Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

Regulation of electron transport in Photosystem-II fragments by magnesium ions

In Photosystem-II reaction-center particles (TSF-IIa) fractionated from spinach chloroplasts by Triton X-100 treatment, divalent cations appear to regulate electron-transport reactions. Oxidation of cytochrome b-559 after illumination of the particles was accelerated by the presence of Mg²⁺, whereas photoreduction of 2,6-dichlorophenolindophenol (DCIP) by diphenyl carbazide was inhibited, both at a half-effective concentration of Mg²⁺ of approx. 0.1 mM.The site of regulation was shown to be on the oxidizing side of Photosystem II, near P-680, based on the effects of actinic-light intensity and nature of the electron donors on DCIP photoreduction. Mg²⁺ was effective in quenching chlorophyll fluorescence in TSF-IIa particles, but the quenching was sensitive to the presence of 3(3,4-dichloropheny)-1,1-dimethylurea. In the reactioncenter (core) complex of Photosystem II, where the light-harvesting chlorophyll-protein complex is absent, there seems to be no regulation by Mg²⁺ on excitation-energy distribution.     

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559_HP). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.     

Further characterization of the two Photosystem II reaction center complex preparations from the thermophilic cyanobacterium Synechococcus sp.

Photochemical and chemical properties of two Photosystem II reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus sp. were examined. (1) The intact reaction center complexes contain each one of photoreducible pheophytin, secondary electron acceptor (Q_A) and cytochrome b-559 per 32–46 chlorophyll a molecules. (2) The reaction center complexes which lack the chlorophyll-binding 40 kDa polypeptide (CP2-b) showed photoaccumulation of reduced pheophytin and photoreduction of Q_A, indicating that the complexes can carry out not only the primary-charge separation, but also the stabilization of the separated charges. The contents of pheophytin, Q_A and cytochrome b-559 were, however, considerably reduced in CP2-b. (3) The two complexes contained very small amounts of manganese. (4) CP2-b was partially deprived of the small polypeptides: the ratios of the peak areas (corrected for molecular weight) of the 47/40/31 plus 28/9 kDa polypeptide bands resolved in sodium dodecyl sulfate gels after electrophoresis under denaturating conditions were approx. 1:1:2:2 in the intact complexes and 1:0:0.4:1 in CP2-b. The results were discussed in terms of the functional molecular organization of the Photosystem II reaction center complexes.     

Characterisation of a PS II reaction centre isolated from the chloroplasts of Pisum sativum

A photosystem II reaction centre has been isolated from peas and found to consist of D1, D2 polypeptides and the apoproteins of cytochrome b-559, being similar to that reported for spinach by Nanba and Satoh [(1987) Proc. Natl. Acad. Sci. USA 84, 109–112]. The complex binds chlorophyll a, pheophytin and the haem of cytochrome b-559 in an approximate ratio of 4:2:1 and also contains about one molecule of β-carotene. It binds no plastoquinone-9 or manganese but does contain at least one non-haem iron. In addition to a light-induced signal due to Pheo⁻ seen under reducing conditions, a light-induced P680⁺ signal is seen when the reaction centre is incubated with silicomolybdate. In the presence of diphenylcarbazide, the P680⁺ signal is partially inhibited and net electron flow to silicomolybdate occurs. This net electron flow is insensitive to o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene but is inhibited by proteolysis with trypsin and by other treatments. Fluorescence, from the complex, peaks at 682 nm at room temperature and at 685 nm at 77 K. This emission is significantly quenched when either the P680⁺Pheo or P680Pheo⁻ states are established indicating that the fluorescence emanates from the back reaction between P680⁺ and Pheo⁻.     

