The psbO mutant of Chlamydomonas reinhardtii is capable of assembling stable, photochemically active reaction center of photosystem II

The functional status of photosystem II (PSII) complex in the dark-grown PsbO-deficient mutant of green alga Chlamydomonas reinhardtii was studied. It was found that ΔpsbO mutant cells of C. reinhardtii grown under heterotrophic conditions (dark + acetate) were capable of assembling stable, photochemically-competent reaction centers of PSII (as confirmed by immunological analysis of D1 protein level, pigments content and photoinduced changes of PSII chlorophyll fluorescence yield), while O₂-evolution activity was not revealed. The ratio F _v/F _m for the dark-grown ΔpsbO mutant C. reinhardtii was 0.37 and that for the dark-grown wild type cells was 0.56. Analysis of chlorophyll fluorescence induction curve indicated that the absence of oxygen-evolving activity could be due to some defects in the organization of the PSII catalytic manganese cluster. Decrease of the rate of the electron donation from water-oxidizing complex to the PSII reaction center as well as the appearance of an additional transient fluorescence peak during the dark relaxation of F _v testify to the damages to the PSII donor side. The data obtained suggest that the dark-grown PsbO-deficient cells of C. reinhardtii are able to form stable, photochemically active PSII reaction center, unable to oxidize water due to probable defects in the assembly of the manganese cluster.     

Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O₂ evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111–1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.     

Polynucleotide kinase from a T4 mutant which lacks the 3'' phosphatase activity.

Polynucleotide kinase from E. coli infected with the PseT 1 mutant of bacteriophage T4 has been isolated. The PseT 1 enzyme purifies similarly to normal polynucleotide kinase and effectively transfers the gamma phosphate of ATP to the 5' terminal hydroxyl of DNA and RNA. The PseT 1 and normal enzymes require similar magnesium ion concentrations, have the same pH optima and are both inhibited by inorganic phosphate. However, the PseT 1 enzyme is totally lacking the 3' phosphatase activity associated with normal polynucleotide kinase. The PseT 1 enzyme is a useful tool for the preparation of oligonucleotides with 3' and 5' terminal phosphates for use as susbstrates for RNA ligase.     

Organization of the photosystem II centers and their associated antennae in the thylakoid membranes: a comparative ultrastructural,biochemical, and biophysical study of Chlamydomonas wild type and mutants lacking in photosystem II reaction centers

We investigated the ultrastructure of thylakoid membranes that lacked either some or all of their Photosystem II centers in the F34SU3 and F34 mutants of Chlamydomonas reinhardtii. We obtained the following results: (a) There are no particles of the 160-A size class on the EF faces of the thylakoids in the absence of Photosystem II centers (as in F34); the F34SU3 contains 50% of the wild-type number of PSII centers and EF particles. (b) The density of the particles on the PF faces of the thylakoids is higher in the mutants than in the wild type. (c) The fluorescence analysis shows that the organization of the pigments is the same regardless of whether 50% of the PSII centers are temporarily inactivated (by preilluminating the wild type) or are actually missing from the thylakoid membrane (F34SU3). Our results, therefore, support a model in which: (a) each 160-A EF particle has only one PSII center surrounded by light-harvesting complexes and (b) part of the PSH antenna is associated with 80-A PF particles in both of the mutants and the wild type.     

Comparative analysis of the biogenesis of photosystem II in the wild- type and Y-1 mutant of Chlamydomonas reinhardtii

