Biochemical and functional properties of photosystem II in agranal membranes from maize mesophyll and bundle sheath chloroplasts

We have studied the occurrence and organization of photosystem II (PSII) in bundle sheath thylakoids and stroma lamellae from maize. As shown by non-denaturing lauryl beta-D- iminopropionidate (Deriphat)/PAGE, PSII exists in a dimeric form in grana membranes. In bundle sheath and stroma lamellae, however, only a monomeric form was found. Based on immunotitration data, we estimated the stoichiometry of the individual components of the PSII core complex and antenna systems. In stroma lamellae, all PSII antenna complexes had a stoichiometry similar to that in grana membranes, with the exception of light-harvesting complex II (LHCII) that was somewhat over-represented, while the minor antenna complexes CP26 and CP29 were under-represented. In bundle sheath, the amount of LHCII was approximately eight times higher than expected with respect to D1. The 33-kDa protein of the oxygen-evolving enhancer polypeptides was not detectable nor was the ferredoxin-NADP+ reductase, thus strongly suggesting that no significant linear electron transport occurs in bundle sheath thylakoids. Fluorescence induction data suggest that most of the PSII reaction centers in bundle sheath and stroma lamellae sustain electron transport towards a secondary acceptor pool. Stromal PSII centers are only weakly inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), whereas, unexpectedly, dichlorobenzoquinone and methyl viologen had a pronounced inhibitory effect of the QA- reoxidation. An additional specificity of these centers is the slow rate (50-ms range) of the QA to QB electron transfer. The amplitude of variable fluorescence found in stroma lamellae can only account for a small fraction (1-2%) of the variable fluorescence of whole thylakoids. This suggests that stromal PSII cannot be solely responsible for the slow beta-phase of the induction kinetics.     

Mutational studies on conserved histidine residues in the chlorophyll-binding protein CP43 of photosystem II

Two chlorophyll-binding antenna proteins in the photosystem II core, CP43 and CP47, are structurally similar and are thought to have evolved from a common ancestor. Several conserved histidine residues in hydrophobic regions of CP47 have been shown to be important for photosystem II structure, function, and energy transfer. The purpose of this study was to determine whether similarly located histidine residues in CP43 function in a similar way. Three conserved histidine residues in presumed membrane-spanning regions of CP43, His40, His105, and His119, were mutated to glutamine (Q) and tyrosine (Y). The strains H105Q, H119Q, and H119Y were photoautotrophs whereas H40Q, H40Y, and H105Y were obligate photoheterotrophs. The H40Y and H105Y strains lacked detectable amounts of photosystem II reaction centers and hence could not evolve oxygen whereas H40Q retained a significant amount of photosystem II and oxygen evolution capacity. The observation that mutation of histidine residues to tyrosine has more drastic effects than mutation of these residues to glutamine is in agreement with results obtained for CP47 and suggests the involvement of these residues in chlorophyll binding. The drastic functional changes observed upon mutating His40 and His105 of CP43 are similar to those observed when mutating the corresponding histidine residues in CP47, thus suggesting that the similarity between CP43 and CP47 extends to the relative importance of functionally relevant residues. Interestingly, the His40-->Gln mutation in CP43 had significant effects on photosystem II electron transfer in that it affected the thermodynamics of Q(A)- oxidation by Q(B) and increased the charge recombination rate between Q(A)- and donor side components. This indicates that relatively minor changes in CP43 can significantly impact the properties of the photosystem II reaction center. The implications of this finding are discussed.     

Isolation of photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer. The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural charasteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H2O to phenyl-p-benzoquinone and ascorbate-Cl2Ind to NADP+ electron transport rates indicate a ratio of around six between Photosystem II and I. The total fractionation procedure could be completed within 2-3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP+. These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C3 plants. Agranal bundle sheath chloroplasts (Chl a/Chl b greater than 5.0) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl a/b light-harvesting complex (LHC), and most of the cyt b559. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl a/b LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P700 are closely coordinated. Thus, it is likely that both cyt f and P700 are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II alpha from the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II beta. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.     

