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PubMed1991

PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp. PCC 6301.

The psaC gene product from Synechococcus sp. PCC 7002 and the psaD gene product from Nostoc sp. PCC 8009 were synthesized in Escherichia coli and purified to homogeneity. Incubation of the PsaC apoprotein with the Synechoccus sp. PCC 6301 photosystem I core protein in the presence of FeCl3, Na2S, and beta-mercaptoethanol resulted in a time-dependent transition in the flash-induced absorption change from a 1.2-ms, P700+ FX- back-reaction to a long-lived, P700+ [FA/FB]- back-reaction. ESR studies showed that FB and FA were photoreduced about equally at 19 K, and while the resonances were shifted upfield, they remained as broad as in the free PsaC holoprotein. When the reconstituted complex was purified in a sucrose gradient containing 0.1% Triton X-100, most of the optical absorption transient reverted to that characteristic of the P700+ FX- back-reaction. Addition of purified PsaD to the incubation mixture led to a greater extent of recovery of electron flow to FA/FB for any given concentration of PsaC. ESR studies showed that FA, rather than FB, became the preferred electron acceptor at 19 K; moreover, the resonances moved upfield and sharpened to become nearly identical with those of a control photosystem I complex. When the sample was purified in a sucrose gradient containing 0.1% Triton X-100, the long-lived P700+ [FA/FB]- optical transient remained stable. Analysis by denaturing polyacrylamide gel electrophoresis showed that the PsaC and PsaD proteins had rebound to the photosystem I core. The data indicate that although PsaC can bind loosely, the presence of PsaD leads to a stable, isolatable photosystem I complex which is spectroscopically indistinguishable from the native complex. Since a PsaC1 fusion protein which contains an amino-terminal extension of five amino acids (MEHSM...) does not bind in the absence of PsaD [Zhao, J., et al. (1990) FEBS Lett. 276, 175-180], the N-terminus of the PsaC protein could provide a site of interaction with the photosystem I core. We propose that the binding of PsaC to the PsaA/PsaB heterodimer is potentiated by insertion of the FA/FB clusters into PsaC, and stabilized by the presence of PsaD.

Amino Acid Sequence↗

PubMed1990

Molecular weight determination of an active photosystem I preparation from a thermophilic cyanobacterium, Synechococcus elongatus.

An active photosystem I (PSI) complex was isolated from the thermophilic cyanobacterium Synechococcus elongatus by a procedure consisting of three steps: First, extraction of photosystem II from the thylakoids by a sulfobetaine detergent yields PSI-enriched membranes. Second, the latter are treated with Triton X-100 to extract PSI particles, which are further purified by preparative isoelectric focusing. Third, anion-exchange chromatography is used to remove contaminating phycobilisome polypeptides. The purified particles show three major bands in sodium dodecyl sulfate gel electrophoresis of apparent molecular mass of 110, 15, and 10 kDa. Charge separation was monitored by the kinetics of flash-induced absorption changes at 820 nm. A chlorophyll/P700 ratio of 60 was found. When the particles are stored at 4 degrees C, charge separation was stable for weeks. The molecular mass of the PSI particles, determined by measurement of zero-angle neutron scattering intensity, was 217,000 Da. The PSI particles thus consist of one heterodimer of the 60-80-kDa polypeptides and presumably one copy of the 15- and 10-kDa polypeptides, respectively.

Amino Acids↗

PubMed1981

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus.

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗

PubMed2001

Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c(6) in Synechococcus sp. PCC 7002 in vivo.

