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1.
CP43, a component of Photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbC gene. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII characteristics including lower oxygen-evolving activity, fewer assembled reaction centers, higher sensitivity to photoinactivation, etc. [Biochemistry 38 (1999) 1582]. Additionally, it was determined that the mutant exhibited an enhanced stability of its S2 state. Recently, we observed a significant chloride effect under chloride-limiting conditions. The mutant essentially lost the ability to grow photoautotrophically, assembled fewer fully functional PSII reaction centers and exhibited a very low rate of oxygen evolution. Thus, the observed phenotype of this mutation is very similar to that observed for the Delta(psb)V mutant, which lacks cytochrome c550 (Biochemistry 37 (1998) 1551). A His-tagged version of the R305S mutant was produced to facilitate the isolation of PSII particles. These particles were analyzed for the presence of cytochrome c550. Reduced minus oxidized difference spectroscopy and chemiluminescence examination of Western blots indicated that cytochrome c550 was absent in these PSII particles. Whole cell extracts from the R305S mutant, however, contained a similar amount of cytochrome c550 to that observed in the control strain. These results indicate that the mutation R305S in CP43 prevents the strong association of cytochrome c550 with the PSII core complex. We hypothesize that this residue is involved in the formation of the binding domain for the cytochrome. 相似文献
2.
CP 47, a component of photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbB gene. Site-specific mutagenesis has been used to alter a portion of the psbB gene encoding the large extrinsic loop E of CP 47 in the cyanobacterium Synechocystis 6803. Alteration of a lysine residue occurring at position 321 to glycine produced a strain with altered PSII activity. This strain grew at wild-type rates in complete BG-11 media (480 µM chloride). However, oxygen evolution rates for this mutant in complete media were only 60% of the observed wild-type rates. Quantum yield measurements at low light intensities indicated that the mutant had 66% of the fully functional PSII centers contained in the control strain. The mutant proved to be extremely sensitive to photoinactivation at high light intensities, exhibiting a 3-fold increase in the rate of photoinactivation. When this mutant was grown in media depleted of chloride (30 µM chloride), it lost the ability to grow photoautotrophically while the control strain exhibited a normal rate of growth. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 µM bromide to the chloride-depleted BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates in either chloride-containing or chloride-depleted media. Oxygen evolution rates for the mutant were further depressed (28% of control rates) under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. Measurements of the variable fluorescence yield indicated that the mutant assembled fewer functional centers in the absence of chloride. These results indicate that the mutation K321G in CP 47 affects PSII stability and/or assembly under conditions where chloride is limiting. 相似文献
3.
We have shown previously that a mutant which contained the alteration (448)R --> (448)S (R448S) in the CP47 protein of photosystem II exhibited a defect in its ability to grow and assemble functional photosystem II reaction centers under chloride-limiting conditions [Wu, J., Masri, N., Lee, W., Frankel, L. K., and Bricker, T. M. (1999) Plant Mol. Biol. 39, 381-386]. In this paper we have examined the function of the oxygen-evolving complex under chloride-sufficient (480 microM) and chloride-limiting (< 20 microM) conditions. When placed under chloride-limiting conditions, both the control strain K3 and R448S cells exhibit a loss of steady-state oxygen evolution, with t(1/2) of 16 and 17 min, respectively. Upon the addition of chloride, both recover their oxygen-evolving capacity relatively rapidly. However, R448S exhibits a much slower reactivation of oxygen evolution than does K3 (t(1/2) of 308 and 50 s, respectively). This may indicate a defect at the low-affinity, rapidly exchanging chloride-binding site [Lindberg, K., and Andréasson, L.-E. (1996) Biochemistry 35, 14259-14267]. Additionally, alterations in the distribution of S states and S-state lifetimes were observed. Under chloride-sufficient conditions, the R448S mutant exhibits a significant increase in the proportion of reaction centers in the S(3) state and a greatly increased lifetime of the S(3) state. Under chloride-limiting conditions, the proportion of reaction centers in both the S(2) and S(3) states increases significantly, and there is a marked increase in the lifetime of the S(2) state. These alterations are not observed in the control strain K3. Our observations support the hypothesis that (448)R of CP47 may participate in the formation of the binding domain for chloride in photosystem II and/or in the functional interaction with the 33 kDa protein with the photosystem. 相似文献
4.
