Regeneration of NADPH by Frozen-thawed Cells of a Blue-green Alga,Synechococcus sp.

The photoreduction of NADP ⁺ and its associated reactions were studied in a blue-green algal preparation that was frozen in liquid nitrogen and thawed at room temperature. The preparation was capable of photoreducing exogeneous NADP⁺. Water was the ultimate electron donor for the reduction. The optimum pH was 7.5 ~ 8.0, and optimum temperature, around 50°C. Light saturation for NADP⁺ photoreduction was reached at 50 μEinstein/m²/sec. Factors limiting the stability of the preparation were examined and a possible application of this cofactor regeneration system is discussed.     

Crystallization of Photosystem I Complexes from the Thermophilic Cyanobacterium, Synechococcus vulcanus

PSI complexes were isolated from the thermophilic cyanobacterium,Synechococcus vulcanus, by mild detergent treatment of the thylakoidmembranes, purified by sucrose-density gradient centrifugation,and then crystallized. High resolution SDS-PAGE revealed thepresence of the product of the psaI gene in S. vulcanus PSIcomplexes and crystals. Crystals of the PSI complexes retainedall of the components of electron carriers and subunit polypeptides(including PsaX) known in cyanobacteria. (Received July 22, 1998; Accepted October 19, 1998)     

Modulating Effect of Far-Red Light on Activities of Alternative Electron Transport Pathways Related to Photosystem I

Barley (Hordeum vulgare L.) leaves were irradiated with far-red (FR) light of various intensities after different periods of dark adaptation in order to investigate activities of alternative electron transport pathways related to photosystem I (PSI). Photooxidation of P700, the primary electron donor of PSI, was saturated at FR light intensity of 0.15 μmol quanta/(m² s). As the photon flux density was raised in this range, the slow and middle components in the kinetics of P700⁺ dark reduction increased, whereas the fast component remained indiscernible. The amplitudes of the slow and middle components diminished upon further increase of FR photon flux density in the range 0.15–0.35 μmol quanta/(m² s) and remained constant at higher intensities. The fast component of P700⁺ reduction was only detected after FR irradiation with intensities above 0.15 μmol quanta/(m² s); the light-response curve for this component was clearly sigmoid. In dark-adapted barley leaves, three stages were distinguished in the kinetics of P700 photooxidation, with the steady state for P700⁺ achieved within about 3 min. In leaves predarkened for a short time, the onset of FR irradiation produced a very rapid photooxidation of P700. As the duration of dark exposure was prolonged, the amplitude of the first peak in the kinetic curve of photoinduced P700 photooxidation was diminished and the time for attaining the steady-state oxidation level was shortened. After a brief dark adaptation of leaves, ferredoxin-dependent electron flow did not appreciably contributed to the kinetics of P700⁺ dark reduction, whereas the components related to electron donation from stromal reductants were strongly retarded. It is concluded that FR light irradiation, selectively exciting PSI, suffices to modulate activities of alternative electron transport routes; this modulation reflects the depletion of stromal reductants due to continuous efflux of electrons from PSI to oxygen under the action of FR light. __________ Translated from Fiziologiya Rastenii, Vol. 52, No. 6, 2005, pp. 805–813. Original Russian Text Copyright ? 2005 by Egorova, Drozdova, Bukhov.     

Polypeptide composition of the Photosystem I complex and the Photosystem I core protein from Synechococcus sp. PCC 6301.

The polypeptide composition of the Photosystem I complex from Synechococcus sp. PCC 6301 was determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. The PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaK and PsaL proteins, as well as three polypeptides with apparent masses less than 8 kDa and small amounts of the 12.6 kDa GlnB (PII) protein, wee present in the Photosystem I complex. No proteins homologous to the PsaG and PsaH subunits of eukaryotic Photosystem I complexes were detected. When the Photosystem I complex was treated with 6.8 M urea and ultrafiltered using a 100 kDa cutoff membrane, the resulting Photosystem I core protein was found to be depleted of the PsaC, PsaD and PsaE proteins. The filtrate contained the missing proteins, along with five proteolytically-cleaved polypeptides with apparent masses of less than 16 kDa and with N-termini identical to that of the PsaD protein. The PsaF and PsaL proteins, along with the three less than 8 kDa polypeptides, were not released from the Photosystem I complex to any significant extent, but low-abundance polypeptides with N-termini identical to those of PsaF and PsaL were found in the filtrate with apparent masses slightly smaller than those found in the native Photosystem I complex. When the filtrate was incubated with FeCl3, Na2S and beta-mercaptoethanol in the presence of the isolated Photosystem I core protein, the PsaC, PsaD and PsaE proteins were rebound to reconstitute a Photosystem I complex functional in light-induced electron flow from P700 to FA/FB. In the absence of the iron-sulfur reconstitution agents, there was little rebinding of the PsaC, psaD or PsaE proteins to the Photosystem I core protein. No binding of the truncated PsaD polypeptides occurred, either in the presence or absence of the iron-sulfur reagents. The reconstitution of the FA/FB iron-sulfur clusters thus appears to be a necessary precondition for rebinding of the PsaC, psaD and psaE proteins to the Photosystem I core protein.     

