Role of the PB-loop in ApcE and phycobilisome core function in cyanobacterium Synechocystis sp. PCC 6803

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (L_CM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus.     

Changes in cyclic and respiratory electron transport by the movement of phycobilisomes in the cyanobacterium Synechocystis sp. strain PCC 6803

Phycobilisomes (PBS) are the major accessory light-harvesting complexes in cyanobacteria and their mobility affects the light energy distribution between the two photosystems. We investigated the effect of PBS mobility on state transitions, photosynthetic and respiratory electron transport, and various fluorescence parameters in Synechocystis sp. strain PCC 6803, using glycinebetaine to immobilize and couple PBS to photosystem II (PSII) or photosystem I (PSI) by applying under far-red or green light, respectively. The immobilization of PBS at PSII inhibited the increase in cyclic electron flow, photochemical and non-photochemical quenching, and decrease in respiration that occurred during the movement of PBS from PSII to PSI. In contrast, the immobilization of PBS at PSI inhibited the increase in respiration and photochemical quenching and decrease in cyclic electron flow and non-photochemical quenching that occurred when PBS moved from PSI to PSII. Linear electron transport did not change during PBS movement but increased or decreased significantly during longer illumination with far-red or green light, respectively. This implies that PBS movement is completed in a short time but it takes longer for the overall photosynthetic reactions to be tuned to a new state.     

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ?sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H₂O₂, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ?sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ?sigCDE than in the control strain. These results indicate that ?sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ?sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ?sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ?sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ?sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.     

Phycobilisomes of wild type and pigment mutants of the cyanobacterium Synechocystis PCC 6803

Mutants affected in their pigment content and in the structure of their phycobilosomes (PBS) were isolated in the cyanobacterium Synechocystis PCC 6803 by enriching a population with the inhibitor p-hydroxymercuribenzoate. Three of these mutants, PMB 2, PMB 10 and PMB 11, with original phenotypes, are described. Applying several criteria of analysis (77K absorption and fluorescence, protein electrophoretic patterns, electron microscopy), it was possible to assign the component polypeptides to each substructure of the phycobilisome. The model structure obtained fits with those described in other species PMB 10 and PMB 11, completely lacking PC, are the first source of pure PBS cores available, in which no contamination by residual PC can be feared, and are thus particularly interesting for further biochemical studies. The capacity of genetic transformation of Synechocystis PCC 6803 by chromosomal DNA makes this system very convenient for the analysis of the regulation of synthesis of the PBS constituents.Abbreviations PSI, PSII photosystems I, II - PBS phycobilisomes - PC phycocyanin - APC allophycocyanin - APC-B alophycocyanin B - PE phycoerythrin - PEC phycoerythrocyanin - WT wind type - Chl chlorophyll Present address: Service de Physiologie Microbienne Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15, France     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE⁻ mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with - presumably - allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.     

Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803

The dxr gene encoding the 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp. PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR. The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration. The purified recombinant native and histidine-tagged enzymes each displayed a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, corresponding to the calculated subunit molecular weights of 42,500 and 46,700, respectively. By native PAGE, both enzymes were dimers under reducing conditions. The kinetic properties for the enzymes were characterized and only minor variations were observed, demonstrating that the N-terminal histidine tag does not greatly affect the activity of the enzyme. Both enzymes had similar properties to previously characterized reductoisomerases from other sources. The K_m's for the metal ions Mn²⁺, Mg²⁺, and Co²⁺ were determined for native DXR for the first time, with the K_m for Mg²⁺ being approximately 200-fold higher than the K_m's for Mn²⁺ and Co²⁺.     

Spectrally decomposed dark-to-light transitions in a PSI-deficient mutant of Synechocystis sp. PCC 6803

Cyanobacterial thylakoid membranes are known to host photosynthetic and respiratory complexes. This hampers a straight forward interpretation of the highly dynamic fluorescence originating from photosynthetic units. The present study focuses on dark-to-light transitions in whole cells of a PSI-deficient mutant of the cyanobacterium Synechocystis sp. PCC 6803. The time-dependent cellular fluorescence spectrum has been measured, while having previously exposed the cells to different conditions that affect respiratory activity. The analysis method used allows the detected signal to be decomposed in a few components that are then assigned to functional emitting species. Additionally, we have worked out a minimal mathematical model consisting of sensible postulated species to interpret the recorded data. We conclude that the following two functional complexes play a major role: a phycobilisome antenna complex coupled to a PSII dimer with either two or no closed reaction centers. Crucially, we present evidence for an additional species capable of strongly quenching fluorescence, whose formation requires the presence of oxygen.     

Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803

Geranylgeranyl reductase catalyses the reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate required for synthesis of chlorophylls, phylloquinone and tocopherols. The gene chlP (ORF sll1091) encoding the enzyme has been inactivated in the cyanobacterium Synechocystis sp. PCC 6803. The resulting ΔchlP mutant accumulates exclusively geranylgeranylated chlorophyll a instead of its phytylated analogue as well as low amounts of α-tocotrienol instead of α-tocopherol. Whereas the contents of chlorophyll and total carotenoids are decreased, abundance of phycobilisomes is increased in ΔchlP cells. The mutant assembles functional photosystems I and II as judged from 77 K fluorescence and electron transport measurements. However, the mutant is unable to grow photoautotrophically due to instability and rapid degradation of the photosystems in the absence of added glucose. We suggest that instability of the photosystems in ΔchlP is directly related to accumulation of geranylgeranylated chlorophyll a. Increased rigidity of the chlorophyll isoprenoid tail moiety due to three additional CC bonds is the likely cause of photooxidative stress and reduced stability of photosynthetic pigment-protein complexes assembled with geranylgeranylated chlorophyll a in the ΔchlP mutant.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E_m) value of − 165 mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of − 220 mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8 Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient ΔpgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/ΔpgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A₁ and A₂ or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.     