Mutational analysis of the genes encoding the photosystem II proteins in Cyanobacterium synechocystis sp. 6803

Chlorophyll--binding protein CP43 and cytochrome b559, encoded by psbC and psbE/F genes, are the components of photosystem II (PS II). Three psbC- and four psbE/F- mutants were isolated from the collection of PS II-deficient mutants of the cyanobacterium Synechocystis sp. 6803. Restoration of photosynthetic activity was achieved by transformation of psbE/F- mutants with cloned psbE/F gene cluster from wild type cells and each of psbC- mutants--with specific part of wild type psbC gene. DNA fragments carrying the mutations were isolated from mutant cells and sequenced. The mutations which affect PS II activity were identified in psbC gene as "frameshift" mutation, stop-codon formation, or as deletion of three nucleotides resulting in loss of one of three Phe residues in position 422-424 of CP43. Sequence of mutant psbE/F genes revealed single mutations resulting in deletion of Phe-36 or substitution of Pro-63 for Leu in alpha-subunit and Val-29 for Phe in beta-subunit of cytochrome b559.     

Fluorescence quenching by chlorophyll cations in photosystem II

Although fluorescence is widely used to study photosynthetic systems, the mechanisms that affect the fluorescence in photosystem II (PSII) are not completely understood. The aim of this study is to define the low-temperature steady-state fluorescence quenching of redox-active centers that function on the electron donor side of PSII. The redox states of the electron donors and acceptors were systematically varied by using a combination of pretreatments and illumination to produce and trap, at low temperature, a specific charge-separated state. Electron paramagnetic resonance spectroscopy and fluorescence intensity measurements were carried out on the same samples to obtain a correlation between the redox state and the fluorescence. It was found that illumination of PSII at temperatures between 85 and 260 K induced a fluorescence quenching state in two phases. At 85 K, where the fast phase was most prominent, only one electron-transfer pathway is active on the donor side of PSII. This pathway involves electron donation to the primary electron donor in PSII, P680, from cytochrome b559 and a redox-active chlorophyll molecule, ChlZ. Oxidized ChlZ was found to be a potent quencher of chlorophyll fluorescence with 15% of oxidized ChlZ sufficient to quench 70% of the fluorescence intensity. This implies that neighboring PSII reaction centers are energetically connected, allowing oxidized ChlZ in a few centers to quench most of the fluorescence. The presence of a well-defined quencher in PSII may make it possible to study the connectivity between antenna systems in different sample preparations. The other redox-active components on the donor side of PSII studied were the O2-evolving complex, the redox-active tyrosines (YZ and YD), and cytochrome b559. No significant changes in fluorescence intensity could be attributed to changes in the redox state of these components. The fast phase of fluorescence quenching is attributed to the rapid photooxidation of ChlZ, and the slow phase is attributed to multiple turnovers providing for further oxidation of ChlZ and irreversible photoinhibition. Significant photoinhibition only occurred at Chl concentrations below 0.7 mg/mL and above 150 K. The reversible oxidation of ChlZ in intact systems may function as a photoprotection mechanism under high-light conditions and account for a portion of the nonphotochemical fluorescence quenching.     

Spatial relationship of photosystem I, photosystem II, and the light-harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

The peculiarities of pigment-protein composition of intergranular thylakoid fragments from maize chloroplasts

The pigment-protein composition of two fractions of intergrana fragments from maize inbred lines F 7 and II 346 chloroplast had been investigated. It was shown that under electrophoretic separation of fraction 70,000 g from both lines and fraction 100,000 g from line F 7 the new band of pigment-protein complexes had been observed. It was determined that its polypeptide composition is the same as light-harvesting complex of PS II one, but it differs by low electrophoretical mobility. The conclusion was made that this is a new form of light-harvesting complex of PS II.     