PsaE Is Required for in Vivo Cyclic Electron Flow around Photosystem I in the Cyanobacterium Synechococcus sp. PCC 7002

Electron transfer rates to P700+ have been determined in wild-type and three interposon mutants (psaE-, ndhF-, and psaE- ndhF-) of Synechococcus sp. PCC 7002. All three mutants grew significantly more slowly than wild type at low light intensities, and each failed to grow photoheterotrophically in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and a metabolizable carbon source. The kinetics of P700+ reduction were similar in the wild-type and mutant whole cells in the absence of DCMU. In the presence of DCMU, the P700+ reduction rate in the psaE mutant was significantly slower than in the wild type. In the presence of DCMU and potassium cyanide, added to inhibit the outflow of electrons through cytochrome oxidase, P700+ reduction rates increased for both the psaE- and ndhF- strains. The reduction rates for these two mutants were nonetheless slower than that observed for the wild-type strain. The further addition of methyl viologen caused the rate of P700+ reduction in the wild type to become as slow as that for the psaE mutant in the absence of methyl viologen. Given the ability of methyl viologen to intercept electrons from the acceptor side of photosystem I, this response reveals a lesion in cyclic electron flow in the psaE mutant. In the presence of DCMU, the rate of P700+ reduction in the psaE ndhF double mutant was very slow and nearly identical with that for the wild-type strain in the presence of 2,4-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a condition under which physiological electron donation to P700+ should be completely inhibited. These results suggest that NdhF- and PsaE-dependent electron donation to P700+ occurs only via plastoquinone and/or cytochrome b6/f and indicate that there are three major electron sources for P700+ reduction in this cyanobacterium. We conclude that, although PsaE is not required for linear electron flow to NADP+, it is an essential component in the cyclic electron transport pathway around photosystem I.     

Modeling of Alternative Pathways of Electron Transport to Photosystem I in Isolated Thylakoids

Kinetics of dark decay of absorbance changes at 830 nm (₈₃₀) was examined in thylakoids isolated from leaves of pea seedlings at various concentrations of exogenous NADPH or NADH. Absorbance changes were induced by far-red light to avoid electron donation from photosystem II. In the presence of either biological reductant, the kinetics of ₈₃₀ decay reflecting dark reduction of 700⁺, the primary electron donor of photosystem I, was fitted by a single exponential term. The rate of 700⁺ reduction increased with the rise in the concentration of both NADPH and NADH. The values of K _M and V _max for 700⁺ reduction estimated from concentration dependences were 105 ± 21 M and 0.32/s for NADPH or 21 ± 8 M and 0.12/s for NADH. The rate of P700⁺ reduction by either NADPH or NADH significantly increased in the presence of rotenone, a specific inhibitor of chloroplast reductase. The value of V _max was changed only in the presence of rotenone, whereas K _m was practically unaffected. Unlike the chloroplasts of intact leaves, the only enzyme mediating the input of reducing equivalents from NADPH or NADH to the electron transport chain was concluded to be present in thylakoids.     

Cross-Linking Studies on the Membrane Topography of Photosystem I Reaction Center Complex in Synechococcus sp.