Cytochrome c(6) donates electrons to photosystem I (PS I) in Synechococcus sp. PCC 7002. In this work, we provide evidence for rapid electron transfer (t(1/2) = 3 micros) from cytochrome c(6) to PS I in this cyanobacterium in vivo, indicating prefixation of the reduced donor protein to the photosystem. We have investigated the cytochrome c(6)-PS I interaction by laser flash-induced spectroscopy of intact and broken cells and by redox titrations of membrane and supernatant fractions. Redox studies revealed the expected membrane-bound cytochrome f, b(6), and b(559) species and two soluble cytochromes with alpha-band absorption peaks of 551 and 553 nm and midpoint potentials of -100 and 370 mV, respectively. The characteristics and the symmetrical alpha-band spectrum of the latter correspond to typical cyanobacterial cytochrome c(6) proteins. Rapid oxidation of cytochrome c(6) by PS I in vivo results in a unique, asymmetric oxidation spectrum, which differs significantly from the spectra obtained for cytochrome c(6) in solution. The basis for the unusual cytochrome c(6) spectrum and possible mechanisms of cytochrome c(6) fixation to PS I are discussed. The occurrence of rapid electron transfer to PS I in cyanobacteria suggests that this mechanism evolved before the endosymbiotic origin of chloroplasts. Its selective advantage may lie in protection against photo-oxidative damage as shown for Chlamydomonas.

Cyanobacteria↗

PubMed2003

Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus.

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.

Cations↗

PubMed2003

Interaction of ferredoxin:NADP+ oxidoreductase with phycobilisomes and phycobilisome substructures of the cyanobacterium Synechococcus sp. strain PCC 7002.

The enzyme ferredoxin-NADP(+) oxidoreductase (FNR) from Synechococcus sp. PCC 7002 has an extended structure comprising three domains (FNR-3D) (Schluchter, W. M., and Bryant, D. A. (1992) Biochemistry 31, 3092-3102). Phycobilisome (PBS) preparations from wild-type cells contained from 1.0 to 1.6 molecules of FNR-3D per PBS, with an average value of 1.3 FNR per PBS. A maximum of two FNR-3D molecules could be specifically bound to wild-type PBS via the N-terminal, CpcD-like domain of the enzyme when exogenous recombinant FNR-3D (rFNR-3D) was added. To localize the enzyme within the PBS, the interaction of PBS and their substructures with rFNR-3D was further investigated. The binding affinity of rFNR-3D for phycocyanin (PC) hexamers, which contained a 22-kDa proteolytic fragment derived from CpcG, the L(RC)(27) linker polypeptide, was higher than its affinity for PC hexamers containing no linker protein. PBS from a cpcD3 mutant, which lacks the 9-kDa, PC-associated rod linker, incorporated up to six rFNR-3D molecules per PBS. PBS of a cpcC mutant, which has peripheral rods that contain single PC hexamers, also incorporated up to six rFNR-3D molecules per PBS. Direct competition binding experiments showed that PBS from the cpcD3 mutant bound more enzyme than PBS from the cpcC mutant. These observations support the hypothesis that the enzyme binds preferentially to the distal ends of the peripheral rods of the PBS. These data also show that the relative affinity order of the PC complexes for FNR-3D is as follows: (alpha(PC)beta(PC))(6)-L(R)(33) > (alpha(PC)beta(PC))(6)-L(RC)(27) > (alpha(PC)beta(PC))(6). The data suggest that, during the assembly of the PBS, FNR-3D could be displaced to the periphery according to its relative binding affinity for different PC subcomplexes. Thus, FNR-3D would not interfere with the light absorption and energy transfer properties of PC in the peripheral rods of the PBS. The implications of this localization of FNR within the PBS with respect to its function in cyanobacteria are discussed.

Amino Acid Sequence↗

PubMed2003

Functional differences of photosystem II from Synechococcus elongatus and spinach characterized by flash induced oxygen evolution patterns.

