Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits. The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex. We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring. The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring.     

Targeted deletion of psaJ from the cyanobacterium Synechocystis sp. PCC 6803 indicates structural interactions between the PsaJ and PsaF subunits of photosystem I

Photosystem I catalyzes the light-driven oxidation of plastocyanin or cytochrome c ₆ and the reduction of ferredoxin or flavodoxin. PsaJ is a 4.4 kDa hydrophobic subunit of photosystem I from cyanobacteria and chloroplasts. To investigate the function of PsaJ, we generated a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 in which the psaJ gene is replaced by a gene for chloramphenicol resistance. Deletion of psaJ led to a reduction in the steady state RNA level from psaF which is located upstream from psaJ. Immunoquantification using an anti-PsaF antibody revealed a significant decrease in the amount of PsaF in membranes of the mutant strain. Trimeric photosystem I complexes isolated from the mutant strain using n-dodecyl -D-maltoside lacked PsaJ, contained ca. 80% less PsaF, but maintained wild-type levels of other photosystem I subunits. In contrast, the photosystem I purified using Triton X-100 contained less than 2% PsaF when compared to the wild type, showing the more extractable nature of PsaF in PsaJ-less photosystem I in the presence of Triton X-100. PsaE was more accessible to removal by NaI in a mutant strain lacking PsaF and PsaJ than in the wild type. The presence of PsaF in photosystem I from the PsaJ-less strain did not alter the increased susceptibility of PsaE to removal by NaI. These results indicate an interaction between PsaJ and PsaF in the organization of the complex.     

A comparative fluorescence kinetics study of Photosystem I monomers and trimers from Synechocystis PCC 6803

Picosecond time-resolved fluorescence measurements have been performed as a function of emission wavelengths in order to investigate the possible functional differences between monomeric and trimeric Photosystem I (PS I) particles from a cyanobacterium Synechocystis. Applying global analysis, four kinetic components were found necessary to describe the fluorescecne decay for both monomers and trimers of PS I. The lifetimes and spectra of the respective components are quite similar, indicating that they can be attributed to identical processes in both the monomers and trimers. It is concluded that both forms of PS I are capable of efficient energy transfer and charge separation, in agreement with a physiological role of both forms. Small differences in the fluorescence decays are discussed in terms of a slightly higher ratio of red emitting pigments per reaction centre in trimers of PS I. A comparison to Synechococcus PS I particles reveals the higher red chlorophyll content of the latter.Abbreviations -DM- -dodecyl-maltoside - Chl- chlorophyll - CMC- critical micellar concentration - DAS- decay-associated spectrum - DCM- 4-dicyano-methylene-2-methyl-6-(-dimethyl-aminostyryl)-4h-pyran - FWHM- full-width at half-maximum - P700- primary electron donor of Photosystem I - PS- photosystem - RC- reaction centre     

Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function.

To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 microE m-2 sec-1), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 microE m-2 sec-1 (doubling time approximately 28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-less/apcE- strain grew photoheterotrophically at normal light intensity (50 microE m-2 sec-1) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-less/apcE- strains had an approximately sixfold enrichment of PSII on a chlorophyll basis and were as active in oxygen evolution (on a per PSII basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSII studies and as background strains to analyze site- and region-directed PSII mutants in vivo.     

Temperature dependence and polarization of fluorescence from Photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