Expression of the genes of the photosystem II (PSII) core polypeptides D1 and D2, of three proteins of the oxygen evolving complex of PSII and of the light harvesting chlorophyll a/b binding proteins (LHCP) has been compared in wild-type (wt) and in the y-1 mutant of Chlamydomonas reinhardtii. Since wt, but not y-1 cells produce a fully developed photosynthetic system in the dark, comparison of the two has allowed us to distinguish the direct effect of light from the influence of plastid development on gene expression. The PSII core polypeptides and LHCP are nearly undetectable in dark-grown y-1 cells but they accumulate progressively during light induced greening. The levels of these proteins in wt are the same in the light and the dark. The amounts of the proteins of the oxygen evolving complex do not change appreciably in the light or in the dark for both wt and y-1. Steady state levels of chloroplast mRNA encoding the core PSII polypeptides remain nearly constant in the light or the dark and are not affected by the developmental stage of the plastid. Levels of nuclear encoded mRNAs for the oxygen evolving proteins and of LHCP increase during light growth in wt and y-1. In contrast to wt, synthesis of LHCP proteins is not detectable in y-1 cells in the dark but starts immediately after transfer to light, indicating that LHCP synthesis is controlled by a light-induced factor or process. While the rates of synthesis of D1 and D2 are immediately enhanced by light in wt, this increase occurs only after a lag in y-1 and thus must be dependent on an early light-induced event in the plastid. These results show that the biosynthesis of PSII is affected by light directly, by the stage of plastid development, and by the interaction of light and events associated with plastid development.     

Isolation of a psaF-deficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit.

The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cw15-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles a functional PSI complex and is capable of photoautotrophic growth. However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex. These results contrast with those obtained previously with a cyanobacterial psaF-, psaJ- double mutant where no phenotype was apparent.     

Quality control of photosystem II. Cleavage of reaction center D1 protein in spinach thylakoids by FtsH protease under moderate heat stress

When spinach thylakoids were subjected to moderate heat stress (40 degrees C for 30 min), oxygen evolution was inhibited, and cleavage of the reaction center-binding protein D1 of photosystem II took place, producing 23-kDa N-terminal fragments. The D1 cleavage was greatly facilitated by the addition of 0.15 mM ZnCl2 and 1 mM ATP and was completely inhibited by 1 mM EDTA, indicating the participation of an ATP-dependent metalloprotease(s) in the D1 cleavage. Herbicides 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, bromoxynil, and ioxynil, all of which bind to the Q(B) site, inhibited the D1 cleavage, suggesting that the DE-loop of the D1 protein is the heat-sensitive cleavage site. We solubilized the protease by treating the thylakoids with 2 M KSCN and detected a protease activity in the supernatant by gelatin activity gel electrophoresis in the 70-80-kDa region. The antibodies against tobacco FtsH and Arabidopsis FtsH2 reacted with a 70-80-kDa band of the KSCN-solubilized fraction, which suggests the presence of FtsH in the fraction. In accordance with this finding, we identified the homolog to Arabidopsis FtsH8 in the 70-80-kDa region by matrix-assisted laser desorption ionization time-of-flight mass analysis of the thylakoids. The KSCN-solubilized fraction was successively reconstituted with thylakoids to show heat-induced cleavage of the D1 protein and production of the D1 fragment. These results strongly suggest that an FtsH protease(s) is involved in the primary cleavage of the D1 protein under moderate heat stress.     

Oxidation of the two beta-carotene molecules in the photosystem II reaction center

We present a spectroscopic characterization of the two nonequivalent beta-carotene molecules in the photosystem II reaction center. Their electronic and vibrational properties exhibit significant differences, reflecting a somewhat different configuration for these two cofactors. Both carotenoid molecules are redox-active and can be oxidized by illumination of the reaction centers in the presence of an electron acceptor. The radical cation species show similar differences in their spectroscopic properties. The results are discussed in terms of the structure and unusual function of these carotenoids. In addition, the attribution of resonance Raman spectra of photosystem II preparations excited in the range 800-900 nm is discussed. Although contributions of chlorophyll cations cannot be formally ruled out, our results demonstrate that these spectra mainly arise from the cation radical species of the two carotenoids present in photosystem II reaction centers.     