Use of gel filtration to examine the distribution of calcium among serum proteins

Gel filtration of serum by use of dextran bisacrylamide beads ("Sephacryl S-200," fractionation range: 5000-250 000 daltons), separates the serum proteins into three distinct peaks. We used an eluent containing (per liter) 140 mmol of sodium, 1.10 mmol of calcium, 0.50 mmol of magnesium, and 10 mmol of 2-([2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino)ethanesulfonate buffer (pH 7.43 at 37 degrees C) to provide the physiological conditions necessary to maintain the equilibrium of bound calcium in serum. We examined the calcium binding by each of the three protein groups. Calculating the calcium bound per gram of protein in each peak, we found that proteins in the first peak (alpha2-macroglobulin, IgM, haptoglobins) bind about the same amount of calcium per gram as does albumin. The proteins of the second peak (largely IgG and IgA) bind less calcium than does albumin, which is the protein accounting for 90% or more of the third peak. We also were able to calculate intrinsic association constants for calcium/albumin under physiological conditions.     

Role of the RCII-D1 protein in the reversible association of the oxygen-evolving complex proteins with the lumenal side of photosystem II

The nuclear-encoded proteins of the oxygen-evolving complex (OEC) of photosystem II are bound on the lumenal side of the thylakoid membrane and stabilize the manganese ion cluster forming the photosystem II electron donor side. The OEC proteins are released from their binding site(s) following light-induced degradation of reaction center II (RCII)-D1 protein in Chlamydomonas reinhardtii. The kinetics of OEC proteins release correlates with that of RCII-D1 protein degradation. Only a limited amount of RCII-D2 protein is degraded during the process, and no loss of the core proteins CP43 and CP47 is detected. The release of the OEC proteins is prevented when the photoinactivated RCII-D1 protein degradation is retarded by addition of 3-(3,5-dichlorophenyl)-1,1-dimethylurea or by a high PQH2/PQ ratio prevailing in membranes of the plastocyanin-deficient mutant Ac208. The released proteins are not degraded but persist in the thylakoid lumen for up to 8 h and reassociate with photosystem II when new D1 protein is synthesized in cells exposed to low light, thus allowing recovery of photosystem II function. Reassociation also occurs following D1 protein synthesis in darkness when RCII activity is only partially recovered. These results indicate that (i) the D1 protein participates in the formation of the lumenal OEC proteins binding site(s) and (ii) the photoinactivation of RCII-D1 protein does not alter the conformation of the donor side of photosystem II required for the binding of the OEC proteins.     

Three-dimensional electron microscopy of the photosystem II core complex

Risk factors for central venous catheter (CVC)-related bacteraemia among infants admitted to a neonatal intensive care unit (NICU) were analysed and the impact of surveillance and continuing education on the incidence of this complication investigated. Among patients admitted to a NICU, CVC-related bacteraemia increased from 1/15 (7%) in 1987 to 11/26 (42%) in 1988 (P = 0.01). Coagulase-negative staphylococci isolated from bacteraemia patients showed clonal diversity by plasmid and chromosomal fingerprinting. A review of CVC care procedures suggested breaches in aseptic techniques. Catheter-care technique was revised to ensure maximal aseptic precautions, including the use of sterile gloves, gown and drapes. The new policy was promoted by a continuing education programme and regular feed-back of CVC-related bacteraemia incidence to NICU staff. In the four-year follow-up period, the attack-rate of CVC-related bacteraemia decreased to 18/156 (12%) patients [relative risk (RR): 0.27, 95% confidence interval (CI); 0.15-0.51; P < 0.001 vs the previous period]. By using the Cox's model proportional hazards, very low birthweight and the period before use of strict aseptic CVC care were found to be predictors of increased risk of catheter-related bacteraemia after adjustment for duration of catheterization. These data provide further evidence that strict aseptic precautions during the maintenance and utilization of CVC can contribute to lower the risk of catheter infection in critically ill neonates. Regular feedback of surveillance data was associated with a progressive decrease in incidence of infection, suggesting that it improved staff compliance with aseptic precautions.     

