Genetic analysis of the Photosystem I subunits from the red alga, Galdieria sulphuraria

Currently, there are very little data available regarding the photosynthetic apparatus of red algae. We have analyzed the genes for Photosystem I in the recently sequenced genome of the red alga Galdieria sulphuraria. All subunits that are conserved between plants and cyanobacteria were unambiguously identified in the Galdieria genome: PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK and PsaL. From the plant specific subunits, PsaN and PsaO were identified but the sequence homology was much lower than for the subunits that are present in plants and cyanobacteria. The subunit PsaX, which is specific for thermophilic cyanobacteria, is not present in the Galdieria genome, whereas PsaM is a plastid-encoded protein as in other red algae. The sequences of the core subunits of PSI were further analyzed by mapping of the conserved areas in the crystal structures of cyanobacterial and plant PSI. The structural comparison shows that PSI from the red alga Galdieria may represent a common ancestral structure at the interface between cyanobacterial and plant PSI. Some subunits have a “zwitter” structure that contains structural elements that show similarities with either plant or cyanobacterial PSI. The structure of PsaL, which is responsible for the trimerization of PSI in cyanobacteria, lacks a short helix and the Ca²⁺ binding site, which are essential for trimer formation indicating that the Galdieria PSI is a monomer. However the sequence homology to plant PsaL is low and lacks strong conservation of the interaction sites with PsaH. Furthermore, the sites for interaction of plant PSI with the LHCI complex are not well conserved between plants and Galdieria, which may indicate that Galdieria may contain a PSI that is evolutionarily much more ancient than PSI from green algae, plants and the current cyanobacteria.     

Isolation and characterization of CAC antenna proteins and photosystem I supercomplex from the cryptophytic alga Rhodomonas salina

In the present paper, we report an improved method combining sucrose density gradient with ion‐exchange chromatography for the isolation of pure chlorophyll a/c antenna proteins from the model cryptophytic alga Rhodomonas salina. Antennas were used for in vitro quenching experiments in the absence of xanthophylls, showing that protein aggregation is a plausible mechanism behind non‐photochemical quenching in R. salina. From sucrose gradient, it was also possible to purify a functional photosystem I supercomplex, which was in turn characterized by steady‐state and time‐resolved fluorescence spectroscopy. R. salina photosystem I showed a remarkably fast photochemical trapping rate, similar to what recently reported for other red clade algae such as Chromera velia and Phaeodactylum tricornutum. The method reported therefore may also be suitable for other still partially unexplored algae, such as cryptophytes.     

A model for the 77 K excited state dynamics in Chlamydomonas reinhardtii in state 1 and state 2

The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core. In a simultaneous analysis of the data from all strains under S1 or S2 conditions a unified model for the excited state dynamics at 77 K was created. This yielded the completely resolved LHCII-LHCI-PSI and LHCII-PSII dynamics and quantified the state transitions. In WT cells the fraction of light absorbed by LHCII connected to PSII decreases from 45% in S1 to 29% in S2, while it increases from 0% to 16% for LHCII connected to PSI. Thus (16/45 =) 36% of all LHCII is involved in the state transition. In the mutant strains deficient in the PSI core, the red most species peaking at 716 nm disappears completely, indicating that this far red Chl pigment is located in the PSI core. In the mutant strain deficient in the PSII core, red shifted species with maxima at 684 and 686 nm appear in the LHCII antenna. LHCII-684 is quenched and decays with a rate of (310 ps)^? 1.     

Action spectra of photosystems II and I and quantum yield of photosynthesis in leaves in State 1

The spectral global quantum yield (Y_II, electrons/photons absorbed) of photosystem II (PSII) was measured in sunflower leaves in State 1 using monochromatic light. The global quantum yield of PSI (Y_I) was measured using low-intensity monochromatic light flashes and the associated transmittance change at 810 nm. The 810-nm signal change was calibrated based on the number of electrons generated by PSII during the flash (4 · O₂ evolution) which arrived at the PSI donor side after a delay of 2 ms. The intrinsic quantum yield of PSI (y_I, electrons per photon absorbed by PSI) was measured at 712 nm, where photon absorption by PSII was small. The results were used to resolve the individual spectra of the excitation partitioning coefficients between PSI (a_I) and PSII (a_II) in leaves. For comparison, pigment–protein complexes for PSII and PSI were isolated, separated by sucrose density ultracentrifugation, and their optical density was measured. A good correlation was obtained for the spectral excitation partitioning coefficients measured by these different methods. The intrinsic yield of PSI was high (y_I = 0.88), but it absorbed only about 1/3 of quanta; consequently, about 2/3 of quanta were absorbed by PSII, but processed with the low intrinsic yield y_II = 0.63. In PSII, the quantum yield of charge separation was 0.89 as detected by variable fluorescence F_v/F_m, but 29% of separated charges recombined (Laisk A, Eichelmann H and Oja V, Photosynth. Res. 113, 145–155). At wavelengths less than 580 nm about 30% of excitation is absorbed by pigments poorly connected to either photosystem, most likely carotenoids bound in pigment–protein complexes.     