Characterization of Nuclear Mutants of Maize Which Lack the Cytochrome f/b-563 Complex

Two high fluorescent, nuclear recessive mutants of maize (Zea mays L.), designated hcf-2 and hcf-6, are described which are missing the chloroplast cytochrome f/b-563 complex. Thylakoids from the mutants show a block in whole chain electron transport activity (H₂O to methyl viologen), while retaining activities associated with photosystem II (H₂O to phenylenediamine) and photosystem I (diaminodurene to methyl viologen). Chemically induced, optical difference spectra indicate a loss of cytochromes f and b-563. Cytochrome b-559 is present in both high and low potential forms. EPR analyses of thylakoid membranes of hcf-6 reveals the lack of a signal (g = 1.90) associated with the Rieske Fe-S center. Additionally, hcf-6 is lacking EPR signals at g = 6 (attributable to the high spin ferric heme of cytochrome b-563) and g = 2.5 (unidentified). The mutant retains signals at g = 2.9 (cytochrome b-559) and at g = 4.3 and 9 (both signals probably arising from a storage form of ferric iron).

Thylakoid polypeptides are examined using polyacrylamide gel electrophoresis. hcf-2 and hcf-6 have identical profiles, showing losses of polypeptides with apparent molecular masses of 33 (cytochrome f), 23 (cytochrome b-563), and 17.5 kilodaltons. The protein associated with the Rieske Fe-S center could not be determined from the gel profiles. Additionally, both mutants show an increase in a band with a molecular mass of 31 kilodaltons.

     

The photochemical activities and electron carriers of developing barley leaves

The development of photochemical activities in isolated barley plastids during illumination of dark-grown plants has been studied and compared with the behaviour of plastocyanin, cytochromes f, b-559_LP, b-563 and b-559_HP and pigments P546 (C550) and P700. Electron-transport activity dependent on Photosystem 1 and cyclic photophosphorylation dependent on N-methylphenazonium methosulphate (phenazine methosulphate) were very active relative to the chlorophyll content after only a few minutes of illumination of etiolated leaves, and then rapidly declined during the first few hours of greening. By contrast, Photosystem 2 activity (measured with ferricyanide as electron acceptor) and non-cyclic photophosphorylation were not detectable during the first 2½h of greening, but then increased in total amount in parallel with chlorophyll. The behaviour of the electron carriers suggested their association with either Photosystem 1 or 2 respectively. In the first group were plastocyanin, cytochrome f and cytochrome b-563, whose concentrations in the leaf did not change during greening, and cytochrome b-559_LP whose concentration fell to one-half its original value, and in the second group were cytochrome b-559_HP and pigment P546, the concentrations of which closely followed the activities of Photosystem 2. Pigment P700 could not be detected during the first hour, during which time some other form of chlorophyll may take its place in the reaction centre of Photosystem 1. The plastids started to develop grana at about the time that Photosystem 2 activity became detectable.     

Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II

The D1-D2-cytochrome b-559 reaction center complex and the 47 kDa antenna chlorophyll protein isolated from spinach Photosystem II were characterized by means of low temperature absorption and fluorescence spectroscopy. The low temperature absorption spectrum of the D1-D2-cytochrome b-559 complex showed two bands in the Q_y region located at 670 and 680 nm. On the basis of its absorption maximum and orientation the latter component may be attributed at least in part to P-680, the primary electron donor of Photosystem II. The 47 kDa antenna complex showed absorption bands at 660, 668 and 677 nm and a minor component at 690 nm. The latter transition appeared to be associated with the characteristic low temperature 695 nm fluorescence band of Photosystem II. The 695 nm emission band was absent in the D1-D2 complex, which indicates that it does not originate from the reaction center pheophytin, as earlier proposed. The transition dipole responsible for the main fluorescence at 684 nm from this complex had a parallel orientation with respect to the membrane plane in the native structure. The reaction center preparation contains two spectrally distinct carotenoids with different orientations.     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using ³²P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.     