The 23 and 17 kDa extrinsic proteins of photosystem II modulate the magnetic properties of the S1-state manganese cluster

An S1-state parallel polarization "multiline" EPR signal arising from the oxygen-evolving complex has been detected in spinach (PSII) membrane and core preparations depleted of the 23 and 17 kDa extrinsic polypeptides, but retaining the 33 kDa extrinsic protein. This S1-state multiline signal, with an effective g value of 12 and at least 18 hyperfine lines, has previously been detected only in PSII preparations from the cyanobacterium sp. Synechocystis sp. PCC6803 [Campbell, K. A., Peloquin, J. M., Pham, D. P., Debus, R. J., and Britt, R. D. (1998) J. Am. Chem. Soc. 120, 447-448]. It is absent in PSII spinach membrane and core preparations that either fully retain or completely lack the 33, 23, and 17 kDa extrinsic proteins. The S1-state multiline signal detected in spinach PSII cores and membranes has the same effective g value and hyperfine spacing as the signal detected in Synechocystis PSII particles. This signal provides direct evidence for the influence of the extrinsic PSII proteins on the magnetic properties of the Mn cluster.     

Modification of pigment composition in the isolated reaction center of photosystem II

The pigment content of isolated reaction centers of photosystem II was modified using an exchange protocol similar to that used for purple bacterial reaction centers. With this method, which is based on incubation of reaction centers at elevated temperature with an excess of chemically modified pigments, it was possible to incorporate [3-acetyl]-chlorophyll a and [Zn]-chlorophyll a into photosystem II reaction centers. Pigment exchange has been verified by absorption, circular dichroism and fluorescence spectroscopy, and quantitated by HPLC analysis of pigment extracts.     

A highactivity ATP translocator in mesophyll chloroplasts of Digitaria sanguinalis, a plant having the C-4 dicarboxylic acid pathway of photosynthesis

The effect of exogenous adenine nucleotides on CO2 fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C4 plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate+oxalacetate induced CO2 fixation, as well as reverse the inhibition of CO2 fixation of carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO2 fixation varied from 40 to 70 mumol CO2 fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80-140 mumol/mg chlorophyll per h, since 2 ATP are required for each CO2 fixed. Fixation of CO2 could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts...     

Investigation of the structure of spinach photosystem II reaction center complex

The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi-twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.     

Characterization of the interaction between manganese and tyrosine Z in acetate-inhibited photosystem II

When acetate-inhibited photosystem II (PSII) membranes are illuminated at temperatures above 250 K and quickly cooled to 77 K, a 240 G-wide electron paramagnetic resonance (EPR) signal is observed at 10 K. This EPR signal arises from a reciprocal interaction between the spin 1/2 ground state of the S2 state of the Mn4 cluster, for which a multiline EPR signal with shifted 55Mn hyperfine peaks is observed, and the oxidized tyrosine residue, YZ*, for which a broadened YZ* EPR spectrum is observed. The S2YZ* EPR signal in acetate-inhibited PSII is the first in which characteristic spectral features from both paramagnets can be observed. The observation of distinct EPR signals from each of the paramagnets together with the lack of a half-field EPR transition indicates that the exchange and dipolar couplings are weak. Below 20 K, the S2YZ* EPR signal in acetate-inhibited PSII is in the static limit. Above 20 K, the line width narrows dramatically as the broad low-temperature S2YZ* EPR signal is converted to a narrow YZ* EPR signal at room temperature. The line width narrowing is interpreted to be due to averaging of the exchange and dipolar interactions between YZ* and the S2 state of the Mn4 cluster by rapid spin-lattice relaxation of the Mn4 cluster as the temperature is increased. Decay of the S2YZ* intermediate at 200 K shows that the g = 4.1 form of the S2 state is formed and that a noninteracting S2-state multiline EPR signal is not observed as an intermediate in the decay. This result shows that a change in the redox state of YZ induces a spin-state change in the Mn4 cluster in acetate-inhibited PSII. The interconversion between spin states of the Mn4 cluster in acetate-inhibited PSII supports the idea that YZ oxidation or YZ* reduction is communicated to the Mn4 cluster through a direct hydrogen-bonding pathway, possibly involving a ligand bound to the Mn4 cluster.