Amino acid sequence of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis

The complete nucleotide sequence of the gene encoding the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequence are presented. The nucleotide sequence of the gene reveals the existence of a typical bacterial signal peptide of 20 amino acid residues which is not found in the mature cytochrome subunit. The gene encoding the cytochrome subunit is preceded by the gene encoding the M subunit. Both genes overlap by 1 bp. The mature cytochrome subunit consists of 336 amino acid residues; 73% of its amino acid sequence was confirmed by protein sequencing work. The mol. wt of the cytochrome subunit including the covalently bound fatty acids and the bound heme groups is 40 500. The internal sequence homology is low, despite the symmetric structure of the cytochrome subunit previously shown by X-ray crystallographic analysis of the intact photosynthetic reaction centre. Sequence homologies to other cytochromes were not found.     

The photosynthetic reaction centre from the purple bacteriumRhodopseudomonas viridis

We first describe the history and methods of membrane protein crystallization, and show how the structure of the photosynthetic reaction centre from the purple bacteriumRhodopseudomonas viridis was solved. The structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure.Published inLes Prix Nobel—The Nobel Prizes 1988 (Nobel Foundation, Stockholm, 1989) and republished here with the permission of the Nobel Foundation the copyright holders.     

Pigment-protein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis

An X-ray structure analysis of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis provides structural details of the pigment-binding sites. The photosynthetic pigments are found in rather hydrophobic environments provided by the subunits L and M. In addition to apolar interactions, the bacteriochlorophylls of the primary electron donor (`special pair') and the bacteriopheophytins, but not the accessory bacteriochlorophylls, form hydrogen bonds with amino acid side chains of these protein subunits. The two branches of pigments which originate at the primary electron donor, and which mark possible electron pathways across the photosynthetic membrane, are in different environments and show different hydrogen bonding with the protein: this may help to understand why only one branch of pigments is active in the light-driven electron transfer. The primary electron acceptor, a menaquinone (Q_A), is in a pocket formed by the M subunit and interacts with it by hydrophobic contacts and hydrogen bonds. Competitive inhibitors of the secondary quinone Q_B (o-phenanthroline, the herbicide terbutryn) are bound into a pocket provided by the L subunit. Apart from numerous van der Waals interactions they also form hydrogen bonds to the protein.     

Three-dimensional crystals of a membrane protein complex: The photosynthetic reaction centre from Rhodopseudomonas viridis

Reaction centres of photosynthesis isolated from the photosynthetic bacterium Rhodopseudomonas viridis by a single step of molecular sieve chromatography were crystallized. The isolated and the crystallized reaction centres contain four different protein subunits, including a membrane-bound cytochrome. Crystallization was achieved by salt precipitation using 2 to 3 m-ammonium sulphate as a precipitating agent in the presence of N,N-dimethyldodecylamine N-oxide as detergent and heptane-1,2,3-triol. Large tetragonal crystals of space group P4₁2₁2 or its enantiomorph diffracting to beyond 2.5 Å were obtained.     

Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.     

Study of QB- stabilization in herbicide-resistant mutants from the purple bacterium Rhodopseudomonas viridis

The pH dependences of the rate constants of P+QB- (kBP) and P+QA- (kAP) charge recombination decays have been studied by flash-induced absorbance change technique, in chromatophores of three herbicide-resistant mutants from Rhodopseudomonas (Rps.) viridis, and compared to the wild type. P, QA, and QB are the primary electron donor and the primary and the secondary quinone acceptors, respectively. The triazine resistant mutants T1 (Arg L217----His and Ser L223----Ala), T3 (Phe L216----Ser and Val M263----Phe), and T4 (Tyr L222----Phe), all mutated in the QB binding pocket of the reaction center, have previously been characterized (Sinning, I., Michel, H., Mathis, P., & Rutherford, A. W. (1989) Biochemistry 28, 5544-5553). The pH dependence curves of kBP in T4 and the wild type are very close. This confirms that the sensitivity toward DCMU of T4 is mainly due to a structural rearrangement in the QB pocket rather than to a change in the charge distribution in this part of the protein. In T3, a 6-fold increase of kAP is observed (kAP = 4200 +/- 300 s-1 at pH 8) compared to that of the wild type (kAP = 720 +/- 50 s-1 at pH 8). We propose that the Val M263----Phe mutation induces a free energy decrease between P+QA- and P+I- (delta G zero IA) (I is the primary electron acceptor) of about 49 meV. The very different pH dependence of kAP in T3 suggests a substantial change in the QA pocket. The 2.5 times increase of kAP above pH 9.5 in the wild type is no longer detected in T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitrogen fixation and ammonia switch-off in the photosynthetic bacterium Rhodopseudomonas viridis.