Two functionally distinct manganese clusters formed by introducing a mutation in the carboxyl terminus of a photosystem II reaction center polypeptide, D1, of the green alga Chlamydomonas reinhardtii

To study the function of the carboxyl-terminal domain of a photosystem II (PSII) reaction center polypeptide, D1, chloroplast mutants of the green alga Chlamydomonas reinhardtii have been generated in which Leu-343 and Ala-344 have been simultaneously or individually replaced by Phe and Ser, respectively. The mutants carrying these replacements individually, L343F and A344S, showed a wild-type phenotype. In contrast, the double mutant, L343FA344S, evolved O2 at only 20-30% of the wild-type rate and was unable to grow photosynthetically. In this mutant, PSII accumulated to 60% of the wild-type level, indicating that the O2-evolving activity per PSII was reduced to approximately half that of the wild-type. However, the amount of Mn atom detected in the thylakoids suggested that a normal amount of Mn cluster was assembled. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that the electron transfer from Q(-)(A) to Q(B) was not affected. When a back electron transfer from Q(-)(A) to a donor component was measured in the presence of 3-(3,4-dichlorophenol)-1,1-dimethylurea, a significantly slower component of the Q(-)(A) oxidation was detected in addition to the normal component that corresponds to the back electron transfer from the Q(-)(A) to the S(2)-state of the Mn cluster. Thermoluminescence measurements revealed that L343FA344S cells contained two functionally distinct Mn clusters. One was equivalent to that of the wild-type, while the other was incapable of water oxidation and was able to advance the transition from the S(1)-state to the S(2)-state. These results suggested that a fraction of the Mn cluster had been impaired by the L343FA344S mutation, leading to decreased O2 evolution. We concluded that the structure of the C-terminus of D1 is critical for the formation of the Mn cluster that is capable of water oxidation, in particular, transition to higher S-states.     

Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

Non-photochemical fluorescence quenching in photosystem II antenna complexes by the reaction center cation radical

In direct experiments, rate constants of photochemical (k_P) and non-photochemical (k_P⁺) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the “dark” reaction center (RC) state, antenna fluorescence decay kinetics were measured under lowintensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a “closed” [P680⁺Q_A^–] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680⁺Q_A^–] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680⁺) state was found to be ～1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ₁ obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680⁺Pheo^– charge recombination. Thus, for the first time the ratio of k_P⁺/k_P = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.     

A reassessment of the use of herbicide binding to measure photosystem II reaction centres in plant thylakoids

The binding of the herbicide atrazine to thylakoid membranes is often used to quantify Photosystem II reaction centres. Two atrazine binding sites, with high and low affinities, have been observed on the D1 and D2 polypeptides of Photosystem II, respectively (McCarthy S., Jursinic P. and Stemler A. (1988) Plant Physiol. 86S:46). We have observed that the accessibility of the low-affinity binding sites is variable, being limited in freshly isolated thylakoids or in fresh frozen-thawed thylakoids, but increasing during storage of the membranes on ice. In contrast, the accessibility of the high-affinity binding sites, which are titratable at low concentrations (< 500 nM) of herbicide, is much less variable, although the dissociation constant is greatly influenced by ethanol. We conclude that to quantify Photosystem II reaction centres by atrazine binding, it is sufficient and more reliable to assay only the high-affinity binding sites.     

Reduction of photosystem I reaction center, P-700, by plastocyanin in stroma thylakoids from spinach: Lateral diffusion of plastocyanin

