexus aurantiacus by chromatography on Sephadex LH-60, Whatman DE-32 and Bio Gel P-10. The complete amino acid sequence of this 4.9 kDa polypeptide (44 amino acid residues) was determined. The polypeptide shows a 3-domain structure, similar to the domain structure of the antenna BChI polypeptides of purple photosynthetic bacteria, and sequence homologies (27–39%) to the light-harvesting α-polypeptides of the B870 (890) antenna complexes from purple bacteria. Therefore, the 4.9 kDa polypeptide is designated B(808-866)-α. The typical His residue (conserved His residue identified in all antenna polypeptides of purple bacteria as possible BChI binding site) is found within the hydrophobic domain, which extends from Asn 10 to Leu 30.     

Photosynthetic and phylogenetic primers for detection of anoxygenic phototrophs in natural environments.

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

Photosynthetic reaction centre of Chloroflexus aurantiacus. I. Primary structure of L-subunit

The L-subunit primary structure of the reaction centre from Chloroflexus aurantiacus composed of 310 amino acid residues has been determined by parallel analysis of the protein and corresponding DNA. Significant homology between this protein and L-subunits from reaction centres of purple bacteria is observed. This implies close similarity in the tertiary structure of these proteins.     

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.     

Structure of the H subunit of the photosynthetic reaction center from the thermophilic purple sulfur bacterium, Thermochromatium tepidum Implications for the specific binding of the lipid molecule to the membrane protein complex.

The photosynthetic reaction center (RC) is a transmembrane protein complex that catalyzes light-driven electron transport across the photosynthetic membrane. The complete amino-acid sequence of the H subunit of the RC from a thermophilic purple sulfur bacterium, Thermochromatium tepidum, has been determined for the first time among purple sulfur bacteria. The H subunit consists of 259 amino acids and has a molecular mass of 28 187. The deduced amino-acid sequences of this H subunit showed a significant (40%) degree of identity with those from mesophilic purple nonsulfur bacteria. The determined primary structure of the H subunit was compared with the structures of mesophilic B. viridis and R. sphaeroides based on the three-dimensional structure of the H subunit from T. tepidum, which has been recently determined by X-ray crystallography. One lipid molecule was found in the crystal structure of the T. tepidum RC, and the head group of the lipid appears to be stabilized by the electrostatic interactions with the conserved basic residues in the H subunit. The above comparison has suggested the existence of a lipid-binding site on the molecular surface at which a lipid molecule can interact with the RC in a specific manner.     

Effect of low temperatures on photochemical activity of PS1 reaction centers from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. frozen under illumination

After cooling of Synechocystis sp. photosystem 1 (PS1) reaction centers (RC) to 160 K under illumination most of the photoactive pigment is fixed for a long time in the oxidized state. The same effect is observed in purple bacteria RC. The dark reduction kinetics of PS1 P700 chlorophyll, which still retains its photochemical activity, in these samples was similar to that in samples cooled in the dark. We suggest that the photoinduced charge separation in PS1 RC, as well as in purple bacteria RC, is accompanied by conformational changes that can be fixed in samples cooled under illumination. As a result, the electrons photomobilized in RC cooled under illumination are unable to return backward the process of electron transfer to P700(+) after cessation of actinic illumination. Such irreversible trapping of electrons can take place in different parts of the PS1 RC electron acceptor chain.     

Structural basis of bacterial photosynthetic reaction centers

The photosynthetic reaction center (RC) is the first membrane protein whose three-dimensional structure was revealed at the atomic level by X-ray crystallograph more than fifteen years ago. Structural information about RC made a great contribution to the understanding of the reaction mechanism of the complicated membrane protein complex. High-resolution structures of RCs from three photosynthetic bacteria are now available, namely, those from two mesophilic purple non-sulfur bacteria, Blastochloris viridis and Rhodobacter sphaeroides, and that from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. In addition, a variety of structural studies, mainly by X-ray crystallography, are still being performed to give more detailed insight into the reaction mechanism of this membrane protein. This review deals with structural studies of bacterial RC complexes, and a discussion about the electron transfer reaction between RCs and electron donors is the main focus out of several topics addressed by these structural studies. The structural data from three RCs and their electron donors provided reliable models for molecular recognition in the primary step of bacterial photosynthesis.     

A comparative look at structural variation among RC–LH1 ‘Core’ complexes present in anoxygenic phototrophic bacteria

Photosynthesis Research - All purple photosynthetic bacteria contain RC–LH1 ‘Core’ complexes. The structure of this complex from Rhodobacter sphaeroides, Rhodopseudomonas...     

Kinetics and energetics of electron transfer in reaction centers of the photosynthetic bacterium Roseiflexus castenholzii

The kinetics and thermodynamics of the photochemical reactions of the purified reaction center (RC)-cytochrome (Cyt) complex from the chlorosome-lacking, filamentous anoxygenic phototroph, Roseiflexus castenholzii are presented. The RC consists of L- and M-polypeptides containing three bacteriochlorophyll (BChl), three bacteriopheophytin (BPh) and two quinones (Q(A) and Q(B)), and the Cyt is a tetraheme subunit. Two of the BChls form a dimer P that is the primary electron donor. At 285K, the lifetimes of the excited singlet state, P*, and the charge-separated state P(+)H(A)(-) (where H(A) is the photoactive BPh) were found to be 3.2±0.3 ps and 200±20 ps, respectively. Overall charge separation P*→→ P(+)Q(A)(-) occurred with ≥90% yield at 285K. At 77K, the P* lifetime was somewhat shorter and the P(+)H(A)(-) lifetime was essentially unchanged. Poteniometric titrations gave a P(865)/P(865)(+) midpoint potential of +390mV vs. SHE. For the tetraheme Cyt two distinct midpoint potentials of +85 and +265mV were measured, likely reflecting a pair of low-potential hemes and a pair of high-potential hemes, respectively. The time course of electron transfer from reduced Cyt to P(+) suggests an arrangement where the highest potential heme is not located immediately adjacent to P. Comparisons of these and other properties of isolated Roseiflexus castenholzii RCs to those from its close relative Chloroflexus aurantiacus and to RCs from the purple bacteria are made.     

A new approach to experimental investigation of dynamic self-organization in reaction centers of purple bacteria

The results of a study of molecular self-organization processes in the reaction centers (RC) ofRb. Sphaeroides purple bacteria by the method of pulsed optical excitation is presented. The existence of a bistability domain for the parameters of RC recovery kinetics is shown. A good agreement between the theory and experimental results is obtained.     

The reaction centre from green sulphur bacteria: progress towards structural elucidation

The reaction centre (RC) of green sulphur bacteria is a FeS-type RC, as are the RCs of Photosystems I (PS I) of oxygenic photosynthetic organisms and of heliobacteria. The core domains of both green sulphur bacterial and heliobacterial RCs are considered to be homodimeric, in contrast to those of purple bacteria, PS I and Photosystem II (PS II). This paper briefly describes the techniques of electron microscopy and image processing suited to investigate the structure of these proteins. Recent advances in the study of the structure of the green sulphur bacterial RC, primarily achieved by the application of scanning transmission electron microscopy, are reviewed.This revised version was published online in October 2005 with corrections to the Cover Date.