The deletion of the amino acids between Gly-351 and Thr-365 within the large, lumen-exposed, hydrophilic region (loop E) of the photosystem II (PSII) chlorophyll a-binding protein CP47 produced a strain of Synechocystis sp. PCC 6803 that failed to assemble stable PSII centers [Eaton-Rye, J. J., and Vermaas, W. F. J. (1991) Plant Mol. Biol. 17, 1165-1177]. The importance of two conserved Phe residues at positions 362 and 363 within this deletion has been investigated. The F363R strain had impaired photoautotrophic growth and an enhanced sensitivity to photoinactivation, demonstrating that Phe is required at position 363 for normal PSII function. In contrast, photoautotrophic growth in strains N361K and F362R was unaffected. Uniquely, among the mutant strains tested, F363R was unable to grow under chloride-limiting conditions, and this effect was reversed by replacing chloride with bromide. The removal of the manganese-stabilizing protein (PSII-O), the 12 kDa extrinsic protein (PSII-U), and cytochrome c-550 (PSII-V) was investigated in each mutant in vivo. In N361K and F362R, removal of PSII-V produced a more deleterious effect than the removal of PSII-O, but even so, all strains remained photoautotrophic. In contrast, the absence of PSII-V and PSII-O in F363R produced obligate photoheterotrophic strains. The removal of PSII-U increased the susceptibility of PSII to heat inactivation and further decreased the stability of PSII in F363R, demonstrating that PSII-U can contribute to the stabilization of mutations that have been introduced into CP47. The order of importance of the selective removal of the extrinsic proteins in strains carrying mutations in loop E of CP47 was found to be as follows: DeltaPSII-V >/= DeltaPSII-O > DeltaPSII-U. 相似文献
5.
Gennady Ananyev Tuan Nguyen Cindy Putnam-Evans G Charles Dismukes 《Photochemical & photobiological sciences》2005,4(12):991-998
The chlorophyll-binding protein CP43 is an inner subunit of the Photosystem II (PSII) reaction center core complex of all oxygenic photoautotrophs. X-Ray structural evidence places the guanidinium cation of the conserved arginine 357 residue of CP43 within a few Angstroms to the Mn(4)Ca cluster of the water-oxidizing complex (WOC) and has been implicated as a possible carbonate binding site. To test the hypothesis, the serine mutant, CP43-R357S, from Synechocystis PCC 6803 was investigated by PSII variable fluorescence (F(v)/F(m)) and simultaneous flash O(2) yield measurements in cells and thylakoid membranes. The R357S mutant assembles PSII-WOC centers, but is unable to grow photoautotrophically. Reconstitution of O(2) evolution by photoactivation and the occurrence of period-four oscillations of F(v)/F(m) establishes that the R357S mutant contains an assembled Mn(4)Ca cluster, but turnover is impaired as seen by an 11-fold larger Kok double miss parameter and faster decay of upper S states. Using pulsed light to avoid photoinactivation, wild-type cells and thylakoid membranes exhibit a 2-4-fold loss in O(2) evolution rate upon partial bicarbonate depletion under multiple turnover conditions, while the R357S mutant is unaffected by bicarbonate. Arginine R357 appears to function in binding a (bi)carbonate ion essential to normal catalytic turnover of the WOC. The quantum yield of electron donation from the WOC into PSII increases with decreasing turnover rate in R357S mutant cells and involves an aborted two-flash pathway that is distinct from the classical four-flash pattern. We speculate that an altered photochemical mechanism for O(2) production occurs via formation of hydrogen peroxide, by analogy to other treatments that retard the kinetics of proton release into the lumen. 相似文献
6.