The subunit arrangement of the photosystem I reaction centercomplex in the thylakoid membranes of the thermophilic cyanobacteriumSynechococcus sp. was examined using three cross-linking reagents.(1) Treatments of osmotically shocked and NaBr-washed protoplastswith low concentrations of hydrophilic cross-linking reagents,dimethyladipimidate and glutaraldehyde, preferentially decreased62, 60, 14 and 13 kDa polypeptides of the photosystem I reactioncenter complex resolved by SDS-polyacrylamide gel electrophoresis,together with the anchor protein and allophycocyanin which areassociated with the outer surface of the thylakoid membranes.This suggests that these four subunits of the photosystem Icomplex are exposed on the stromal surface of thylakoid membranes.In contrast, a hydrophobic cross-linker, hexamethylenediisocyanate,unspecifically cross-linked most of the membrane polypeptides.(2) The 13 and 14 kDa polypeptides decreased always in parallelto each other on treatment of the protoplasts or isolatd CP1-awith the three cross-linking reagents, and the disappearanceof the two polypeptides was accompanied by the appearance ofa cross-linked product(s), when fixed with glutaraldehyde andhexamethylenediisocyanate. The results suggest that the 13 and14 kDa polypeptides are neighboring polypeptides in the complex. (Received June 7, 1986; Accepted November 13, 1986)     

Size of the Plastoquinone Pool Functioning in Photosynthetic and Respiratory Electron Transport of Synechococcus sp.

The size of the plastoquinone pool on the reducing side of photosystem2 in the cyanobacterium Synechococcus sp. was estimated by measuringthe area over the fluorescence induction curve in the presenceof 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone relativeto the area in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea.Plastoquinone was found mostly in the reduced state in freshlyharvested cells but was oxidized by aeration of the cells inthe dark. The pool in the starved cells usually consisted offive or six plastoquinone molecules with a maximum of eightper photosystem 2 reaction center. Addition of glucose or fructoseto the starved cells completely reduced the plastoquinone poolunder anaerobic conditions or in the presence of KCN. The quinonereduced by brief illumination was rapidly and completely oxidizedin the dark. The dark oxidation proceeded at a rate comparableto that of respiratory O₂ uptake in the cyanobacterium and wasstrongly inhibited by KCN. It is concluded that a major populationof the plastoquinone molecules present in the cells functionsas the acceptor pool of photosystem 2 and that the pool is entirelyshared by respiratory electron transport in the cyanobacterium. (Received June 22, 1983; Accepted August 20, 1983)     

Light adaptation of cyclic electron transport through Photosystem I in the cyanobacterium Synechococcus sp. PCC 7942

Photosystem I-driven cyclic electron transport was measured in intact cells of Synechococcus sp PCC 7942 grown under different light intensities using photoacoustic and spectroscopic methods. The light-saturated capacity for PS I cyclic electron transport increased relative to chlorophyll concentration, PS I concentration, and linear electron transport capacity as growth light intensity was raised. In cells grown under moderate to high light intensity, PS I cyclic electron transport was nearly insensitive to methyl viologen, indicating that the cyclic electron supply to PS I derived almost exclusively from a thylakoid dehydrogenase. In cells grown under low light intensity, PS I cyclic electron transport was partially inhibited by methyl viologen, indicating that part of the cyclic electron supply to PS I derived directly from ferredoxin. It is proposed that the increased PSI cyclic electron transport observed in cells grown under high light intensity is a response to chronic photoinhibition.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES energy storage - MV methyl viologen - PA_m photoacoustic thermal signal with strong non-modulated background light added - PA_s photoacoustic thermal signal without background light added CIW/DPB Publication No. 1205.     

Characterization of a Synechococcus sp. strain PCC 7002 mutant lacking Photosystem I. Protein assembly and energy distribution in the absence of the Photosystem I reaction center core complex