The PsaE subunit of photosystem I prevents light-induced formation of reduced oxygen species in the cyanobacterium Synechocystis sp. PCC 6803

The PsaE protein is located at the reducing side of photosystem I (PSI) and is involved in docking the soluble electron acceptors, particularly ferredoxin. However, deletion of the psaE gene in the cyanobacterium Synechocystis sp. strain PCC 6803 inhibited neither photoautotrophic growth, nor in vivo linear and cyclic electron flows. Using photoacoustic spectroscopy, we detected an oxygen-dependent, PSI-mediated energy storage activity in the ΔpsaE null mutant, which was not present in the wild type (WT). The expression of the genes encoding catalase (katG) and iron superoxide dismutase (sodB) was upregulated in the ΔpsaE mutant, and the increase in katG expression was correlated with an increase in catalase activity of the cells. When catalases were inhibited by sodium azide, the production of reactive oxygen species was enhanced in ΔpsaE relative to WT. Moreover, sodium azide strongly impaired photoautotrophic growth of the ΔpsaE mutant cells while WT was much less sensitive to this inhibitor. The katG gene was deleted in the ΔpsaE mutant, and the resulting double mutant was more photosensitive than the single mutants, showing cell bleaching and lipid peroxidation in high light. Our results show that the presence of the PsaE polypeptide at the reducing side of PSI has a function in avoidance of electron leakage to oxygen in the light (Mehler reaction) and the resulting formation of toxic oxygen species. PsaE-deficient Synechocystis cells can counteract the chronic photoreduction of oxygen by increasing their capacity to detoxify reactive oxygen species.     

Temperature-dependent hyper-activation of monoglucosyldiacylglycerol synthase is post-translationally regulated in Synechocystis sp. PCC 6803

The mechanism of monoglucosyldiacylglycerol (MGlcDG) increase following heat shock in Synechocystis sp. PCC 6803 was examined by measuring MGlcDG synthase (Sll1377) activity. Temperature-dependent activation of Sll1377 was observed in the membrane fraction of Synechocystis sp. PCC 6803, whereas the Sll1377 protein level remained unchanged, suggesting that the activity is post-translationally regulated without covalent modification of Sll1377 by soluble enzymes. Four individual mutations introduced into recombinant Sll1377 (D147, D200, R329, and R331) significantly reduced the activity and blocked temperature-dependent activation, suggesting that these amino acid residues are essential for Sll1377 activity at both normal growth temperature and the higher temperature.     

Structural organisation of phycobilisomes from Synechocystis sp. strain PCC6803 and their interaction with the membrane

In cyanobacteria, the harvesting of light energy for photosynthesis is mainly carried out by the phycobilisome — a giant, multi-subunit pigment-protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides such that light absorption and energy transfer to photosystem II are optimised. In this work we have studied, using single particle electron microscopy, the phycobilisome structure in mutants lacking either two or all three of the phycocyanin hexamers. The images presented give much greater detail than those previously published, and in the best two-dimensional projection maps a resolution of 13 Å was achieved. As well as giving a better overall picture of the assembly of phycobilisomes, these results reveal new details of the association of allophycocyanin trimers within the core. Insights are gained into the attachment of this core to the membrane surface, essential for efficient energy transfer to photosystem II. Comparison of projection maps of phycobilisomes with and without reconstituted ferredoxin:NADP oxidoreductase suggests a location for this enzyme within the complex at the rod-core interface.     

Lack of digalactosyldiacylglycerol increases the sensitivity of Synechocystis sp. PCC 6803 to high light stress

The physiological role of digalactosyldiacylglycerol (DGDG) in photosynthesis was examined using a dgdA mutant of Synechocystis sp. PCC 6803 that is defective in the biosynthesis of DGDG. The dgdA mutant cells showed normal growth under low light (LL) conditions. However, their growth was retarded under high light (HL) conditions and under Ca²⁺- and/or Cl⁻-limited conditions compared to wild-type cells. The retardation in growth of the mutant cells was recovered by exogenous supply of DGDG in the growth medium. The dgdA mutant showed increased sensitivity to photoinhibition. Although both photodamage and repair processes of photosynthesis were affected, the repair process was more severely affected than the photodamage process, suggesting that DGDG plays an important role in the photosynthetic repair cycle.     

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni²⁺-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b₆f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.     

Heat- and light-induced reorganizations in the phycobilisome antenna of Synechocystis sp. PCC 6803. Thermo-optic effect

By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 °C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 °C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F₆₈₀/F₆₅₅ fluorescence intensity ratio. In contrast, heat treatment at 60 °C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F₆₅₅), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F₆₈₀ originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600-7500 μmol photons m⁻² s⁻¹) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 °C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS.     

Ferredoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803.

The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.     

Growth arrest of Synechocystis sp. PCC6803 by superoxide generated from heterologously expressed Rhodobacter sphaeroides chlorophyllide a reductase

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR-mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.     

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates,yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP⁺ to NADPH by ferredoxin–NADP⁺-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8 ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2 ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP⁺ + NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.