Rhodopseudomonas viridis ATCC 19567 grows by means of nitrogen fixation in yeast extract-N2 or nitrogen-free medium when sparged with 5% CO2 and 95% N2 in the light at 30 degrees C. Acetylene reduction assays for nitrogenase activity revealed an initially high level of activity during early-logarithmic growth phase, a lower plateau during mid- to late-logarithmic phase, and a dramatic reduction of activity at the beginning of the stationary phase. When viewed by electron microscopy, nitrogen-fixing R. viridis cells appeared to be morphologically and ultrastructurally similar to cells grown on nitrogen-rich media. Whole cells prepared under reducing conditions in the dark for electron spin resonance spectroscopy yielded g4.26 and g3.66 signals characteristic of the molybdenum-iron protein of nitrogenase. During growth on N2 in the absence of fixed-nitrogen sources, the nitrogenase activity of R. viridis measured by acetylene reduction stopped rapidly in response to the addition of NH4Cl as has been observed in other Rhodospirillaceae. However, unlike the nitrogenase of Rhodopseudomonas palustris or Rhodospirillum rubrum, which recover from this treatment within 40 min, the nitrogenase activity of R. viridis was not detectable for nearly 4 h.     

Localisation of reaction centre and light harvesting complexes in the photosynthetic unit of Rhodopseudomonas viridis

The photosynthetic unit of Rhodopseudomonas viridis contains a reaction centre (P960) and a light harvesting complex (B1015). Immune electron microscopy combined with image processing has allowed the central core of the photosynthetic unit to be identified as the reaction centre and the surrounding protein ring as the light harvesting complex. This light harvesting complex, subdivided into twelve subunits was shown to contain 24 bacteriochlorophyll b molecules. A model is presented which may account for the far red shift of the Qy absorption of the bacteriochlorophyll b molecules in vivo.     

Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microaerophilic growth and induction of the photosynthetic reaction center in Rhodopseudomonas viridis.

Rhodopseudomonas viridis was grown in liquid culture at 30 degrees C anaerobically in light (generation time, 13 h) and under microaerophilic growth conditions in the dark (generation time, 24 h). The bacterium could be cloned at the same temperature anaerobically in light (1 week) and aerobically in the dark (3 to 4 weeks) if oxygen was limited to 0.1%. Oxygen could not be replaced by dimethyl sulfoxide, potassium nitrate, or sodium nitrite as a terminal electron acceptor. No growth was observed anaerobically in darkness or in the light when air was present. A variety of additional carbon sources were used to supplement the standard succinate medium, but enhanced stationary-phase cell density was observed only with glucose. Conditions for induction of the photosynthetic reaction center upon the change from microaerophilic to phototrophic growth conditions were investigated and optimized for a mutant functionally defective in phototrophic growth. R. viridis consumed about 20-fold its cell volume of oxygen per hour during respiration. The MICs of ampicillin, kanamycin, streptomycin, tetracycline, 1-methyl-3-nitro-1-nitrosoguanidine, and terbutryn were determined.     

Primary structure of a high potential iron-sulfur protein from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas gelatinosa.

