The complete nucleotide sequence of two Chlorojlexus aurantiacus reaction-center genes has been obtained. The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N-chlorosuccinimide peptide fragments isolated from the purified reaction-center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2-propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.The initial steps of photosynthesis, the conversion of light into chemical energy, occur within a pigment-protein complex, called the reaction center. Reaction centers of several photosynthetic bacteria, belonging primarily to the family of Rhodospirillaceae, are composed of three polypeptides, M, L and H, with approximate molecular masses of 34.4 kDa, 31.6 kDa, and 28.5 kDa, respectively. The subunits are present in the native pigment-protein in a 1 : 1 : 1 stoichiometry. In addition, the reaction center of Rhodopseudon~onas viridis contains a fourth subunit, a four-heme cytochrome (40 kDa). Noncovalently bound to the protein entity is a set of cofactors: four bacteriochlorophylls (Bchl), two bacteriopheophytins (Bphe), two quinones and one non-heme iron. Crystallization and X-ray structure analysis of the Rps. viridis and Rhodobacter sphaeroides reaction centers have led to the elucidation of the three-dimensional structure of the pigment-protein moiety [l -41. The core of the reaction center is formed by the L and M subunits through which the special Bchl dimer, the accessory Bchl, Bphe and quinones form a branched electrontransport chain with twofold symmetry [I -41. These data, in conjunction with numerous spectroscopic data, have greatly augmented our understanding of the primary photochemical charge separation; for recent reviews, see [5 -71.Recently, we have reported the biochemical characterization of the reaction center of the thermophilic green bacterium Chlorojlexus aurantiacus. Although C. aurantiacus has a slightly different pigment composition, 3 Bchl : 3 Bphe/reaction-center molecule compared to a 4: 2 ratio in Rhodospirillaceae reaction centers [8, 91, the photochemical and early electron-transfer reactions, as determined by picosecond spectroscopy and circular dichroism in C. aurantiacus reactioncenter preparations, seem to be analogous to those of other purple bacteria reaction centers [6, 91. In contrast, the protein compone...
A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chlorojlexus aurantiacus. The procedure consisted of three chromatography steps. The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl-N-methylglucamide (Mega-9). The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less. Under non-denaturing conditions, a pigmented protein band having a M , of 52000 -55000 was observed in sodium dodecyl sulfate gels. When the isolated complex was heat-dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar M , (24000 and 24500) were observed. Two protein bands were also observed in two-dimensional isoelectric focusing/sodium-dodecyl-sulfate polyacrylamide gel electropheresis ; the PI values of the two polypeptides were 6.5 and 6.7. Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides.The N-terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria.The Chlorojlexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins. Hence, it appears to be the smallest photochemically active reaction center isolated to date.The first steps of photosynthesis, the conversion of light energy into chemical energy, occours within pigment-protein complexes called reaction centers. The reaction center of several photosynthetic bacteria, primarily belonging to the family Rhodospirillaceae, have been isolated and biochemically characterized. Most reaction center complexes are comprised of three polypeptides having apparent M , = 28 000, 24000 and 21 000 as determined by NaDodS0,-PAGE. The polypeptides are present in the complex in a 1 : 1 : 1 molar stoichiometry. The reaction center from Rps. viridis contains an additional four heme cytochrome subunits ( M , 40000). Non-covalently bound to the protein moiety are four bacteriochlorophylls, two bacteriopheophytins, one to two quinones and non-heme iron (for reviews, see [I, 21). The recent success in crystallizing reaction center from Rps. viridis and the subsequent X-ray diffraction analysis has led to a three-dimensional molecular model of this pigment-protein [3, 41. Extensive optical and time-resolved spectroscopical analyses have revealed the primary photochemical electron-transfer steps in isolated reaction centers. While there is general agreement on the 'late' photochemical processes (> lo-" s), there are conflicting data on the late femtoseconds to early picoseconds events [5 -1 I].The subcellular organization and location of the components of the photosynthetic apparatus and t...
The light‐harvesting bacteriochlorophyll‐carotenoid‐protein complex B800–850 was been isolated from membranes of the phototroph‐negative mutant strain Y5 of Rhodopseudomonas capsulata. The three polypeptides of the complex have been found to be soluble in chloroform‐methanol (1:1, v/v) in the presence of 0.1 M ammonium acetate. They were extracted from the complex and separated by gel filtration on Sephadex LH‐60 in the same solvent mixture. Minimum molecular weights based on amino acid composition are 12000, 9300, and 5100. Values previously determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are 14000, 10000 and 8000. The two smaller polypeptides (polarities 31% and 39%) are completely soluble in chloroform/methanol/ammonium acetate while the largest and most polar (41%) polypeptide is only partially soluble. The largest polypeptide contains no tryptophan. The middle polypeptide contains no cysteine and arginine, while the small polypeptide lacks cysteine. Methionine is shown to be the amino terminus for the small and middle polypeptides by two independent methods (Edman degradation and dansylation). Both methods also indicated that the N terminus of the 14000 polypeptide seems to be blocked. Partial N‐terminal amino acid sequences were obtained for the two smaller polypeptides. No homology between the two proteins was observed.
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