The reduction kinetics of the photooxidized photosystem I reaction center (P-700⁺) by plastocyanin was studied in the stroma thylakoids prepared by the Yeda press treatment. The kinetics of the P-700⁺ reduction after flash excitation were biphasic and separated into two independent first-order reactions, the fast phase with a half-time of about 4 ms and the slow phase with a half-time of about 18 ms. Only the fast phase of the P-700⁺ reduction was sensitive to KCN and glutaraldehyde treatments of the thylakoids which block the plastocyanin site in the photosynthetic electron flow indicating that the fast phase is mediated by plastocyanin. However, the content of plastocyanin in the stroma thylakoids used was greatly decreased by the Yeda press treatment to only half that of P-700⁺ reduced in the fast phase. This indicates that one plastocyanin molecule turns over more than once in the single turnover of P-700⁺ rather than forming a fixed complex with P-700. On the other hand, the slow phase was not affected by KCN or glutaraldehyde treatment and its apparent rate constant linearly depended on the concentration of reduced dichlorophenolindophenol. These results indicate that the slow phase shows direct reduction of P-700⁺ by dichlorophenolindophenol. A second-order rate constant of 3.96 × 10⁵m^?1 s^?1 was obtained for the slow phase at pH 7.6, 25 °C. Analysis of reaction kinetics in the initial portion of the fast phase indicated initial interaction between P-700⁺ and the reduced plastocyanin and gave a half-time of 0.53 ms for the bimolecular reaction. We assumed the lateral diffusion of plastocyanin on the thylakoid membrane and calculated the two-dimensional diffusion coefficient for plastocyanin from the half-time of the initial reduction of P-700⁺ as about 2 × 10^?9 cm² s^?1.     

Triplet formation on a monomeric chlorophyll in the photosystem II reaction center as studied by time-resolved infrared spectroscopy

The process of formation of the triplet state of chlorophyll in the photosystem II (PS II) reaction center complex was studied by means of time-resolved infrared (IR) spectroscopy. Using a dispersive-type IR spectrometer with a time resolution of approximately 55 ns, transient spectra in the C=O stretching region (1760--1600 cm(-1)) were measured at 77 K. The data were analyzed by singular-value decomposition and subsequent least-squares fitting. Two distinct spectral components having different kinetic behaviors were resolved. One had spectral features characterized by negative peaks at 1740 and 1680 cm(-1) and an overall positive background and was assigned to the P(680)(+)Phe(-)/P(680)Phe radical pair by static FTIR measurements of the P(680)(+)/P(680) and Phe(-)/Phe differences. The other had prominent negative and positive peaks at 1668 and 1628 cm(-1), respectively, which were previously assigned to the keto C==O change upon triplet formation of the monomeric chlorophyll denoted as Chl(T) [Noguchi, T., Tomo, T., and Inoue, Y. (1998) Biochemistry 37, 13614-13625]. The former component of P(680)(+)Phe(-)/P(680)Phe exhibited a multiphasic decay with time constants of 77 ns (75%), 640 ns (18%), 8.3 micros (4%), and 0.3 ms (3%), while the latter component of (3)Chl(T)/Chl(T) was formed with a single-exponential rise with a time constant of 57 ns and had a lifetime of 1.5 ms. From the observations that only the two spectral components were resolved without any other triplet intermediates and the time constant of (3)Chl(T) formation roughly agreed with or seemed even faster than that of the major phase of the P(680)(+)Phe(-) decay, two alternative mechanisms of triplet formation are proposed. (i) (3)Chl(T) is directly formed from P(680)(+)Phe(-) by charge recombination at Chl(T), and (ii) (3)P(680) is formed, and then the triplet is transferred to Chl(T) with a time constant of much less than 50 ns. The location of Chl(T) in the D1 subunit as the monomer chlorophyll corresponding to the accessory bacteriochlorophyll in the L subunit of purple bacteria is favored to explain the former mechanism as well as the triplet properties reported in the literature. The physiological role of the triplet formation on Chl(T) is also discussed.     