O2 reduction by photosystem I involves phylloquinone under steady-state illumination

O₂ reduction was investigated in photosystem I (PS I) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone-binding site A₁. PS I complexes from WT and menB mutant exhibited different dependencies of O₂ reduction on light intensity, namely, the values of O₂ reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light-induced O₂ reduction by PS I.     

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.     

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom,Chaetoceros gracilis

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.     

Serum profile in preeclampsia and intra-uterine growth restriction revealed by iTRAQ technology

In order to identify new protein markers modified in placental diseases, high-throughput analysis of proteins in the plasma of pregnant women was carried out for normal and pathological pregnancies (Preeclampsia and/or Intra-Uterine Growth Restriction) using iTRAQ technology. We could identify 166 proteins that were modified (p < 0.05) and the technique used allowed the detection of previously undetected factors, such as various members of the SERPINA clade. The modifications of two proteins (C reactive protein and antichymotrypsin, SERPINA3) were validated on individual samples. Complement and coagulation cascades proteins were significantly enriched among modified protein clusters in the case of intra-uterine growth restriction (p < 2.6 · 10^? 11). Several proteins were specifically enriched in isolated preeclampsia and depleted when preeclampsia was complicated by intra-uterine growth restriction. These findings suggest that the growth restricted foeto-placental unit is able to moderate some changes in maternal plasma composition. Overall, the use of iTRAQ technology, for the first time on this subject, enabled us to provide a new list of proteins modified in placental diseases, among which proteins expressed at a low level that were not accessible by other methods.     

Phylomineralogy of the Coralline red algae: Correlation of skeletal mineralogy with molecular phylogeny

The coralline algae in the orders Corallinales and Sporolithales (subclass Corallinophycidae), with their high degree of mineralogical variability, pose a challenge to projections regarding mineralogy and response to ocean acidification. Here we relate skeletal carbonate mineralogy to a well-established phylogenetic framework and draw inferences about the effects of future changes in sea-water chemistry on these calcified red algae. A collection of 191 coralline algal specimens from New Zealand, representing 13 genera and 28 species, included members of three families: Corallinaceae, Hapalidiaceae, and Sporolithaceae. While most skeletal specimens were entirely calcitic (range: 73–100 wt.% calcite, mean 97 wt.% calcite, std dev = 5, n = 172), a considerable number contained at least some aragonite. Mg in calcite ranged from 10.5 to 16.4 wt.% MgCO₃, with a mean of 13.1 wt.% MgCO₃ (std dev = 1.1, n = 172). The genera Mesophyllum and Lithophyllum were especially variable. Growth habit, too, was related to mineralogy: geniculate coralline algae do not generally contain any aragonite. Mg content varied among coralline families: the Corallinaceae had the highest Mg content, followed by the Sporolithaceae and the Hapalidiaceae. Despite the significant differences among families, variation and overlap prevent the use of carbonate mineralogy as a taxonomic character in the coralline algae. Latitude (as a proxy for water temperature) had only a slight relationship to Mg content in coralline algae, contrary to trends observed in other biomineralising taxa. Temperate magnesium calcites, like those produced by coralline algae, are particularly vulnerable to ocean acidification. Changes in biomineralisation or species distribution may occur over the next few decades, particularly to species producing high-Mg calcite, as pH and CO₂ dynamics change in coastal temperate oceans.     

Developing an improved biomonitoring tool for fine sediment: Combining expert knowledge and empirical data

The Proportion of Sediment-sensitive Invertebrates (PSI) index is a biomonitoring tool that is designed to identify the degree of sedimentation in rivers and streams. Despite having a sound biological basis, the tool has been shown to have only a moderate correlation with fine sediment, which although comparable to other pressure specific indices, limits confidence in its application. The aim of this study was to investigate if the performance of the PSI index could be enhanced through the use of empirical data to supplement the expert knowledge and literature which were used to determine the original four fine sediment sensitivity ratings. The empirical data used, comprised observations of invertebrate abundance and percentage fine sediment, collected across a wide range of reference condition temperate stream and river ecosystems (model training dataset n = 2252). Species were assigned sensitivity weights within a range based on their previously determined sensitivity rating. Using a range of weights acknowledges the breadth of ecological niches that invertebrates occupy and also their differing potential as indicators. The optimum species-specific sensitivity weights were identified using non-linear optimisation, as those that resulted in the highest Spearman's rank correlation coefficient between the Empirically-weighted PSI (E-PSI) scores and deposited fine sediment in the model training dataset. The correlation between percentage fine sediment and E-PSI scores in the test dataset (n = 252) was eight percentage points higher than the correlation between percentage fine sediment and the original PSI scores (E-PSI r_s = −0.74, p < 0.01 compared to PSI r_s = −0.66, p < 0.01). This study demonstrates the value of combining a sound biological basis with evidence from large empirical datasets, to test and enhance the performance of biomonitoring tools to increase confidence in their application.     