The third amino acid sequence of a high potential iron-sulfur protein, that of the non-sulfur purple photosynthetic bacterium Rhodopseudomonas gelatinosa, has been determined. It consists of a single polypeptide chain of 74 amino acid residues, which is slightly smaller than the high potential iron-sulfur proteins from the sulfur purple bacteria Chromatium vinosum (85 residues) and Thiocapsa pfennigii (81 residues). The sequence of the gelatinosa protein is similar to the C. vinosum and T. pfennigii proteins with 38% and 37% identically matching residues, although six gaps are proposed for the comparison (the C. vinosum and T. pfennigii proteins have 44% identically matching residues out of 73 positions compared with only one 4-residue gap). Only 17 redisues, including the 4 cystein residues essential for binding the four-iron-sulfur chromophore, are invariant in the three known sequences. A discussion of the role of conserved residues in maintenance of the three-dimensional structure and in electron transport is presented.     

The ;heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the gene, nucleotide and amino acid sequence

The gene coding for the `heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis was isolated in an expression vector. Expression of the heavy subunit in Escherichia coli was detected with antibodies raised against crystalline reaction centres. The entire subunit, and not a fusion protein, was expressed in E. coli. The protein coding region of the gene was sequenced and the amino acid sequence derived. Part of the amino acid sequence was confirmed by chemical sequence analysis of the protein. The heavy subunit consists of 258 amino acids and its mol. wt. is 28 345. It possesses one membrane-spanning α-helical segment, as was revealed by the concomitant X-ray structure analysis.     

The 'light' and 'medium' subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the genes, nucleotide and amino acid sequence

The 'light' (L) and the 'medium' (M) subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis were isolated and their amino-terminal sequences, as well as the sequences of several chymotryptic peptides, determined. Rps. viridis DNA was cloned in the Escherichia coli plasmid pBR322. Mixed oligonucleotide probes derived from the amino acid sequences were synthesized and utilised to isolate one clone which contained the genes for the L and M subunits of the reaction centre as well as the alpha and beta subunits of the light-harvesting complex and part of the gene for the reaction centre cytochrome. The nucleotide sequences of the L and M subunit genes and teh derived amino acid sequences are presented. The L subunit consists of 273 amino acids and has a mol. wt of 30 571. The M subunit consists of 323 amino acids and has a mol. wt of 35 902. The primary structure is discussed in the light of the recently published secondary and tertiary structure which has shown that both subunits contain five membrane-spanning helices.     

A system for site-specific mutagenesis of the photosynthetic reaction center in Rhodopseudomonas viridis.

The purple non-sulfur bacterium Rhodopseudomonas viridis contains a photosynthetic reaction center which has been structurally resolved to 2.3 A providing a unique basis for the study of biological electron transfer processes by the method of site-specific mutagenesis. Here we report the construction of a puf operon deleted mutant strain incapable of photosynthetic growth. The deletion was introduced with the help of a newly constructed suicide vector by electroporation which is with conjugation another gene transfer system for R. viridis. The deletion strain was complemented by conjugational gene transfer with wild-type (WT) and mutated LM genes of the puf operon. The complemented WT and mutations YL162F and HL153F grew photosynthetically, expressed and assembled the four subunits L, M, H and Cyt c of the reaction center correctly. These first mutations already demonstrate the value of the R. viridis system for a detailed structure-function analysis of photosynthetic electron transfer.     

Heterogeneous profile in thermal denaturation of DNA preparation from the non-sulfur purple photosynthetic bacterium Rhodopseudomonas spheroides

Thermal denaturation profiles of DNA preparations from fourstrains of Rhodopseudomonas spheroides were comparatively studied.All the melting curves in 0.1 ? SSC displayed Tm at 82 ?0.5?and a faint bimodal transition near 78?. The differential meltingrates as a function of temperature exhibited several peaks,suggesting that inter- or intra-molecular structural heterogeneitymay be present in R. spheroides DNA. ¹ Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Japan. (Received February 15, 1975; )     

Composition and localization of bacteriochlorophyll a intermediates in the purple photosynthetic bacterium Rhodopseudomonas sp. Rits