To determine the fluorescence properties of cyanobacterial Photosystem I (PS I) in relatively intact systems, fluorescence emission from 20 to 295 K and polarization at 77 K have been measured from phycobilisomes-less thylakoids of Synechocystis sp. PCC 6803 and a mutant strain lacking Photosystem II (PS II). At 295 K, the fluorescence maxima are 686 nm in the wild type from PS I and PS II and at 688 nm from PS I in the mutant. This emission is characteristic of bulk antenna chlorophylls (Chls). The 690-nm fluorescence component of PS I is temperature independent. For wild-type and mutant, 725-nm fluorescence increases by a factor of at least 40 from 295 to 20 K. We model this temperature dependence assuming a small number of Chls within PS I, emitting at 725 nm, with an energy level below that of the reaction center, P700. Their excitation transfer rate to P700 decreases with decreasing temperature increasing the yield of 725-nm fluorescence.Fluorescence excitation spectra of polarized emission from low-energy Chls were measured at 77 and 295 K on the mutant lacking PS II. At excitation wavelengths longer than 715 nm, 760-nm emission is highly polarized indicating either direct excitation of the emitting Chls with no participation in excitation transfer or total alignment of the chromophores. Fluorescence at 760 nm is unpolarized for excitation wavelengths shorter than 690 nm, inferring excitation transfer between Chls before 760-nm fluorescence occurs.Our measurements illustrate that: 1) a single group of low-energy Chls (F725) of the core-like PS I complex in cyanobacteria shows a strongly temperature-dependent fluorescence and, when directly excited, nearly complete fluorescence polarization, 2) these properties are not the result of detergent-induced artifacts as we are examining intact PS I within the thylakoid membrane of S. 6803, and 3) the activation energy for excitation transfer from F725 Chls to P700 is less than that of F735 Chls in green plants; F725 Chls may act as a sink to locate excitations near P700 in PS I.Abbreviations Chl chlorophyll - BChl bacteriochlorophyll - PS Photosystem - S. 6803 Synechocystis sp. PCC 6803 - PGP potassium glycerol phosphate     

Fluorescence from low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at physiological temperatures

Fluorescence spectra from Photosystem I (PS I) are measured from 25 to –5 °C on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Emission from antenna chlorophylls (Chls) with energy levels below that of the reaction center, or low-energy Chls (LE Chls), is resolved verifying their presence at physiological temperatures. The 25°C spectrum is characterized by peaks at 688 and 715 nm. As temperature decreases, fluorescence at 688 nm decreases while at 715 nm it increases. The total fluorescence yield does not change. The temperature dependent spectra are fit to a sum of two basis spectra. At 25°C, the first basis spectrum has a major peak at 686 nm and a minor peak at 740 nm. This is attributed to fluorescence from the majority or bulk antenna Chls. The second basis spectrum has a major peak at 712 nm, with shoulders at 722 and 770 nm. It characterizes fluorescence from a small number of LE Chls. A progressive shift to the red in the fluorescence spectra occurs as the temperature is decreased. The temperature dependence in the relative amount of fluorescence from the bulk and LE Chls is fit using a two-component energy transfer model at thermal equilibrium.     

Excitation wavelength dependence of the fluorescence kinetics in Photosystem I particles from Synechocystis PCC 6803 and Synechococcus elongatus

The excitation-wavelength dependence of the excited-state dynamics of monomeric and trimeric Photosystem I (PSI) particles from Synechocystis PCC 6803 as well as trimeric PSI particles from Synechococcus elongatus has been studied at room temperature using time-resolved fluorescence spectroscopy. For aselective (400 nm), carotenoid (505 nm), and bulk chlorophyll (approximately 650 nm) excitation in all species, a downhill energy-transfer component is observed, corresponding to a lifetime of 3.4-5.5 ps. For selective red excitation (702-719 nm) in all species, a significantly faster, an approximately 1-ps, uphill transfer component was recorded. In Synechococcus PSI, an additional approximately 10-ps downhill energy-transfer component is found for all wavelengths of excitation, except 719 nm. Each of the species exhibits its own characteristic trap spectrum, the shape of which is independent of the wavelength of excitation. This trap spectrum decays in approximately 23 ps in both monomeric and trimeric Synechocystis PSI and in approximately 35 ps in trimeric Synechococcus PSI. The data were simulated based on the 2.5 A structural model of PSI of Synechococcus elongatus using the F?rster equation for energy transfer, and using the 0.6-1-ps charge-separation time and the value of 1.2-1.3 for the index of refraction that were obtained from the dynamics of a hypothetical PSI particle without red chls. The experimentally obtained lifetimes and spectra were reproduced well by assigning three of the chlorophyll-a (chla) dimers observed in the structure to the C708/C702RT pool of red chls present in PSI from both species. Essential for the simulation of the dynamics of Synechococcus PSI is the assignment of the single chla trimer in the structure to the C719/C708RT pool present in this species.     