The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes

A photosystem I (PSI)-fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI-FCP sample revealed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae.     

Community level physiological study of algicidal bacteria in the phycospheres of Skeletonema costatum and Scrippsiella trochoidea

Bacteria in the phycosphere have a unique ecological relationship with host algae due to their utilization of algal extracellular products as nutrients. Some bacteria control the growth of algal cells and even lyse them. The diversity of bacteria and their community dynamics in the phycosphere of microalgae are still relatively little understood, especially of those associated with red tide-causing algae. In this study, scanning electron microscope (SEM) images of algal cell morphology revealed that the phycosphere bacteria of the red tide-causing algae, Skeletonema costatum and Scrippsiella trochoidea, could lyse them within 72 h. The community level physiology of the algicidal bacteria was studied using Biolog ECO microplates, a common method for the ecological study of microbial communities. The average well color development (AWCD) values of bacteria in the phycospheres of both species were low, indicating that the bacteria had low metabolic activity overall. The diversity indices were both lower than the bacterial diversity from natural environments. However, the bacteria associated with S. trochoidea demonstrated a higher AWCD value and diversity than those in the phycosphere of S. costatum. The utilization of carbon sources significantly changed at different lytic times, reflecting that the bacterial community structure changed during the algae-lysing process. These results revealed that the bacterial communities in phycospheres had a simple structure and low diversity. When the balance between algae and bacteria broke down, the total bacterial density increased while the algicidal bacteria accumulated and became the dominant species, changing the bacterial community structure in this micro-ecosystem.     

The effects of applying sewage sludge into Jiangxi red soil on the growth of vegetables and the migration and enrichment of Cu and Zn

Jiangxi red soil was used as the tested soil and water spinach (Ipomoea aquatic) and Chinese chive (Allium tuberosum) were used as the tested vegetables in this study to investigate the effects of different amounts of sewage-sludge application on the growth of vegetables and the migration and enrichment patterns of Cu and Zn in vegetables using the potted method. The results indicated that the application of sewage sludge could improve the properties of red soil and promote vegetable growth. The dry weight of water spinach and Chinese chive reached the maximal levels when treated with the amount of sewage sludge at 4% and 10%, which was 4.38 ± 0.82 g and 1.56 ± 0.31 g, respectively. The dry weights after the application of sewage sludge were all larger than control treatment (CK) without sludge application. With increases in the applied amount of sewage sludge, the concentrations of Cu and Zu in red soil continued to increase, and the peak value was not reached. After the two vegetables were planted, the concentrations of Cu and Zn in red soil decreased by different degrees. The degrees of decrease of Zn were generally higher than those of Cu. The enrichment coefficient of water spinach on Cu showed a trend of increase followed by a decrease and reached the peak value of 1.04 ± 0.38 when the applied amount was 4%. The enrichment coefficient of Chinese chive on Cu overall showed a decreasing trend and did not reach the peak value under the treatment levels used in this experiment. The enrichment pattern of Chinese chive on Zn was not obvious, and the differences among all treatment levels were not significant (p < 0.05). However, the enrichment coefficient after the application of sewage sludge was significantly lower than that without the application of sludge.     

PSI-O, a new 10-kDa subunit of eukaryotic photosystem I

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate–polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N-terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI-O. The mature protein has two transmembrane helices and a calculated mass of 10 104 Da. The PSI-O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae.     

Microbial–meiofaunal interrelationships in coastal sediments of the Red Sea

Population density and biomass of bacteria and meiofauna were investigated seasonally in the sediments of the north-western bank of Red Sea. Samples of sediments were collected seasonally from three different stations to determine microphytobenthic biomass (chlorophyll a), protein, lipid, carbohydrate, and total organic matter concentrations. These investigations revealed that microbial components tended to increase their dominancy, whereas sensitive meiofauna were extremely reduced during the entire study period. Thus a very low density of the total meiofauna (with an annual average of 109 ± 26 ind./10 cm²) was recorded whilst the benthic microbial population densities exhibited higher values (ranging from 0.31 ± 0.02 × 10⁸ to 43.67 ± 18.62 × 10⁸/g dry sediment). These changes in the relative importance analysis of benthic microbial components versus meiofaunal ones seem to be based on the impact of organic matter accumulation on the function and structure of these benthic communities. Proteins, lipids and carbohydrates showed very low concentration values, and the organic matter mostly consisted of carbohydrates, reflecting lower nutritional values for benthic fauna in general and meiofauna in particular. The distribution of microbial and meiofaunal communities seems to be dependent on the quality of the organic matter rather than on its quantity. Total organic matter concentrations varied between 5.8 and 7.6 mg/g, with organic carbon accounting for only 32% of the total organic matter. Chlorophyll a attained very low values, fluctuating between 0.11 and 0.56 μg/g, indicating the oligotrophy of the studied area. The very low concentration of chlorophyll a in the Red Sea sediment suggests that the sedimentary organic matter, heterotrophic bacteria and/or protozoa constitute an alternative resource that is consumed by meiofauna when algae are less abundant. Protozoa, therefore, represent the “missing link in bacteria–meiofauna interaction in the Red Sea marine sediment ecosystem.     