Detailed comparative studies of flash induced oxygen evolution patterns in thylakoids from the thermophilic cyanobacterium Synechococcus elongatus (S. elongatus; also referred to as Thermosynechococcus elongatus) and from spinach led to the following results: (i) the miss parameter alpha of S. elongatus thylakoids exhibits a pronounced temperature dependence with a minimum of 7% at 25 degrees C and values of 17 and 10% at 3 and 35 degrees C, respectively, while for spinach thylakoids alpha decreases continuously from 18% at 35 degrees C down to 8% at 3 degrees C; (ii) at all temperatures, the double hit probability beta exceeds in S. elongatus the corresponding values of spinach by an increment Delta beta of about 3%; (iii) at 20 degrees C the slow relaxation of the oxidation states S(2) and S(3) is about 15 and 30 times, respectively, slower in S. elongatus than in spinach, while the reduction of these S states by tyrosine Y(D) is 2-3 times faster; (iv) the reaction S(0)Y(D)(ox) --> S(1)Y(D) is slower by a factor of 4 in S. elongatus as compared to spinach; and (v) the activation energies of S state dark relaxations in S. elongatus are all within a factor of 1.5 as compared to the previously reported values from spinach thylakoids [Vass, I., Deak, Z., and Hideg, E. (1990) Biochim. Biophys. Acta 1017, 63-69; Messinger, J., Schröder, W. P., and Renger, G. (1993) Biochemistry 32, 7658-7668], but the difference between the activation energies of the slow S(2) and S(3) decays is significantly larger in S. elongatus than in spinach. These results are discussed in terms of differences between cyanobacteria and higher plants on the acceptor side of PSII and a shift of the redox potential of the couple Y(D)/Y(D)(ox). The obtained data are also suitable to address questions about effects of the redox state of Y(D) on the miss probability and the possibility of an S state dependent miss parameter.

Adaptation, Physiological↗

PubMed2004

Removal of PsaF alters forward electron transfer in photosystem I: evidence for fast reoxidation of QK-A in subunit deletion mutants of Synechococcus sp. PCC 7002.

Recent studies of point mutations in photosystem I have suggested that the two kinetic phases of phylloquinone reoxidation represent electron transfer in the two branches of cofactors. This interpretation implies that changes in the relative amplitudes of the two kinetic phases represent a change in the extent of electron transfer in the two branches. Using time-resolved electron paramagnetic resonance (EPR), this issue is investigated in subunit deletion mutants of Synechococcus sp. PCC 7002. The spin-polarized EPR signals of P(700)(+)A(1)(-) and P(700)(+)FeS(-), both at room temperature and in frozen solution, are altered by deletion of PsaF and/or PsaE, and the differences from the wild type are much more pronounced in PS I complexes isolated from the mutants using Triton X-100 rather than n-dodecyl beta-d-maltopyranoside. The changes in the transient EPR data for the mutant complexes are consistent with a significant fraction of reaction centers showing (i) faster electron transfer from A(1)(-) to F(X), (ii) slower forward electron transfer from A(0)(-) to A(1), and (iii) slightly altered quinone hyperfine couplings, possibly as a result of a change in the hydrogen bonding. The fraction of fast electron transfer and its dependence on the isolation procedure are estimated approximately from simulations of the room temperature EPR data. The results are discussed in terms of possible models for the electron transfer. It is suggested that the detergent-induced fraction of fast electron transfer is most likely due to alteration of the environment of the quinone in the PsaA branch of cofactors and is not the result of a change in the directionality of electron transfer.

Chlorophyll↗

PubMed1996

Laser flash absorption spectroscopy study of flavodoxin reduction by photosystem I in Synechococcus sp. PCC 7002.

The photoreduction of flavodoxin by trimeric photosystem I, both from the cyanobacterium Synechococcus sp. PCC 7002, was investigated by flash absorption spectroscopy. After addition of flavodoxin in darkness, single flash experiments show that the transient signals change between individual flashes. This behavior is assigned to a progressive accumulation of flavodoxin semiquinone, which is relatively stable under most experimental conditions. Different conditions were devised in order to study the reduction of the oxidized and semiquinone forms of flavodoxin separately. Both processes were identified by their differential spectra measured between 460 and 630 nm. Detailed kinetic characteristics of flavodoxin reduction were obtained at pH 8.0 in the presence of salts. The kinetics of reduction of oxidized flavodoxin displays a single-exponential component. The rate of this component increases with the flavodoxin concentration up to an asymptotic value of about 600 s-1. The semiquinone form of flavodoxin being protonated, this rate corresponds to a rate-limiting reaction which could be either an electron transfer reaction or a protonation reaction. In contrast, the reduction of flavodoxin semiquinone is biphasic. A fast first-order phase with t 1/2 approximately 10 microseconds is interpreted as an electron transfer process within a preformed complex. A dissociation constant of 2.64 microM is calculated for this complex by assuming a simple binding equilibrium between photosystem I and flavodoxin semiquinone. The slower phase observed for semiquinone reduction is concentration dependent, and a second-order rate constant of 1.7 x 10(8) M-1 s-1 is calculated. For both one-electron reduction steps, different optimal salt concentrations are observed indicating slightly different interactions between photosystem I and flavodoxin in its oxidized and semiquinone states.