Isolation and characterisation of the Photosystem two reaction centre complex from a double mutant of Chlamydomonas reinhardtii

A rapid procedure has been developed for the isolation of the photosystem two reaction centre complex (PS II RC) from a double mutant of Chlamydomonas reinhardtii, F54-14, which lacks the Photosystem one complex and the chloroplast ATPase. Thylakoid membranes are solubilised with 1.5% (w/v) Triton X-100 and the PS II RC purified by anion-exchange chromatography using TSK DEAE-650(S) (Merck). The complex has a pigment stoichiometry of approximately six chlorophyll a: two pheophytin a: one cytochrome b-559: one to two -carotene. It photoaccumulates reduced pheophytin and oxidised P680 in the presence of sodium dithionite and silicomolybdate, respectively. Immunoblotting experiments have confirmed the presence of the D1 and D2 polypeptides in this complex. The -subunit of cytochrome b-559 was identified by N-terminal sequencing. Comparison of the complex with the PS II RC from pea using SDS-polyacrylamide gel electrophoresis showed that their polypeptide compositions were similar. However, the -subunit of cytochrome b-559 from C. reinhardtii has a lower apparent molecular weight than the pea counterpart whereas the -subunit is larger.Abbreviations DM n-dodecyl -d-maltoside - RC reaction centre - SiMo silicomolybdate, SiMo₁₂O₄₀ ^4– - TAP Tris-acetate-phosphate     

A pathway for the reduction of cytochrome b-559 by Photosystem II in chloroplasts

Cytochrome b-559, which is normally reduced in the dark, was oxidized by preillumination in the presence of N-methyl-phenazonium methosulfate with low intensity far-red light. The average half-time for the photoreduction of oxidized cytochrome b-559 by a long actinic flash ranged from 90 to 110 ms. In the presence of 0.25 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea the half-time for the photoreduction increased to 230 ms although the extent of the absorbance increase was unchanged. Under similar conditions inhibition of electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and the increase in the chlorophyll fluorescence show that a large fraction of the Photosystem II reaction centers are blocked. These results are consistent with the concept that electrons are shared between different photosynthetic units by a common pool of plastoquinone and imply that the principle pathway for the reduction of cytochrome b-559 by Photosystem II occurs through plastoquinone. In the presence of the uncoupler gramicidin which stimulates non-cyclic electron transport, the rate of photoreduction of cytochrome b-559 is slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 180}\text{ms}$), from which it is inferred that cytochrome b-559 competes with cytochrome f for electrons out of this pool. Comparison of cytochrome b-559 photoreduction and electron transport rates using untreated and KCN-treated chloroplasts indicate that, under conditions of basal electron transport from water to ferricyanide, approximately one-fifth of the electrons from Photosystem II go through cytochrome b-559 to ferricyanide. Further support for this pathway is provided by a comparison of the effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone) on the rates of reduction of cytochrome b-559 and ferricyanide.     

The Cytochromes of Prototheca zopfii

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a₃. Cytochromes a and a₃ could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a₃ complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a₃, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).     

Resolution of the light-harvesting chlorophyll ab-protein of Vicia faba chloroplasts into two different chlorophyll-protein complexes

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll $\text{a}\text{b}$ ratio of 0.9 and is associated with polypeptides of 24 000 and 23 000 daltons. The 24 000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23 000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll $\text{a}\text{b}$ ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23 000. The two 23 000 dalton polypeptides occurring in both complexes are not identical.     

Reorganization of Thylakoid Components during Chloroplast Development in Higher Plants after Transfer to Darkness : Changes in Photosystem I Unit Components, and in Cytochromes