Role of phosphatidylglycerol in the function and assembly of Photosystem II reaction center, studied in a cdsA-inactivated PAL mutant strain of Synechocystis sp. PCC6803 that lacks phycobilisomes

To analyze the role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria we used two mutants of Synechocystis sp. PCC6803: the PAL mutant which has no phycobilisomes and shows a high PSII/PSI ratio, and a mutant derived from it by inactivating its cdsA gene encoding cytidine 5'-diphosphate diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the PAL/DeltacdsA mutant cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a slowdown of electron transfer from the acceptor Q(A) to Q(B) in PSII and (c) in a modification of chlorophyll fluorescence curve. The depletion of PG affected neither the redox levels of Q(A) nor the S(2) state of the oxygen-evolving manganese complex, as indicated by thermoluminescence studies. Two-dimensional PAGE showed that in the absence of PG (a) the PSII dimer was decomposed into monomers, and (b) the CP43 protein was detached from a major part of the PSII core complex. [(35)S]-methionine labeling confirmed that PG depletion did not block de novo synthesis of the PSII proteins. We conclude that PG is required for the binding of CP43 within the PSII core complex.     

High field EPR study of the pheophytin anion radical in wild type and D1-E130 mutants of photosystem II in Chlamydomonas reinhardtii

The intermediate electron acceptor in photosystem II is a pheophytin molecule. The radical anion of this molecule was studied using high field electron paramagnetic resonance in a series of Chlamydomonas reinhardtii mutants. Glutamic acid 130 of the D1 polypeptide is thought to hydrogen bond the ring V carbonyl group of this radical. Mutations at this site, designed to weaken or remove this hydrogen bond, strongly affected the g tensor of the radical. The upward shift of the g(x) component followed the decreasing hydrogen bonding capacity of the amino acid introduced. This behavior is similar to that of tyrosyl and semiquinone radicals. It is also consistent with the optical spectra of the pheophytin in similar mutants. Density functional calculations were used to calculate the g tensors and rationalize the observed trend in the variation of the g(x) value for pheophytin and bacteriopheophytin radical. The theoretical results support the experimental observations and demonstrate the sensitivity of g values to the electrostatic protein environment for these types of radicals.     

Protein-pigments and the photosystem II reaction center: a glimpse into the history of research and reminiscences

This article provides a glimpse into the dawning of research on chlorophyll-protein complexes and a brief recollection of the path that led us to the identification of the photosystem II reaction center, i.e., the polypeptides that carry the site of primary charge separation in oxygenic photosynthesis. A preliminary version of the personal review on the latter topic has already appeared in this journal (Satoh Photosynth Res 76:233-240, 2003).     

Electric field effects on the chlorophylls,pheophytins, and beta-carotenes in the reaction center of photosystem II

We present an electric field modulated absorption spectroscopy (Stark effect) study of isolated photosystem II reaction center complexes, including a preparation in which the inactive pheophytin H(B) was exchanged for 13(1)-deoxo-13(1)-hydroxy-pheophytin. The results reveal that the Stark spectrum of the Q(x) and Q(y) transitions of the pheophytins has a second-derivative line shape, indicating that the Stark effect is dominated by differences in the dipole moment between the ground and the electronically excited states of these transitions (Delta mu). The Delta mu values for the Q(x) and Q(y) transitions of H(B) are small (Delta mu = 0.6-1.0 D f(-1)), whereas that of the Q(x) transition of the active pheophytin H(A) is remarkably large (Delta mu = 3 D f(-1)). The Stark spectrum of the red-most absorbing pigments also shows a second-derivative line shape, but this spectrum is considerably red-shifted as compared to the second derivative of the absorption spectrum. This situation is unusual but has been observed before in heterodimer special pair mutants of purple bacterial reaction centers [Moore, L. J., Zhou, H., and Boxer, S. G. (1999) Biochemistry 38, 11949-11960]. The red-shifted Stark spectra can be explained by a mixing of exciton states with a charge-transfer state of about equal energy. We conclude that the charge transfer state involves H(A) and its immediate chlorophyll neighbor (B(A)), and we suggest that this (B(A)(delta+)H(A)(delta-)) charge transfer state plays a crucial role in the primary charge separation reaction in photosystem II. In contrast to most other carotenes, the two beta-carotene molecules of the photosystem II reaction center display a very small Delta mu, which can most easily be explained by excitonic coupling of both molecules. These results favor a model that locates both beta-carotene molecules at the same side of the complex.