The psbC gene encodes the intrinsic chlorophyll protein CP 43, a component of photosystem II in higher plants, green algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into the portion of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Three mutations, E293Q, E339Q, and E352Q, each produced a strain with impaired photosystem II activity. The E293Q mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of several PS II components indicated that this mutant accumulated normal quantities of PS II proteins. However, this mutant evolved oxygen to only 56% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 60% of the fully functional PS II centers found in the control strain. The E339Q mutant grew photoautotrophically at a severely reduced rate. Both immunological analysis and variable fluorescence yield experiments indicated that E339Q assembled a normal complement of PS II centers. However, this mutant was capable of evolving oxygen to only 20% of control rates. Variable fluorescence yield experiments demonstrated that this mutant was inefficient at using water as an electron donor. Both E293Q and E339Q strains exhibited an increased (approximately 2-fold) sensitivity to photoinactivation. The E352Q mutant was the most severely affected. This mutant failed to grow photoautotrophically and exhibited essentially no capacity for oxygen evolution. Measurements of total variable fluorescence yield indicated that this mutant assembled no functional PS II centers. Immunological analysis of isolated thylakoid membranes from E352Q revealed a complete absence of CP 43 and reduced levels of both the D1 and manganese-stabilizing proteins. These results suggest that the mutations E293Q and E339Q each produce a defect associated with the oxygen-evolving complex of photosystem II. The E352Q mutation appears to affect the stability of the PS II complex. This is the first report showing that alteration of negatively charged residues in the CP 43 large extrinsic loop results in mutations affecting PS II assembly/function. 相似文献
7.
We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU. This mutant carries an R7L mutation on the α-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 Å PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lα cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lα cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lα cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the α-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress. 相似文献
8.
The PsbY protein is not essential for oxygenic photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803
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A tetra-manganese cluster in the photosystem II (PSII) pigment-protein complex plays a critical role in the photosynthetic oxygen evolution process. PsbY, a small membrane-spanning polypeptide, has recently been suggested to provide a ligand for manganese in PSII (A.E. Gau, H.H. Thole, A. Sokolenko, L. Altschmied, R.G. Herrmann, E.K. Pistorius [1998] Mol Gen Genet 260: 56-68). We have constructed a mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with an inactivated psbY gene (sml0007). Southern-blot and polymerase chain reaction analysis showed that the mutant had completely segregated. However, the DeltapsbY mutant cells grew normally under photoautotrophic conditions. Moreover, growth of the wild-type and mutant cells were similar under high-light photoinhibition conditions, as well as in media without any added manganese, calcium, or chloride, three required inorganic cofactors for the oxygen-evolving complex of PSII. Analysis of steady-state and flash-induced oxygen evolution, fluorescence induction, and decay kinetics, and thermoluminescence profiles demonstrated that the DeltapsbY mutant cells have normal photosynthetic activities. We conclude that the PsbY protein in Synechocystis 6803 is not essential for oxygenic photosynthesis and does not provide an important binding site for manganese in the oxygen-evolving complex of PSII. 相似文献
9.