Electronic spectra of PS I mutants: the peripheral subunits do not bind red chlorophylls in Synechocystis sp. PCC 6803

Steady-state fluorescence and absorption spectra have been obtained in the Qy spectral region (690-780 nm and 600-750 nm, respectively) for several subunit-deficient photosystem I mutants from the cyanobacterium Synechocystis sp. PCC 6803. The 77 K fluorescence spectra of the wild-type and subunit-deficient mutant photosystem I particles are all very similar, peaking at approximately 720 nm with essentially the same excitation spectrum. Because emission from far-red chlorophylls absorbing near 708 nm dominates low-temperature fluorescence in Synechocystis sp., these pigments are not coordinated to any the subunits PsaF, Psa I, PsaJ, PsaK, PsaL, or psaM. The room temperature (wild-type-mutant) absorption difference spectra for trimeric mutants lacking the PsaF/J, PsaK, and PsaM subunits suggest that these mutants are deficient in core antenna chlorophylls (Chls) absorbing near 685, 670, 675, and 700 nm, respectively. The absorption difference spectrum for the PsaF/J/I/L-deficient photosystem I complexes at 5 K reveals considerably more structure than the room-temperature spectrum. The integrated absorbance difference spectra (when normalized to the total PS I Qy spectral area) are comparable to the fractions of Chls bound by the respective (groups of) subunits, according to the 4-A density map of PS I from Synechococcus elongatus. The spectrum of the monomeric PsaL-deficient mutant suggests that this subunit may bind pigments absorbing near 700 nm.     

A large fraction of PsaF is nonfunctional in photosystem I complexes lacking the PsaJ subunit

PsaJ is a small hydrophobic subunit of the photosystem I complex (PSI) whose function is not yet fully understood. Here we describe mutants of the green alga Chlamydomonas reinhardtii, in which the psaJ chloroplast gene has been inactivated either in a wild-type or in a PsaF-deficient nuclear background. Cells lacking one or both subunits grow photoautotrophically and contain normal levels of PSI. Flash-absorption spectroscopy performed with isolated PSI particles isolated from the PsaJ-deficient strain indicates that only 30% of the PSI complexes oxidize plastocyanin (Pc) or cytochrome c6 (Cyt c6) with kinetics identical to wild type, whereas the remaining 70% follow slow kinetics similar to those observed with PsaF-deficient PSI complexes. This feature is not due to partial loss of PsaF, as the PsaJ-less PSI complex contains normal levels of the PsaF subunit. The N-terminal domain of PsaF can be cross-linked to Pc and Cyt c6 indicating that in the absence of PsaJ, this domain is exposed in the lumenal space. Therefore, the decreased amount of functional PsaF revealed by the electron-transfer measurements is best explained by a displacement of the N-terminal domain of PsaF which is known to provide the docking site for Pc and Cyt c6. We propose that one function of PsaJ is to maintain PsaF in a proper orientation which allows fast electron transfer from soluble donor proteins to P700(+).     

Red antenna states of photosystem I from Synechocystis PCC 6803

Single-molecule spectroscopy at low temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the red-shifted chlorophyll a (Chl a) molecules responsible for the fluorescence from photosystem I (PSI). Emission spectra of single PSI complexes from the cyanobacterium Synechocystis PCC 6803 show zero-phonon lines (ZPLs) as well as broad intensity distributions without ZPLs. ZPLs are found most frequently on the blue side of the broad intensity distributions. The abundance of ZPLs decreases almost linearly at longer wavelengths. The distribution of ZPLs indicates the existence of at least two pools with maxima at 699 and 710 nm. The pool with the maximum at 710 nm is assigned to chlorophylls absorbing around 706 nm (C706), whereas the pool with the maximum at 699 nm (F699) can be assigned to chlorophylls absorbing at 692, 695, or 699 nm. The broad distributions dominating the red side of the spectra are made up of a low number of emitters assigned to the red-most pool C714. The properties of F699 show close relation to those of F698 in Synechococcus PCC 7002 and C708 in Thermosynechococcus elongatus. Furthermore, a high similarity is found between the C714 pool in Synechocystis PCC 6803 and C708 in Synechococcus PCC 7002 as well as C719 in T. elongatus.     

Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A fully-segregated mutant (ccaA::kanR) defective in the ccaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kanR, but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed 18O exchange between 13C18O2 and H2O, indicative of CA activity, while ccaA::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kanR cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kanR was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kanR cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.     

Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803

We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 +/- 0.3 chlorophyll (Chl) a and 1.8 +/- 0.1 beta-carotene; one cytochrome b(559) was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and beta-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.     

Supercomplexes of IsiA and Photosystem I in a mutant lacking subunit PsaL

The cyanobacterium Synechocystis PCC 6803 grown under short-term iron-deficient conditions assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 IsiA complexes. Furthermore, it has been shown that single or double rings of IsiA with up to 35 copies in total can surround monomeric PSI. Here we present an analysis by electron microscopy and image analysis of the various PSI-IsiA supercomplexes from a Synechocystis PCC 6803 mutant lacking the PsaL subunit after short- and long-term iron-deficient growth. In the absence of PsaL, the tendency to form complexes with IsiA is still strong, but the average number of complete rings is lower than in the wild type. The majority of IsiA copies binds into partial double rings at the side of PsaF/J subunits rather than in complete single or double rings, which also cover the PsaL side of the PSI monomer. This indicates that PsaL facilitates the formation of IsiA rings around PSI monomers but is not an obligatory structural component in the formation of PSI-IsiA complexes.     

Spectral resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 through direct excitation

We have measured fluorescence spectra from Photosystem I (PS I) on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 at room temperature as a function of excitation wavelength. Our data show a gradual enhancement of long-wavelength fluorescence at 710 nm as the excitation wavelength is increased from 695 to 720 nm. This verifies the presence of low-energy chlorophylls (LE Chls), antenna Chls with energy levels below that of the primary electron donor, P700. The change in fluorescence with excitation wavelength is attributed to the finite time it takes for equilibration of excitations between the bulk and LE Chls. The spectra were deconvoluted into the sum of two basis spectra, one an estimate for fluorescence from the majority or bulk Chls and the other, the LE Chls. The bulk Chl spectrum has a major peak at 688 nm and a lower amplitude vibrational band around 745 nm and is assumed independent of excitation wavelength. The LE Chl spectrum has a major peak at 710 nm, with shoulders at 725 and 760 nm. The relative amplitude of emission at the vibrational side bands increases slightly as the excitation wavelength increases. The ratio of the fluorescence yields from LE Chls to that from bulk Chls ranges from 0.3 to 1.3 for excitation wavelengths of 695 to 720 nm, respectively. These values are consistent with a model where the LE Chls are structurally close to P700 allowing for direct transfer of excitations from both the bulk and LE Chls to P700.     

Chloramphenicol enhances Photosystem II photodamage in intact cells of the cyanobacterium Synechocystis PCC 6803

Photosynthesis Research - The effect of chloramphenicol, an often used protein synthesis inhibitor, in photosynthetic systems was studied on the rate of Photosystem II (PSII) photodamage in the...     

Photosynthesis and photorespiration in a mutant of the cyanobacterium Synechocystis PCC 6803 lacking carboxysomes

A mutant of the cyanobacterium Synechocystis PCC 6803 was obtained by replacing the gene of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) with that of the photosynthetic bacterium Rhodospirillum rubrum. This mutant consequently lacks carboxysomes — the protein complexes in which the original enzyme is packed. It is incapable of growing at atmospheric CO₂ levels and has an apparent photosynthetic affinity for inorganic carbon (C_i) which is 1000 times lower than that of the wild type, yet it accumulates more C_i than the wild type. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis. Unlike the carboxysomal carboxylase activity of Rubisco, which is almost insensitive to inhibition by O₂ in vitro, the soluble enzyme is competitively inhibited by O₂. The photosynthetic rate and C_i compensation point of the wild type were hardly affected by low O₂ levels. Above 100 μM O₂, however, both parameters became inhibited. The CO₂ compensation point of the mutant was linearly dependent on O₂ concentration. The higher sensitivity of the mutant to O₂ inhibition than that expected from in-vitro kinetics parameters of Rubisco, indicates a low capacity to recycle photorespiratory metabolites to Calvin-cycle intermediates.