Site-directed mutagenesis of the basic residues 321 K to 321 G in the CP 47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving … (original) (raw)
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Plant Mol Biol, 1997
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 M 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 M 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 M 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.
Biochemistry Usa, 1994
The intrinsic chlorophyll-protein CP 47 is a component of photosystem II in higher plants, green algae and cyanobacteria. We had shown previously by biochemical methods that the domain 364 E-440 D of CP 47 interacts with the 33 kDa extrinsic protein of photosystem II [Odom, W. R., & Bricker, T. M. (1992) Biochemistry 31, 5616-5620]. In this study, using oligonucleotide-directed mutagenesis in the cyanobacterium Synechocystis 6803, mutations at 17 conserved charged residues were introduced into the domain 364 E-444 R of the CP 47 protein. Only mutations introduced at positions 384 R and 385 R led to a modified PS II phenotype. We previously described a mutation at (RR384385GG) which resulted in a mutant with a defective oxygen-evolving complex [Putnam-Evans, C., & Bricker, T. M. (1992) Biochemistry 31, 11482-11488]. An additional set of mutations, 384 R to 384 G, 385 R to 385 G, and 384,385 RR to 384,385 EE has now been introduced at this site yielding the mutants R384G, R385G, and RR384385EE, respectively. Steady state oxygen evolution measurements and quantum yield measurements demonstrated that these mutants exhibited significant alterations in their ability to evolve oxygen. Total fluorescence yield measurements indicated that all of these mutants contained about 85%-90% of the PS II reaction centers found in the control strain. This decrease was insufficient to explain the oxygen evolution results. Analysis of oxygen flash yield parameters indicated that there was little change in the S-state parameters R, , γ, or δ. Measurement of the S 2 lifetime, however, demonstrated that the S 2 lifetime of the mutants was 2-3 times longer than that of the control. Additionally, examination of the risetime of the oxygen signal indicated that there was a significant retardation (6-7-fold) in the rate of oxygen release, suggesting a retarded S 3-[S 4 ]-S 0 transition. These data reinforce our hypothesis that the positive charge density at positions 384 R and 385 R in the large extrinsic loop of CP 47 is necessary for its function in water oxidation. We speculate that this positive charge density may be an important factor in establishing the proper interaction between CP 47 and the 33 kDa extrinsic protein.
Biochemistry, 2001
We have shown previously that a mutant which contained the alteration 448 R f 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 µM) and chloride-limiting (<20 µM) 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. †
Biochemistry, 1999
The intrinsic chlorophyll protein CP 43, a component of photosystem II (PS II) in higher plants, green algae, and cyanobacteria, is encoded by the psbC gene. Oligonucleotide-directed mutagenesis was employed to introduce mutations into a segment of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Two mutations, R305S and R342S, each produced a strain with impaired photosystem II activity. The R305S mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of a number of PSII components indicated that this mutant accumulated normal quantities of PSII proteins. However, this mutant evolved oxygen to only 70% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 70% of the PSII centers found in the control strain. The R342S mutant failed to grow photoautotrophically and exhibited no capacity for oxygen evolution. However, when grown photoheterotrophically in medium containing both glucose and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), oxygen-evolving activity was observed in the R342S mutant, but at a low level of approximately 10% of the control rate. Immunological analysis of isolated thylakoid membranes from this mutant also indicated that this strain accumulated normal amounts of PSII core proteins. Total variable fluorescence yields for the R342S mutant indicated that it assembled a severely reduced number of fully functional PSII centers. R305S and R342S mutant strains exhibited, respectively, 2.7-and 4-fold increased sensitivity to photoinactivation. The fluorescence rise times for both mutants were comparable to the control when hydroxylamine was used as electron donor. However, both strains exhibited an increase (2.5-and 8-fold, respectively, for R305S and R342S) in fluorescence rise times with water as an electron donor. These results suggest that the mutations R305S and R342S each produce a defect associated with the oxygen-evolving complex of photosystem II. These are the first site-directed mutations in CP 43 to show such an effect.
Biochemistry, 1999
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.
