New and unexpected routes for ultrafast electron transfer in photosynthetic reaction centers.
(25/1996)
In photosynthetic reaction centers, the excitation with light leads to the formation of a charge separated state across the photosynthetic membrane. For the reaction center of purple non-sulphur bacteria, it was previously generally assumed that this primary charge separation could only start with the excitation of the so-called special pair of bacteriochlorophyll molecules located in the heart of the RC. However, recently new and ultrafast pathways of charge separation have been discovered in the bacterial RC that are driven directly by the excited state of the accessory monomeric bacteriochlorophyll present in the active branch of cofactors. These results demonstrate that the route for energy conversion in photosynthesis can be much more flexible than previously thought. We suggest that the existence of multiple charge separation routes is particularly relevant for the mechanism of charge separation in the photosystem II reaction center of higher plants. (+info)
In vitro random mutagenesis of the D1 protein of the photosystem II reaction center confers phototolerance on the cyanobacterium Synechocystis sp. PCC 6803.
(26/1996)
The D1 protein of the photosystem II reaction center is thought to be the most light-sensitive component of the photosynthetic machinery. To understand the mechanisms underlying the light sensitivity of D1, we performed in vitro random mutagenesis of the psbA gene that codes for D1, transformed the unicellular cyanobacterium Synechocystis sp. PCC 6803 with mutated psbA, and selected phototolerant transformants that did not bleach in high intensity light. A region of psbA2 coding for 178 amino acids of the carboxyl-terminal portion of the peptide was subjected to random mutagenesis by low fidelity polymerase chain reaction amplification or by hydroxylamine treatment. This region contains the binding sites for Q(B), D2 (through Fe), and P680. Eighteen phototolerant mutants with single and multiple amino acid substitutions were selected from a half million transformants exposed to white light at 320 micromol m(-2) s(-1). A strain transformed with non-mutagenized psbA2 became bleached under the same conditions. Site-directed mutagenesis has confirmed that one or more substitutions of amino acids at residues 234, 254, 260, 267, 322, 326, and 328 confers phototolerance. The rate of degradation of D1 protein was not appreciably affected by the mutations. Reduced bleaching of mutant cyanobacterial cells may result from continued buildup of photosynthetic pigment systems caused by changes in redox signals originating from D1. (+info)
Transcript cleavage, attenuation, and an internal promoter in the Rhodobacter capsulatus puc operon.
(27/1996)
The stoichiometry of the structural proteins of the photosynthetic apparatus in purple photosynthetic bacteria is achieved primarily by complex regulation of the levels of mRNA encoding the different proteins, which has been studied in the greatest detail in the puf operon. Here we investigated the transcriptional and posttranscriptional regulation of the puc operon, which encodes the peripheral light harvesting complex LHII. We show that, analogous to the puf operon, a primary transcript encoding five puc genes is rapidly processed to generate more stable RNA subspecies. Contrary to previous hypotheses, translational coupling and regulation of puc transcription by puc gene products were found not to occur. A putative RNA stem-loop structure appears to attenuate transcription initiated at the puc operon major promoter. We also found that a minor pucD-internal promoter contributes to the levels of a message that encodes the LHII 14-kDa gamma (PucE) protein. (+info)
Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites.
(28/1996)
The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a-Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins. (+info)
Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.
(29/1996)
The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected. (+info)
Voltammetric detection of superoxide production by photosystem II.
(30/1996)
Oxygen radicals play both pathological and physiological roles in biological systems. The detection of such radicals is difficult due to their transient nature and the presence of highly efficient antioxidant mechanisms. In plants the physiological role of oxygen is twofold, oxygen is produced by the oxidation of water and consumed as an electron acceptor. The direct involvement of oxygen in photosynthetic events exposes the photosynthetic apparatus to a high probability of damage by oxygen radicals. We report here a direct, simple and rapid method for the measurement of superoxide in vitro based on voltammetric detection. It has potential applications for other in vitro systems investigating superoxide production. We show that in addition to the well established production of superoxide from photosystem I, under reducing conditions superoxide is also produced by photosystem II, probably from the Q(A) site. (+info)
Light-induced degradation of cytochrome b559 during photoinhibition of the photosystem II reaction center.
(31/1996)
The behaviour of cytochrome (cyt) b559 during acceptor- and donor-side photoinhibition has been investigated in oxygen-evolving and non-evolving photosystem II (PSII) membranes. Strong illumination at 20 degrees C under aerobiosis induced a strong decrease in the absorbance of the cyt b559 alpha-band in the two preparations. This absorbance decline was observed only in non-oxygen-evolving PSII samples when illumination was performed under aerobiosis but at 4 degrees C, or under anaerobiosis at 20 degrees C. These results suggest that acceptor-side photoinhibition induces the degradation of cyt b559 by a mechanism related to an enzymatic reaction mediated by singlet oxygen. Donor-side photoinhibition may induce, however, a non-enzymatic photocleavage of the protein. (+info)
Chloroplast acclimation in leaves of Guzmania monostachia in response to high light.
(32/1996)
Acclimation of leaves to high light (HL; 650 micromol m(-2) s(-1)) was investigated in the long-lived epiphytic bromeliad Guzmania monostachia and compared with plants maintained under low light (LL; 50 micromol m(-2) s(-1)). Despite a 60% decrease in total chlorophyll in HL-grown plants, the chlorophyll a/b ratio remained stable. Additionally, chloroplasts from HL-grown plants had a much lower thylakoid content and reduced granal stacking. Immunofluorescent labeling techniques were used to quantify the level of photosynthetic polypeptides. HL-grown plants had 30% to 40% of the content observed in LL-grown plants for the light-harvesting complex associated with photosystems I and II, the 33-kD photosystem II polypeptide, and Rubisco. These results were verified using conventional biochemical techniques, which revealed a comparable 60% decrease in Rubisco and total soluble protein. When expressed on a chlorophyll basis, the amount of protein and Rubisco was constant for HL- and LL-grown plants. Acclimation to HL involves a tightly coordinated adjustment of photosynthesis, indicating a highly regulated decrease in the number of photosynthetic units manifested at the level of the content of light-harvesting and electron transport components, the amount of Rubisco, and the induction of Crassulacean acid metabolism. This response occurs in mature leaves and may represent a strategy that is optimal for the resource-limited epiphytic niche. (+info)