Cyanobacteria↗

PubMed1996

Structure of Synechococcus elongatus [Fe2S2] ferredoxin in solution.

Ferredoxins of the [Fe2S2] type function in photosynthetic electron transport as essential electron acceptors of photosystem I. The solution structure of the 97 amino acid ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus was determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. The structure consists of a four-stranded parallel/ antiparallel beta-sheet, a short two-stranded antiparallel beta-sheet, and three short helices. The overall structure is similar to the structure of the ferredoxin from Anabaena. In contrast to related ferredoxins from mesophilic organisms, this thermostable protein contains a salt bridge inside a 17-amino acid hydrophobic core.

Amino Acid Sequence↗

PubMed1998

Effect of near-infrared light on the S2-state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn cluster of Photosystem II (PSII) from Synechococcus elongatus was studied using EPR. A signal with features between g = 5 and g = 9 is reported from the S2-state. The signal is attributed to the manganese cluster in a state with a spin 5/2 state. Spectral simulations of the signal indicate zero field splitting parameters where the |E/D| was 0.13. The new signal is formed by irradiating PSII samples which contain the spin = 1/2 S2-state using 813 nm light below 200 K. This effect is attributed to a spin-state change in the manganese cluster due to absorption of the IR light by the Mn-cluster itself. The signal is similar to that reported recently in PSII of plants [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. In plant PSII the comparable signal is formed at a lower temperature (optimally below 77 K), and gradual warming of the sample in the dark leads to the formation of the state responsible for the well-known g = 4.1 signal prior to formation of the spin 1/2 multiline signal. In the present work using cyanobacterial PSII, warming of the sample in the dark leads to the formation of the spin 1/2 multiline signal without formation of the g = 4 type signal as an intermediate. These observations provide a partial explanation for the long-standing "mystery of the missing g = 4 state" in cyanobacterial PSII. The observations are rationalized in terms of three possible states which can exist for S2: (i) the spin 1/2 multiline signal, (ii) the state responsible for the g = 4.1 signal, and (iii) the new spin 5/2 state. The relative stability of these states differs between plants and cyanobacteria.

Cyanobacteria↗

PubMed1999

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.

Amino Acid Sequence↗

PubMed1999

Detection of an electron paramagnetic resonance signal in the S0 state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.

Cyanobacteria↗

PubMed1998

Expression of psbA genes produces prominent 5' psbA mRNA fragments in Synechococcus sp. PCC 7942.

Expression of the psbA genes, which in the cyanobacterium Synechococcus sp. PCC 7942 encode two different forms of the reaction centre D1 protein of photosystem II (D1:1 and D1:2), was studied under different light and temperature conditions. In addition to the mature 1200 nt psbA messages, three shorter mRNA fragments of 220, 320 and 900 nt were also found. All three mRNA fragments could be recognized by using different gene probes from the coding region of the psbAI gene, whereas the corresponding psbAII/III gene probes recognized only the 220 nt mRNA fragment. The 5' 320 nt mRNA fragment from the psbAI gene probably represents a degradation product, since the corresponding 3' 900 nt psbAI mRNA fragment was also detected. By contrast, the 5' 220 nt mRNA fragment of all psbA messages is suggested to be a truncated psbA transcript, since no corresponding 3' fragment was ever found. Inhibition of translation either by a protein synthesis inhibitor or by a shift of cells to lower temperature, increased the number of 1200 nt psbAII/III messages but the number of 5' 220 nt psbAII/III mRNA fragment increased even more dramatically. The first 66 bp after ATG, where the psbAI and psbAII/III genes mostly differ from each other, also appeared important in determining the amount of produced truncated psbA transcripts, as evidenced by the expression of different tac-psbA constructs in the presence of protein synthesis inhibitor. We suggest that both the psbAI and the psbAII/III genes have a latent intragenic termination site and truncated psbA transcripts are produced at high levels under stress conditions when transcription becomes uncoupled from translation. This is to prevent wasting metabolic energy in the production of unused transcripts.