A Synechococcus sp. strain PCC 7002 psaAB::cat mutant has been constructed by deletional interposon mutagenesis of the psaA and psaB genes through selection and segregation under low-light conditions. This strain can grow photoheterotrophically with glycerol as carbon source with a doubling time of 25 h at low light intensity (10 E m^–2 s^–1). No Photosystem I (PS I)-associated chlorophyll fluorescence emission peak was detected in the psaAB::cat mutant. The chlorophyll content of the psaAB::cat mutant was approximately 20% that of the wild-type strain on a per cell basis. In the absence of the PsaA and PsaB proteins, several other PS I proteins do not accumulate to normal levels. Assembly of the peripheral PS I proteins PsaC,PsaD, PsaE, and PsaL is dependent on the presence of the PsaA and PsaB heterodimer core. The precursor form of PsaF may be inserted into the thylakoid membrane but is not processed to its mature form in the absence of PsaA and PsaB. The absence of PS I reaction centers has no apparent effect on Photosystem II (PS II) assembly and activity. Although the mutant exhibited somewhat greater fluorescence emission from phycocyanin, most of the light energy absorbed by phycobilisomes was efficiently transferred to the PS II reaction centers in the absence of the PS I. No light state transition could be detected in the psaAB::cat strain; in the absence of PS I, cells remain in state 1. Development of this relatively light-tolerant strain lacking PS I provides an important new tool for the genetic manipulation of PS I and further demonstrates the utility of Synechococcus sp. PCC 7002 for structural and functional analyses of the PS I reaction center.Abbreviations ATCC American type culture collection - Chl chlorophyll - DCMU 3-(3,4-dichlorophyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - PCC Pasteur culture collection - PS I Photosystem I - PS II Photosystem II - SDS sodium dodecyl sulfate     

Phycobilisome Mobility in the Cyanobacterium Synechococcus sp. PCC7942 is Influenced by the Trimerisation of Photosystem I

We have constructed a mutant of the cyanobacterium Synechococcus sp. PCC7942 deficient in the Photosystem I subunit PsaL. As has been shown in other cyanobacteria, we find that Photosystem I is exclusively monomeric in the PsaL(-) mutant: no Photosystem I trimers can be isolated. The mutation does not significantly alter pigment composition, photosystem stoichiometry, or the steady-state light-harvesting properties of the cells. In agreement with a study in Synechococcus sp. PCC7002 [Schluchter et al. (1996) Photochem Photobiol 64: 53-66], we find that state transitions, a physiological adaptation of light-harvesting function, occur significantly faster in the PsaL(-) mutant than in the wild-type. To explore the reasons for this, we have used fluorescence recovery after photobleaching (FRAP) to measure the diffusion of phycobilisomes in vivo. We find that phycobilisomes diffuse, on average, nearly three times faster in the PsaL(-) mutant than in the wild-type. We discuss the implications for the mechanism of state transitions in cyanobacteria.     

Purification of C-Type Cytochromes from a Thermophilic Blue-Green Alga, Synechococcus vulcanus, by Hydrophobic Column Chromatography using Toyopearls

Two C-type cytochromes, c-550 and c-553, were extracted by animproved procedure from a thermophilic blue-green alga, Synechococcusvulcanus, and effectively purified by a two-step hydrophobicchromatography. The first step was performed with a ToyopearlHW-65C :ammonium sulfate-66 column and the second with a butyl-Toyopearl650 column. This work is the first to apply butyl-toyopearl650 to protein purification. (Received July 2, 1984; Accepted September 13, 1984)     

Inorganic Carbon Accumulation Stimulates Linear Electron Flow to Artificial Electron Acceptors of Photosystem I in Air-Grown Cells of the Cyanobacterium Synechococcus UTEX 625

The effect of inorganic carbon (Ci) transport and accumulation on photosynthetic electron transport was studied in air-grown cells of the cyanobacterium Synechococcus UTEX 625. When the cells were depleted of Ci, linear photosynthetic electron flow was almost completely inhibited in the presence of the photosystem I (PSI) acceptor N,N-dimethyl-p-nitrosoaniline (PNDA). The addition of Ci to these cells, in which CO2 fixation was inhibited with glycolaldehyde, greatly stimulated linear electron flow and resulted in increased levels of photochemical quenching and O2 evolution. In aerobic conditions substantial quenching resulted from methyl viologen (MV) addition and further quenching was not observed upon the addition of Ci. In anaerobic conditions MV addition did not result in quenching until Ci was added. Intracellular Ci pools were formed when MV was present in aerobic or anaerobic conditions or PNDA was present in aerobic conditions. There was no inhibitory effect of Ci depletion on electron flow to 2,6-dimethylbenzoquinone and oxidized diaminodurene, which accept electrons from photosystem II. The degree of stimulation of PNDA-dependent O2 evolution varied with the Ci concentration. The extracellular Ci, concentration required for a half-maximum rate (K1/2) was 3.8 [mu]M and the intracellular K1/2 was 1.4 mM for the stimulation of PNDA reduction. These values agreed closely with the K1/2 values of extracellular and intracellular Ci for O2 photoreduction. Linear electron flow to artificial electron acceptors of PSI was enhanced by intracellular Ci, which appeared to exert an effect on PSI or on the intersystem electron transport chain.     