It was shown earlier that in etiolated bean (Phaseolus vulgaris, var. red kidney) leaves exposed to continuous light for a short time and then transferred to darkness a reorganization of their photosystem II (PSII) unit components occurs. This reorganization involves disorganization of the light-harvesting complex of PSII (LHC-II), destruction of its chlorophyll b and the 25 kilodalton polypeptide, and reuse of its chlorophyll a for the formation of additional, small in size, PSII units (Argyroudi-Akoyunoglou, Akoyunoglou, Kalosakas, Akoyunoglou 1982 Plant Physiol 70: 1242-1248). The present study further shows that parallel to the PSII unit reorganization a reorganization of the PSI unit components also occurs: upon transfer to darkness the 24, 23, and 21 kilodalton polypeptides, components of the light-harvesting complex of PSI (LHC-I), are decreased, the 69 kilodalton polypeptide, component of the chlorophyll a-rich P700-protein complex (CPI), is increased and new smallsized PSI units are formed. Concomitantly, the cytochrome f/chlorophyll and the cytochrome b/chlorophyll ratios are gradually increased. This suggests that the concentration of the electron transport components is also modulated in darkness to allow for adequate electron flow to occur between the newly synthesized PSII and PSI units.     

The light-harvesting chlorophyll a/b · protein complex of the green alga Acetabularia mediterranea isolation and characterization of two subunits

In the green alga Acetabularia mediterranea a light-harvesting chlorophyll a/b · protein complex of 67 000 daltons has been found which contains two polypeptide chains of 21 500 and 23 000 daltons. These two polypeptides were isolated on a preparative scale and were further characterized by several different methods. Both polypeptides proved to be very similar. While their amino acid and sugar compositions as well as their immunochemical properties were almost identical the tryptic peptides and the cyanogen bromide fragments of the two polypeptides revealed minor but significant differences. The 67 000-dalton chlorophyll a/b · protein complex and its two polypeptide components were compared to the light-harvesting chlorophyll a/b · protein of higher plants.     

Development of the Primary Photochemical Apparatus of Photosynthesis during Greening of Etiolated Bean Leaves

Seven-day-old dark-grown bean leaves were greened under continuous light. The amount of chlorophyll, the ratio of chlorophyll a to chlorophyll b, the O₂ evolving capacity and the primary photochemical activities of Photosystem I and Photosystem II were measured on the leaves after various times of greening. The primary photochemical activities were measured as the photo-oxidation of P₇₀₀, the photoreduction of C-550, and the photo-oxidation of cytochrome b₅₅₉ in intact leaves frozen to −196 C. The results indicate that the reaction centers of Photosystem I and Photosystem II begin to appear within the first few minutes and that Photosystem II reaction centers accumulate more rapidly than Photosystem I reaction centers during the first few hours of greening. The very early appearances of the primary photochemical activity of Photosystem II was also confirmed by light-induced fluorescence yield measurements at −196 C.     

Purification of Photosystem II particles from Phormidium laminosum using the detergent dodecyl-β-d-maltoside. Properties of the purified complex

The purification and properties of a new oxygen-evolving Photosystem (PS) II particle from the thermophilic blue-green alga Phormidium laminosum are described. The activity of the lauryldimethylamine N-oxide PS II-enriched supernatant described previously (Stewart, A.C. and Bendall, D.S. (1979) FEBS Lett. 107, 308–312) was found to be stabilized for several days at 4°;C by the addition of a second detergent, dodecyl-β-d-maltoside (lauryl maltoside). The lauryl maltoside/lauryldimethylamine N-oxide extract could be fractionated by sucrose density gradient centrifugation. Very high rates of oxygen evolution, typically 1900–2400 μmol O₂/mg chlorophyll a per h at pH 7 with dimethylbenzoquinone and ferricyanide as acceptors, were observed for the lowest green band from the gradient. This fraction contained cytochromes b-559 (high-potential) and c-549, but was completely devoid of P-700 and cytochromes b-563 and f. The purified oxygen-evolving particles comprised seven major polypeptides (M_r 58 900, 52 400, 43 200, 33 900, 30 000, 16 000 and 15 000) and approximately five minor polypeptides. The particles contained 3–4 Mn atoms per reaction centre and had a chlorophyll antenna of approx. 50 chlorophyll a. The fast phase of fluorescence induction curves in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) could be described by an exponential, suggesting that no energy transfer was occurring between the PS II units responsible for this phase. Comparison of the area above the fluorescence induction curves in the absence and presence of DCMU suggested an acceptor pool size of 2–3 equivalents per centre.