Biochemistry, 1992
ThepsbB gene encodes the intrinsic chlorophyll-a binding protein CPa-1 (CP-47), a component of photosystem I1 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 far mutagenesis were absolutely conserved basic residue pairs occurring within the large extrinsic loop. One mutation, RR384385GG, produced a strain with impaired photosystem I1 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 a t low light intensities indicated that the mutant had 30% fewer fully functional photosystem I1 centers than do control strains of Synechocystis. Immunological analysis of a number of photosystem I1 protein components indicated that the mutant accumulates normal quantities of photosystem I1 proteins and that the ratio of photosystem I1 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 H20 to 2,6-dichlorophenolindophenol electron transport rate observed in wild-type strains. The 1,5diphenylcarbazide to 2,6-dichlorophenolindophenol electron transport rates of Tris-treated thylakoids from the mutant, however, were comparable to control rates. These results suggest that alteration of this basic residue pair leads to a defect associated with the oxygen-evolving complex of photosystem 11. Photosystem I1 (PS 11)' is a multisubunit thylakoid membrane protein complex which catalyzes the light-driven oxidation of water to molecular oxygen and the reduction of plastoquinone to plastoquinol. This complex consists of both intrinsic and extrinsic protein subunits. Intrinsic polypeptides with apparent molecular masses of 49 (CPa-l), 45 (CPa-2), 34 (Dl), 32 (D2), 9 and 4.5 (a and B subunits of cytochrome b559), and 4 kDa (psbl gene product) appear to form the minimum complex capable of photosynthetic oxygen evolution (Burnap & Sherman, 1991; Philbrick et al., 1991; Bricker, 1992). Extrinsic proteins with apparent molecular masses of 33 (manganese-stabilizing protein), 24, and 17 kDa are required for optimal oxygen evolution rates at in vivo concentrations of calcium and chloride in higher plants and green algae; the cyanobacteria lack the 24-and 17-kDa components. A number of other low molecular weight proteins have been identified which seem to be associated with PS 11; however, their functions have not been clearly defined (Ikeuchi et al., 1989). Oxygen evolution also requires the presence of four manganese, one or two calcium, and several chloride ions This work was sponsored by USDA-NRICGP Grant 91-37036-6350 to T.M.B. 1 Abbreviations: bp, base pairs; chl, chlorophyll; DCBQ, 2,6-dichloropbenzoquinone; DCPIP, 2,6.dichlorophenolindophenol; DPC, 1,5-diphenylcarbazide; DTSP, dithiobis(succinimidy1 propionate); EDC, l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; HEPES, N-(2-hydroxyethyl)-piperazineN'-2-ethane~ulfonic acid; kb, kilo&, MES, 2-(N-morpholino)ethanesulfonic acid; NHS-biotin, N-hydroxysuccinimidobiotin; PCR, polymerase chain reaction; PS I, photosystem I; FS 11, photosystem 11; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane.
Plant Mol Biol, 1999
The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II which functions in both light-harvesting and oxygen evolution. Using the Escherichia coli mutator strain XL-1 Red, we introduced mutations at 14 sites in the large extrinsic loop E of CP 47 and its adjacent transmembrane α-helix VI. Four mutant cell lines were recovered in which the histidyl residues 455 H, 466 H and 469 H were altered. The cell lines H455T, H455Y, H469Y, and the double mutant F432L,H466R exhibited phenotypes that supported the identification of the histidyl residues 455 H, 466 H and 469 H as chlorophyll ligands. Four additional mutant cell lines were recovered which contained mutations at positions 448 R in the large extrinsic loop of CP 47. These mutants, R448K, R448Q, R448S, and R448W, exhibited variable phenotypes ranging from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters. Those mutants exhibiting photoautotrophic growth and oxygen evolution capability under standard conditions were unable to grow photoautotrophically or evolve oxygen when grown at low chloride concentrations. Finally, a mutant cell line exhibiting a substitution at position 342 G was recovered. The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution. In addition to these alterations, mutants were recovered in which deletions and insertions (leading to frame shifts) and stop codons were introduced. These mutants uniformly lacked the ability to either grow photoautotrophically or evolve oxygen.
Plant Physiology, 1987
psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertion of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic Dl polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.
Plant Mol Biol, 1996
The intrinsic chlorophyll-protein CP 47 is a component of photosystem II which functions in both light-harvesting and oxygen evolution. The large extrinsic loop E of this protein has been shown to interact with the oxygen-evolving site. Previously, Vermaas and coworkers have produced a number of deletions within loop E which yielded mutants which were unable to grow photoautotrophically and which could not evolve oxygen at normal rates. During the course of our site-directed mutagenesis program in Synechocystis 6803, we have altered all of the conserved charged residues which were present within six of these deletions. All ten of these mutants were photoautotrophic and evolved oxygen at normal rates. We speculate that the severe phenotypes of the deletion mutants observed by Vermaas and coworkers is due to large structural perturbations in the extrinsic loop E of CP 47.
Photosynth Res, 1999
Photosystem I (PSI) interacts with plastocyanin or cytochrome c 6 on the luminal side. To identify sites of interaction between plastocyanin/cytochrome c 6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein from Synechocystis sp. PCC 6803. The eight mutant strains differed in their photoautotrophic growth. Western blotting with subunit-specific antibodies indicated that the mutations affected the PSI level in the thylakoid membranes. PSI proteins could not be detected in the S600R/G601C/ N602I, N609K/S610C/T611I, and M614I/G615C/W616A mutant membranes. The other mutant strains contained different levels of PSI proteins. Among the mutant strains that contained PSI proteins, the H595C/L596I, Q627H/L628C/I629S, and N638C/N639S mutants showed similar levels of PSI-mediated electron transfer activity when either cytochrome c 6 or an artificial electron donor was used. In contrast, cytochrome c 6 could not function as an electron donor to the W622C/A623R mutant, even though the PSI activity mediated by an artificial electron donor was detected in this mutant. Thus, the W622C/A623R mutation affected the interaction of the PSI complex with cytochrome c 6 . Biotin-maleimide modification of the mutant PSI complexes indicated that His-595, Trp-622, Leu-628, Tyr-632, and Asn-638 in wildtype PsaB may be exposed on the surface of the PSI complex. The results presented here demonstrate the role of an extramembrane loop of a PSI core protein in the interaction with soluble electron donor proteins.