Rhodopseudomonas sp. Rits is a recently isolated new species of photosynthetic bacteria and found to accumulate a significantly high amount of bacteriochlorophyll (BChl) a intermediates possessing non-, di- and tetra-hydrogenated geranylgeranyl groups at the 17-propionate as well as normal phytylated BChl a (Mizoguchi T et al. (2006) FEBS Lett 580:137-143). A phylogenetic analysis showed that this bacterium was closely related to Rhodopseudomonas palustris. The strain Rits synthesizes light-harvesting complexes 2 and 4 (LH2/4), as peripheral antennas, as well as the reaction center and light-harvesting 1 core complex (RC-LH1 core). The amounts of these complexes were dependent upon the incident light intensities, which was also a typical behavior of Rhodopseudomonas palustris. HPLC analyses of extracted pigments indicated that all four BChls a were associated with the purified photosynthetic pigment-protein, as complexes described above. The results suggested that this bacterium could use these pigments as functional molecules within the LH2/4 and RC-LH1 core. Pigment compositional analyses in several purple photosynthetic bacteria showed that such BChl a intermediates were always detected and were more widely distributed than expected. Long chains in the propionate moiety of BChl a would be one of the important factors for assembly of LH systems in purple photosynthetic bacteria.     

The binding of triazine herbicides to the photosynthetic reaction center of Rhodopseudomonas viridis. Energy minimization studies.

The binding of six herbicides of the triazine family to the photosynthetic reaction center of Rhodopseudomonas viridis was investigated with energy-minimization techniques, in order to correlate experimental with calculated data. The inhibitors were modeled in the active site according to the X-ray structure analysis of the complex formed between the triazine terbutryn (2-ethylamino-4-t-butylamino-6-methylthio-s-triazine) and the reaction center of R. viridis [Michel, H., Epp. O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. 40 different energy minimizations were carried out with varying cutoff radii, partial charges on inhibitor atoms and dielectric constants, i.e. 10 different combinations of these were tested. The impact of these parameters on the calculated binding and interaction energy was either examined for all protein/triazine complexes or, in the case of the dielectric constant, a smaller sample was used. The calculated energies are dominated by van der Waals interactions, which change by up to 20% when extending the cutoff radius from 0.8 nm to 1.5 nm. The use of uniform or distance-dependent dielectric constant or partial charges on the inhibitor atoms does not severely influence the resulting structures, but shows a great impact on the calculated energies. In the two groups of triazines, each containing three inhibitors with methoxy or methylthio substituents, correlations of biological and calculated data were found quite often, but only once with all six triazines. The energy-minimized structures were compared and analysed. A third hydrogen bond, not seen in the X-ray analysis of the reaction center/tertubryn complex, was found between the t-butylamino moiety of terbutryn (and equivalent moieties in the other triazines) and the carbonyl oxygen of TyrL222.     

Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.     

The Rhodopseudomonas viridis photosynthetic membrane: arrangement in situ

The organization of photosynthetic membranes in the cytoplasm of the photosynthetic bacterium Rh. viridis has been examined by several techniques for electron microscopy. Thin sections of membrane stacks show that the regular lattice of membrane subunits reported in other studies can be observed in thin section. Tilting of sections in the electron microscope shows that the regular lattices of several membranes overlap in a way that suggests they are in register with each other. This observation can be confirmed by freeze-fracture images in which a regular arrangement of membrane lattices can be observed, each perfectly aligned.Analysis of the spacings of membrane pairs shows that the photosynthetic membranes of Rh. viridis are very closely apposed. The mean diameter of two membranes is 160A, and the average space between two such membranes is only 42A. When a recently developed atomic level model of Rh. viridis reaction center is superimposed against these spacings, each reaction center extends from the surface of its respective membrane far enough to make contact with an apposing membrane. The limited free space between membranes and regular alignment of lattices has a number of implications for how this membrane is organized to carry out the process of energy transfer.