Heat-Stability of Iron-Sulfur Centers and P-700 in Photosystem I Reaction Center Complexes Isolated from the Thermophilic Cyanobacterium Synechococcus elongatus

Stabilities of iron-sulfur centers and reaction center chlorophyllP-700 in Photosystem I reaction center complex (CP1-a), isolatedby sodium dodecyl sulfate treatment from the thermophilic cyanobacteriumSynechococcus elongatus, were studied by EPR and optical spectroscopy.P-700 was destroyed by treatment at temperatures above 80?Cfor 5 minutes with a half inactivation temperature of 93?C.The three iron-sulfur centers F_A, F_B and F_X showed similar thermalstabilities and were half inactivated at about 70?C. Thus, theisolated Photosystem I reaction center complexes of S. elongatusare still highly resistant to heat. (Received May 9, 1990; Accepted June 25, 1990)     

The structure of Photosystem I from the thermophilic cyanobacterium Synechococcus sp. determined by electron microscopy of two-dimensional crystals.

The structure of the Photosystem I (PS I) complex from the thermophilic cyanobacterium Synechococcus sp. has been investigated by electron microscopy and image analysis of two-dimensional crystals. Crystals were obtained from isolated PS I by removal of detergents with Bio-Beads. After negative staining, either single layers or two superimposed layers with a rotational different orientation were observed. The layers have a rectangular unit cell of 16.0 x 15.0 nm, which contains two PS I monomers. The monomers are arranged alternating up and down in each layer. For double-layer crystals, the images of the two layers could be separately processed by a combination of Fourier-peak-filtering and correlation averaging. Features in the two-dimensional plane can be seen with a resolution up to 1.5-1.8 nm. A model for the PS I structure was obtained by combining three-dimensional reconstructions from three tilt-series. The model shows an asymmetric PS I complex. On one side (presumably the stromal side) there is a 3 nm high ridge. This is most likely comprised of the psaC, psaD and psaE subunits. The other side (presumably the lumenal side) is rather flat, but in the center there is a 3 nm deep indentation, which possibly separates partly the two large subunits psaA and psaB.     

Stoichiometry of Photosystem I, Photosystem II, and Phycobilisomes in the Red Alga Porphyridium cruentum as a Function of Growth Irradiance

Cells of the red alga Porphyridium cruentum (ATCC 50161) exposed to increasing growth irradiance exhibited up to a three-fold reduction in photosystems I and II (PSI and PSII) and phycobilisomes but little change in the relative numbers of these components. Batch cultures of P. cruentum were grown under four photon flux densities of continuous white light; 6 (low light, LL), 35 (medium light, ML), 180 (high light, HL), and 280 (very high light, VHL) microeinsteins per square meter per second and sampled in the exponential phase of growth. Ratios of PSII to PSI ranged between 0.43 and 0.54. About three PSII centers per phycobilisome were found, regardless of growth irradiance. The phycoerythrin content of phycobilisomes decreased by about 25% for HL and VHL compared to LL and ML cultures. The unit sizes of PSI (chlorophyll/P₇₀₀) and PSII (chlorophyll/Q_A) decreased by about 20% with increase in photon flux density from 6 to 280 microeinsteins per square meter per second. A threefold reduction in cell content of chlorophyll at the higher photon flux densities was accompanied by a twofold reduction in β-carotene, and a drastic reduction in thylakoid membrane area. Cell content of zeaxanthin, the major carotenoid in P. cruentum, did not vary with growth irradiance, suggesting a role other than light-harvesting. HL cultures had a growth rate twice that of ML, eight times that of LL, and slightly greater than that of VHL cultures. Cell volume increased threefold from LL to VHL, but volume of the single chloroplast did not change. From this study it is evident that a relatively fixed stoichiometry of PSI, PSII, and phycobilisomes is maintained in the photosynthetic apparatus of this red alga over a wide range of growth irradiance.     

Harvesting far-red light: Functional integration of chlorophyll f into Photosystem I complexes of Synechococcus sp. PCC 7002

The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photosynthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cyanobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectroscopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally connected to the reaction center of the PSI complex and their presence does not change the overall pigment organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photosynthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis.