Isolation and amino-terminal sequences of subunits from the photosynthetic reaction center of Rhodopseudomonas capsulata (original) (raw)
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Pure and Applied Chemistry, 2000
The photoprotective function of 15,15'-cis-carotenoids bound to the photosynthetic reaction centers (RCs) of purple bacteria has been studied using carotenoids reconstituted into carotenoidless RCs from Rhodobacter sphaeroides strain R26.1. The triplet-energy level of the carotenoid has been proposed to affect the quenching of the triplet state of special-pair bacteriochlorophyll (P). This was investigated using microsecond flash photolysis to detect the carotenoid triplets as a function of the number of conjugated double bonds, n. The carotenoid triplet signals were extracted by using singular-value decomposition (SVD) of the huge matrices data, and were confirmed for those having n = 8 to 11. This interpretation assumes that the reconstituted carotenoids occupy the same binding site in the RC. We have been able to confirm this assumption using X-ray crystallography to determine the structures of carotenoidless, wild-type carotenoid-containing, and 3,4-dihydro-spheroidene-reconstituted RCs. The X-ray study also emphasized the importance of the methoxy group of the carotenoids for binding to the RCs. Electroabsorption (Stark) spectroscopy was used to investigate the effect of the carotenoid on the electrostatic field around P. This electrostatic field changed by 10 % in the presence of the carotenoid. the determination of the structure of the RC from Rhodobacter (Rba.) sphaeroides . shows the arrangement of the pigments in the RC of Rba. sphaeroides . The RC is composed of H, L, and M subunits, a special pair of bacteriochlorophylls (BChl) (P), two accessory monomeric BChls (B A and B B ), two monomeric bacteriopheophytins (H A and H B ), and two quinones (Q A and Q B ) are bound to the L and M subunits with pseudo two-hold symmetry. After excitation energy is transferred from the antenna system to the RC, an electron is transferred from P to H A and then onto Q A . If excess light energy is supplied to the RC, a triplet excited-state of P ( 3 P) is generated. The presence of 3 P can result in the production of harmful singlet oxygen .
The EMBO Journal, 1986
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 synthesised and utilised to isolate one clone which contained the genes for the L and M subunits of the reaction centre as well as the a and ,B 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 the 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.
Journal of bacteriology, 1995
The carotenoid biosynthesis genes form a cluster within the genome of Rhodobacter sphaeroides, lying in the middle of a larger cluster and 45 kb in length, which contains genes for bacteriochlorophyll biosynthesis and for the reaction center and light-harvesting apoproteins. The positions and approximate limits of the carotenoid genes were determined previously by localized transposon Tn5 mutagenesis and by comparison with the closely related Rhodobacter capsulatus carotenoid gene cluster. In this report, analysis of the DNA and deduced amino acid sequences of the carotenoid genes in R. sphaeroides are presented. Twenty-five Tn5 insertion mutants were used to produce a base-specific Tn5 insertion map of this region, and carotenoid gene assignment was supported by spectroscopic, ultrastructural, and high-pressure liquid chromatography analyses of these mutants. A region in the 3' end of crtD which affects bacteriochlorophyll biosynthesis was discovered, and CrtA was found to poss...
The EMBO Journal, 1987
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 confrmned 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 carotenoid band shift in reaction centers from Rhodopseudomonas sphaeroides
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1977
A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathechromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron accepter of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.
Journal of Biological Chemistry, 1999
The nucleotide sequence of the puf operon, which contains the genes encoding the B870 light-harvesting protein and the reaction center complex of the purple photosynthetic bacterium, Rhodovulum sulfidophilum, was determined. The operon, which consisted of six genes, pufQ, pufB, pufA, pufL, pufM, and pufC, is a new variety in photosynthetic bacteria in the sense that pufQ and pufC coexist. The amino acid sequence of the cytochrome subunit of the reaction center deduced from the pufC sequence revealed that this cytochrome contains only three possible heme-binding motifs; the heme-1binding motif of the corresponding tetraheme cytochrome subunits was not present. This is the first exception of the "tetraheme" cytochrome family in purple bacteria and green filamentous bacteria. The pufC sequence also revealed that the sixth axial ligands to heme-1 and heme-2 irons were not present in the cytochrome either. This cytochrome was actually detected in membrane preparation as a 43-kDa protein and shown to associate functionally with the photosynthetic reaction center as the immediate electron donor to the photo-oxidized special pair of bacteriochlorophyll. This new cytochrome should be useful for studies on the role of each heme in the cytochrome subunit of the bacterial reaction center and the evolution of proteins in photosynthetic electron transfer systems.
Plant and Cell …, 2010
The crtB gene of Synechocystis sp. PCC 6803, encoding phytoene synthase, was inactivated in the ∆ crtH mutant to generate a carotenoidless ∆ crtH/B double mutant. ∆ crtH mutant cells were used because they had better transformability than wild-type cells, most probably due to their adaptation to partial carotenoid defi ciency. Cells of the ∆ crtH/B mutant were light sensitive and could grow only under light-activated heterotrophic growth conditions in the presence of glucose. Carotenoid defi ciency did not signifi cantly affect the cellular content of phycobiliproteins while the chlorophyll content of the mutant cells decreased. The mutant cells exhibited no oxygen-evolving activity, suggesting the absence of photochemically active PSII complexes. This was confi rmed by 2D electrophoresis of photosynthetic membrane complexes. Analyses identifi ed only a small amount of a non-functional PSII core complex lacking CP43, while the monomeric and dimeric PSII core complexes were absent. On the other hand, carotenoid defi ciency did not prevent formation of the cytochrome b 6 f complex and PSI, which predominantly accumulated in the monomeric form. Radioactive labeling revealed very limited synthesis of inner PSII antennae, CP47 and especially CP43. Thus, carotenoids are indispensable constituents of the photosynthetic apparatus, being essential not only for antioxidative protection but also for the effi cient synthesis and accumulation of photosynthetic proteins and especially that of PSII antenna subunits.