Cyanobacteria↗

PubMed1999

A thermophilic cyanobacterium Synechococcus elongatus has three different Class I prenyltransferase genes.

Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C5) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C20) diphosphate synthase, another encodes a farnesyl (C15) diphosphate synthase whose optimal reaction temperature is 60 degrees C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 degrees C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C25), hexaprenyl (C30), and heptaprenyl (C35) diphosphates from dimethylallyl (C5) diphosphate, geranyl (C10) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).

Amino Acid Sequence↗

PubMed2000

Characterization of processes responsible for the distinct effect of herbicides DCMU and BNT on Photosystem II photoinactivation in cells of the cyanobacterium Synechococcus sp. PCC 7942.

Light-induced modification of Photosystem II (PS II) complex was characterized in the cyanobacterium Synechococcus sp. PCC 7942 treated with either DCMU (a phenylurea PS II inhibitor) or BNT (a phenolic PS II inhibitor). The irradiance response of photoinactivation of PS II oxygen evolution indicated a BNT-specific photoinhibition that saturated at relatively low intensity of light. This BNT-specific process was slowed down under anaerobiosis, was accompanied by the oxygen-dependent formation of a 39 kDa D1 protein adduct, and was not related to stable Q(A) reduction or the ADRY effect. In the BNT-treated cells, the light-induced, oxygen-independent initial drop of PS II electron flow was not affected by formate, an anion modifying properties of the PS II non-heme iron. For DCMU-treated cells, anaerobiosis did not significantly affect PS II photoinactivation, the D1 adduct was not observed and addition of formate induced similar initial decrease of PS II electron flow as in the BNT-treated cells. Our results indicate that reactive oxygen species (most likely singlet oxygen) and modification of the PS II acceptor side are responsible for the fast BNT-induced photoinactivation of PS II.

Journal Article↗

PubMed2002

Purification, cofactor analysis, and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase.

The narB gene of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an assimilatory nitrate reductase that uses photosynthetically reduced ferredoxin as the physiological electron donor. This gene was expressed in Escherichia coli and electrophoretically pure preparations of the enzyme were obtained using affinity chromatography with either reduced-ferredoxin or NarB antibodies. The electronic absorption spectrum of the oxidized enzyme showed a shoulder at around 320 nm and a broad absorption band between 350 and 500 nm. These features are indicative of the presence of an iron-sulfur centre(s) and accordingly metal analysis showed ca. 3 atoms of Fe per molecule of protein that could represent a [3Fe-4S] cluster. Further analysis indicated the presence of 1 atom of Mo and 2 molecules of ribonucleotide-conjugated molybdopterin per molecule of protein. This, together with the requirement of a mobA gene for production of an active enzyme, strongly suggests the presence of Mo in the form of the bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor in Synechococcusnitrate reductase. A model for the coordination of the Mo atom to the enzyme is proposed. Four conserved Cys residues were replaced by site-directed mutagenesis. The effects of these changes on the enzyme activity and electronic absorption spectra support the participation of those residues in iron-sulfur cluster coordination.

Journal Article↗

PubMed2002

Synergistic effect of high-light and low temperature on cell growth of the Delta12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002.

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Delta12 fatty acid desaturase mutant strains. Under a light intensity of 250 mumol m(-2) s(-1), wild-type cells could grow exponentially in a temperature range of 20-38 degrees C, but growth was non-exponential below 20 degrees C and ceased at 12 degrees C. The Delta12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25-38 degrees C but grew slowly at 22 degrees C, and no cell growth took place below 18 degrees C. Under a very high-light intensity of 2.5 mmol m(-2) s(-1), wild-type cells could grow exponentially in a temperature range of 30-38 degrees C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Delta12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 degrees C, but were killed at 30 degrees C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Delta12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 degrees C under the normal light intensity of 250 mumol m(-2) s(-1). These results suggest that growth of the Delta12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria.

Journal Article↗