The effects of mitochondrial complex I blockade on ATP and permeability in rat pulmonary microvascular endothelial cells in culture (PMVEC) are overcome by coenzyme Q1 (CoQ1)

In isolated rat lung perfused with a physiological saline solution (5.5 mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (K_f), effects that are overcome using an amphipathic quinone (CoQ₁) [Free Radic. Biol. Med. 65:1455–1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5 mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2 mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8 nmol/mg protein, depolarized the mitochondrial membrane potential (Δψ_m) from −129.0±3.7 (mean±SEM) to −92.8±5.5 mV, and decreased O₂ consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1 nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC–dextran (~40 kDa) continually over a 6 h time course. When CoQ₁ was present with rotenone, normal ATP (17.4±1.4 nmol/mg protein), O₂ consumption (1.5±0.1 nmol/min/mg protein), Δψ_m (−125.2±3.3 mV), and permeability (PS) were maintained. Protective effects of CoQ₁ on rotenone-induced changes in ATP, O₂ consumption rate, Δψ_m, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol–mitochondria electron shuttle [Free Radic. Biol. Med. 65:1455–1463; 2013]. Key rotenone effects without and with CoQ₁ were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2 mM.     

Effect of dietary fish meal replacement by red algae, Gracilaria arcuata, on growth performance and body composition of Nile tilapia Oreochromis niloticus

A 12-week long feeding experiment was initiated to evaluate the effect of dietary supplementation of red algae, Gracilaria arcuata, on the growth performance, feed utilization and body composition of Nile tilapia Oreochromis niloticus (Linnaeus, 1758). The fish were fed with an algae-free control diet (C) and three experimental diets which replaced conventional fish meal with varying levels of dried G. arcuata (20%, 40% and 60%, represented as G20, G40 and G60, respectively). The growth parameters of final weight (FW), weight gain (WG), percentage of weight gain (WG%), daily growth rate (DGR) and specific growth rate (SGR) were significantly reduced (P < 0.05) at all levels of algae incorporation compared to the control diet. Moreover, the negative impact of Gracilaria meal on the growth performance of Nile tilapia increased as the proportion of algae in the diet increased, with fish on diet G20 exhibiting a significantly higher growth performance than the fish on either of the G40 and G60 diets. On the other hand, the feed utilization parameters feed conversion ratio (FCR) and protein efficiency ratio (PER) did not show significant differences between the fish in the control group and those on diet G20, although poorer FCR and PER outcomes were achieved in the case of fish on diet G60. The content of moisture, protein and ash in muscle and carcass increased as the proportion of Gracilaria meal in the diets increased, but the reverse was true for lipid level. These results indicate that incorporation of less than 20% red algae, Gracilaria arcuata, could be feasible in the diet of Nile tilapia and further studies are recommended to optimize the level of algae to improve growth performance.     

Purification and characterization of an azoreductase from Escherichia coli CD-2 possessing quinone reductase activity

An oxygen-insensitive intracellular enzyme that is responsible for the decolorization of azo dyes was purified from Escherichia coli CD-2. The molecular weight of the purified enzyme was estimated as 27,000 ± 500 Da. Protein identification indicated that the enzyme had high sequence homology with E. coli K12 quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. With methyl red as substrate, the optimal pH value and temperature were 6.5 and 37 °C, respectively. The enzyme was stable under different physiochemical conditions. The azoreductase activity was restrained by SDS and was almost completely inhibited by Co²⁺ and Hg²⁺. K_m and V_max values were 0.18 mM and 8.12 U mg^?1 of protein for NADH and 0.05 mM and 6.46 U mg^?1 of protein for methyl red, respectively. The purified enzyme could efficiently decolorize methyl red with both NADH and NADPH as electron donors.     

Replacement of the methionine axial ligand to the primary electron acceptor A0 slows the A0 reoxidation dynamics in Photosystem I

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P₇₀₀, and a sequence of electron acceptors, A, A₀ and A₁, bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A₀, are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A₀⁻ (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A₀⁻ on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A₀⁻ is transient, and that reoxidation of A₀⁻ occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A₁. This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A₀ in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.