The psbB gene encodes the intrinsic chlorophyll-a binding protein CPa-1 (CP-47), a component of photosystem II in higher plants, algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into a segment of the psbB gene encoding the large extrinsic loop region of CPa-1 in the cyanobacterium Synechocystis sp. PCC 6803. Altered psbB genes were introduced into a mutant recipient strain (DEL-1) of Synechocystis in which the genomic psbB gene had been partially deleted. Initial target sites for mutagenesis were absolutely conserved basic residue pairs occurring within the large extrinsic loop. One mutation, RR384385GG, produced a strain with impaired photosystem II activity. This strain exhibited growth characteristics comparable to controls. However, at saturating light intensities this mutant strain evolved oxygen at only 50% of the rate of the control strains. Quantum yield measurements at low light intensities indicated that the mutant had 30% fewer fully functional photosystem II centers than do control strains of Synechocystis. Immunological analysis of a number of photosystem II protein components indicated that the mutant accumulates normal quantities of photosystem II proteins and that the ratio of photosystem II to photosystem I proteins is comparable to that found in control strains. Upon exposure to high light intensities the mutant cells exhibited a markedly increased susceptibility to photoinactivation. However, Tris-treated thylakoid membranes from both the mutant and wild-type exhibited comparable rates of photoinactivation. Thylakoid membranes isolated from RR384385GG exhibited only 15% of the H2O to 2,6-dichlorophenolindophenol electron transport rate observed in wild-type strains.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
Resistance to photoinhibition of photosystem II and catalase and antioxidative protection in high mountain plants 总被引:8,自引:1,他引:7
In leaves of three alpine high mountain plants, Homogyne alpina, Ranunculus glacialis and Soldanella alpina, both photosystem II (PSII) and the enzyme catalase appeared to he highly resistant to photoinactivation under natural field conditions. While the Dl protein of PSII and catalase have a rapid turnover in light and require continuous new protein synthesis in non-adapted plants, little apparent photoinactivation of PSII or catalase was induced in the alpine plants by translation inhibitors or at low temperature, suggesting that turnover of the Dl protein and catalase was slow in these leaves. In vitro PSII was rapidly inactivated in light in isolated thylakoids from H. alpina and R. glacialis. In isolated intact chloroplasts from R. glacialis, photoinactivation of PSII was slower than in thylakoids. Partially purified catalase from R. glacialis and S. alpina was as sensitive to photoinactivation in vitro as catalases from other sources. Catalase from H. alpina had, however, a 10-fold higher stability in light. The levels of xanthophyll cycle carotenoids, of the antioxidants ascorbate and glulathione, and of the activities of catalase, superoxide dismutase and glutathione reductase were very high in S. alpina, intermediate in H. alpina, but very low in R. glacialis. However, isolated chloroplasts from all three alpine species contained much higher concentrations of ascorbate and glutathione than chloroplasts from lowland plants. 相似文献
11.
The effects of photosystem II (PSII) exogenous electron donors and acceptors on the kinetics of weak light photoinhibition of NH2OH/EDTA-extracted spinach PSII membranes were examined. Under aerobic conditions, Mn2+ (approximately 1 Mn/reaction center; Km approximately 400 nM) inhibited photoinactivation and approximately 1 Mn/reaction center plus 100 microM NH2NH2 gave almost complete protection. In the absence of electron donors, strict anaerobiosis greatly inhibited photoinactivation even in the presence of an electron acceptor. Under aerobic conditions, the addition of electron acceptors (FeCN, DCIP), oxyradical scavengers, or superoxide dismutase strongly suppressed rates of photodamages. Increase in the concentrations of superoxide above those produced by illuminated NH2OH/EDTA-photosystem II membranes increased the rates of damage in the light but gave no damage in the dark. Scavengers of hydroxyl radicals and singlet oxygen did not suppress the rates of aerobic photoinhibition. These findings, along with others, lead us to conclude that photodamage of the secondary donors of the PSII reaction center occurs by two mechanisms: (1) a rapid superoxide and tyrosine YZ+ dependent process and (2) a slower process in which P680+/Chl+ catalyze the damages. 相似文献
12.
Irreversible photoinactivation of photosystem II (PSII) results in the degradation of the reaction center II D1 protein. In Synechocystis PCC 6714 cells, recovery of PSII activity requires illumination. The rates of photoinactivation and recovery of PSII activity in the light are similar in cells grown in minimal (MM) or glucose-containing medium (GM). Reassembly of PSII with newly synthesized proteins requires degradation of the D1 protein of the photoinactivated PSII. This process may occur in darkness in both types of cells. The degraded D1 protein is, however, only partially replaced by newly synthesized protein in MM cells in darkness while a high level of D1 protein synthesis occurs in darkness in the GM cells. The newly synthesized D1 protein in darkness appears to be assembled with other PSII proteins. However, PSII activity is not recovered in such cells. Illumination of the cells in absence but not in the presence of protein synthesis inhibitors allows recovery of PSII activity. 相似文献
13.
《Plant Physiology and Biochemistry》2002,40(1):41-49
The Arabidopsis thaliana subunit PsbS of photosystem II (PSII) is essential for the non-photochemical quenching of chlorophyll fluorescence and thus for ΔpH-dependent energy dissipation (qE). As a result of the excision of an En-transposon, a frameshift mutation in the psbS gene was obtained, which results in the complete absence of the PsbS protein and of qE. Two-dimensional gel analyses of thylakoid membranes indicated that the depletion of PsbS has no effect on PSII composition, excluding a structural role for PsbS in the organization of the PSII antenna. The susceptibility of mutant plants to photoinactivation of PSII was significantly increased during exposure to high light for up to 8 h. Divergence of mutant plants from wild-type levels of photoinactivation were most pronounced during the first 2 h of illumination, while after longer exposure times the rate of PSII inactivation were similar in both genotypes. The increased PSII inactivation in the mutant was not accompanied by an increased rate of D1 protein degradation, and recovery of PSII activity in the mutant under low light was similar or even faster in comparison to wild-type plants. However, growth under high light intensities resulted in decreased growth rates of psbs mutant plants. We conclude that energy dissipation in PSII related to qE is not primarily required for the protection of PSII against light-induced destruction, but may rather be involved in reducing the electron pressure on the photosynthetic electron transport chain at saturating light intensities. 相似文献
14.
15.
Antenna Size Dependency of Photoinactivation of Photosystem II in Light-Acclimated Pea Leaves 总被引:3,自引:0,他引:3
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Utilization of absorbed light energy by photosystem (PS) II for O2 evolution depends on the light-harvesting antenna size, but the role of antenna size in the photoinactivation of PSII seems controversial. To address this controversy, pea (Pisum sativum L.) plants were grown in low (50 [mu]mol m-2 s-1) or high (650 [mu]mol m-2 s-1) light. The doubled functional antenna size of PSII in low light allows each PSII to utilize twice as many photons at given flash light energies for O2 evolution. The application of a target theory to depict the photon dose dependency of PSII photoinactivation measured by repetitive-flash O2 yield and the ratio of variable to maximal chlorophyll fluorescence indicates that photoinactivation of PSII is probably a single-hit process in which repair or photoprotective mechanisms are only slightly involved. Furthermore, the exacerbation of photoinactivation of PSII with greater antenna size under anaerobic conditions strongly indicates that photoinactivation of PSII depends on antenna size. 相似文献
16.
In photosynthetic organisms, impairment of the activities of enzymes in the Calvin cycle enhances the extent of photoinactivation of Photosystem II (PSII). We investigated the molecular mechanism responsible for this phenomenon in the unicellular green alga Chlamydomonas reinhardtii. When the Calvin cycle was interrupted by glycolaldehyde, which is known to inhibit phosphoribulokinase, the extent of photoinactivation of PSII was enhanced. The effect of glycolaldehyde was very similar to that of chloramphenicol, which inhibits protein synthesis de novo in chloroplasts. The interruption of the Calvin cycle by the introduction of a missense mutation into the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) also enhanced the extent of photoinactivation of PSII. In such mutant 10-6C cells, neither glycolaldehyde nor chloramphenicol has any additional effect on photoinactivation. When wild-type cells were incubated under weak light after photodamage to PSII, the activity of PSII recovered gradually and reached a level close to the initial level. However, recovery was inhibited in wild-type cells by glycolaldehyde and was also inhibited in 10-6C cells. Radioactive labelling and Northern blotting demonstrated that the interruption of the Calvin cycle suppressed the synthesis de novo of chloroplast proteins, such as the D1 and D2 proteins, but did not affect the levels of psbA and psbD mRNAs. Our results suggest that the photoinactivation of PSII that is associated with the interruption of the Calvin cycle is attributable primarily to the inhibition of the protein synthesis-dependent repair of PSII at the level of translation in chloroplasts. 相似文献
17.
The extent to which PSII photoinactivation affects electron transport (PhiPSII) and CO2 assimilation remains controversial, in part because it frequently occurs alongside inactivation of other components of photosynthesis, such as PSI. By manipulating conditions (darkness versus low light) after a high light/low temperature treatment, we examined the influence of different levels of PSII inactivation at the same level of PSI inactivation on PhiPSII and CO2 assimilation for Arabidopsis. Furthermore, we compared PhiPSII at high light and optimum temperature for wild-type Arabidopsis and a mutant (npq4-1) with impaired capacities for energy dissipation. Levels of PSII inactivation typical of natural conditions (< 50%) were not associated with decreases in PhiPSII and CO2 assimilation at photon flux densities (PFDs) above 150 micromol m(-2) s(-1). At higher PFDs, the light energy being absorbed was in excess of the energy that could be utilized by downstream processes. Arabidopsis plants downregulate PSII activity to dissipate such excess in accordance with the level of PSII photoinactivation that also serves to dissipate absorbed energy. Therefore, the overall levels of non-photochemical dissipation and the efficiency of photochemistry were not affected by PSII inactivation at high PFD. Under low PFD conditions, such compensation is not necessary, because the amount of light energy absorbed is not in excess of that needed for photochemistry, and inactive PSII complexes are dissipating energy. We conclude that moderate photoinactivation of PSII complexes will only affect plant performance when periods of high PFD are followed by periods of low PFD. 相似文献
18.
Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the alpha or the beta subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kalpha mutant grew photoautotrophically, and accumulated stable PSII reaction centers ( approximately 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Ybeta mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers ( approximately 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559alpha and beta polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559beta polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YbetaPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YbetaPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YbetaPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kalpha mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kalpha mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kalpha and H22YbetaPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII. 相似文献
19.
Chung-Hsien Hung 《BBA》2007,1767(6):686-693
Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII. 相似文献
20.
Diatoms host chlorophyll a/c chloroplasts distinct from green chloroplasts. Diatoms now dominate the eukaryotic oceanic phytoplankton, in part through their exploitation of environments with variable light. We grew marine diatoms across a range of temperatures and then analyzed their PSII function and subunit turnover during an increase in light to mimic an upward mixing event. The small diatom Thalassiosira pseudonana initially responds to increased photoinactivation under blue or white light with rapid acceleration of the photosystem II (PSII) repair cycle. Increased red light provoked only modest PSII photoinactivation but triggered a rapid clearance of a subpool of PsbA. Furthermore, PsbD and PsbB content was greater than PsbA content, indicating a large pool of partly assembled PSII repair cycle intermediates lacking PsbA. The initial replacement rates for PsbD (D2) were, surprisingly, comparable to or higher than those for PsbA (D1), and even the supposedly stable PsbB (CP47) dropped rapidly upon the light shift, showing a novel aspect of rapid protein subunit turnover in the PSII repair cycle in small diatoms. Under sustained high light, T. pseudonana induces sustained nonphotochemical quenching, which correlates with stabilization of PSII function and the PsbA pool. The larger diatom Coscinodiscus radiatus showed generally similar responses but had a smaller allocation of PSII complexes relative to total protein content, with nearly equal stiochiometries of PsbA and PsbD subunits. Fast turnover of multiple PSII subunits, pools of PSII repair cycle intermediates, and photoprotective induction of nonphotochemical quenching are important interacting factors, particularly for small diatoms, to withstand and exploit high, fluctuating light. 相似文献