Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH⁻₄). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH⁻₄ in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13¹-deoxo-13¹-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo⁻ and P680⁺. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Q_y and Q_x transitions of this molecule were determined to be located at 5°C at 679.5–680 nm and 542 nm, respectively.     

Photoreduction of pheophytin as a result of electron donation from the water-splitting system to Photosystem-II reaction centers

Reversible photoreduction of pheophytin (Pheo) accompanied by a decrease of chlorophyll-fluorescence yield is observed in subchloroplast oxygen-evolving preparations of Photosystem II (PS II) under anaerobic conditions. This photoreaction is activated at addition of CCCP, inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and reactivated upon subsequent addition of ascorbate. Benzyl viologen as well as methyl viologen accelerates dark oxidation of reduced pheophytin, indicating that they are able to accept an electron from Pheo^⨪. The data on both the photoreduction of pheophytin in the absence of exogenous reductants - when electron donation to reaction centers of PS II occurs only from water - and the inhibition of this photoreaction by DCMU show that the pheophytin photoreduction is sensitized by reaction centers of PS II, and it probably occurs as a result of electron donation from the water-splitting system being in the sate S₃ to P^⨥-680Pheo^⨪Q⁻, producing the long-lived state S₀ P-680Pheo^⨪Q⁻ and O₂. Photoreduction of pheophytin in the presence of ascorbate (and dithionite) evidently occurs as a result of donation of its electrons to P^⨥-680Pheo^⨪Q⁻ by means of the S-states of the water-oxidizing system. It is shown that the photoinduced decrease of fluorescence in chloroplasts under anaerobic conditions is due to two processes: photoreduction of pheophytin in Photosystem II and photooxidation of Q⁻ by Photosystem I. It is suggested that photoreduction of pheophytin takes also place under aerobic conditions when Q is reduced. It may contribute to the P−S fluorescence decrease during fluorescence induction in leaves.     

Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 131-deoxo-131-hydroxy-pheophytin a and comparison of their 6 K absorption spectra

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 13¹-deoxo-13¹-hydroxy-pheophytin a (13¹-OH-Pheo). The absorption bands of 13¹-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC_1×) or twice (RC_2×). HPLC analysis and Pheo Q_X absorption at 543 nm show that in RC_1× about 50% of Pheo is replaced and in RC_2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo⁻ is unmodified in RC_1× and decreased only in RC_2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the Q_Y region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC_1× or RC_2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Formation and decay of P680 (PD1–PD2)PheoD1 radical ion pair in photosystem II core complexes

Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheo⁻) at 460 nm is observed with rise time of ~ 11 ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (Pheo_D1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680⁺Pheo_DI⁻. The subsequent electron transfer from Pheo_D1⁻ to primary plastoquinone electron acceptor (Q_A) was accompanied by relaxation of the 460-nm band and occurred within ~ 250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680⁺ spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of Pheo_DI⁻, which is very similar to that measured earlier by accumulation method. The spectrum of Pheo_DI⁻ formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to Pheo_D1. According to previous measurements in the femtosecond–picosecond time range this Chl-670 was ascribed to Chl_D1 [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45–50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~ 1 ps and ~ 14 ps. These components appear to reflect formation of P680⁺Chl_D1⁻ and P680⁺Pheo_D1⁻, respectively, as found earlier. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Primary radical ion pairs in photosystem II core complexes

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700–710 nm at 278 K. It was found that the initial charge separation between P680* and Chl_D1 (Chl-670) takes place with a time constant of ～1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680^*(1?δ) (P680^δ+1Chl _D1 ^δ? ), where δ ～ 0.5. The subsequent electron transfer from P680^δ+Chl _D1 ^δ? to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (Chl _D1 ^δ? ) and bleaching of the Pheo_D1 bands at 420, 545, and 680 nm with development of the Pheoband at 460 nm. Further electron transfer to Q_A occurs within 250 ps in accordance with earlier data. The spectra of P680⁺ and Pheo^? formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ～1 and ～13 ps due to the formation of P680^δ+Chl _D1 ^δ? and P680⁺Pheo _D1 ^? , respectively.     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu²⁺ ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO₄ and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (A of 430–440 nm) and by the flash induced formation of P680⁺ (A at 820 nm). The relatively slow relaxation kinetics of the latter signal (t_1/2 1 ms) may suggest that in a substantial fraction of the RCs Q_A remains bound to the complex.Abbreviations Car -carotene - Chl a chlorophyll a - CP43, CP47 chlorophyll-proteins, with R_m 43 and 47 kDa - DBMIB dibromothymoquinone,2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone - DM -dodecyl-d-maltoside - HPLC high-performance liquid chromatography - OG n-octyl--d-glucopyranoside - IMAC immobilied metal affinity chromatography - Pheo a pheophytin a - PQ-9 plastoquinone-9 - P680 primary electron donor in PS II - PS II RC Photosystem II reaction centre - Q_A primary electron acceptor in PS II - SB-12 N-dodecyl-N,N-dimethyl-3-amino-1-propanesulphonate, (sulphobetain 12)     

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⁻.     

Photophysical processes of energy conversion in thylakoid membranes of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> mutants D1-R323H,D1-R323D,and D1-R323L

Chlamydomonas reinhardtii mutants D1-R323H, D1-R323D, and D1-R323L showed elevated chlorophyll fluorescence yields, which increased with decline of oxygen evolving capacity. The extra step K ascribed to the disturbance of electron transport at the donor side of PS II was observed in OJIP kinetics measured in mutants with a PEA fluorometer. Fluorescence decay kinetics were recorded and analyzed in a pseudo-wild type (pWt) and in mutants of C. reinhardtii with a Becker and Hickl single photon counting system in pico- to nanosecond time range. The kinetics curves were fitted by three exponentials. The first one (rapid, with lifetime about 300 ps) reflects energy migration from antenna complex to the reaction center (RC) of photosystem II (PS II); the second component (600–700 ps) has been assigned to an electron transfer from P680 to Q_A, while the third one (slow, 3 ns) assumingly originates from charge recombination in the radical pair [P680^+• Pheo^−•] and/or from antenna complexes energetically disconnected from RC II. Mutants showed reduced contribution of the first component, whereas the yield of the second component increased due to slowing down of the electron transport to Q_A. The mutant D1-R323L with completely inactive oxygen evolving complex did not reveal rapid component at all, while its kinetics was approximated by two slow components with lifetimes of about 2 and 3 ns. These may be due to two reasons: a) disconnection between antennae complexes and RC II, and b) recombination in a radical pair [P680^+• Pheo^−•] under restricted electron transport to Q_A. The data obtained suggest that disturbance of oxygen evolving function in mutants may induce an upshift of the midpoint redox potential of Q_A/Q_A⁻ couple causing limitation of electron transport at the acceptor side of PS II.     

D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA₃ gene over the psbA₁ gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at < 10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q_x band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the Pheo_D1 Q_x band induced by Q_A⁻ (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t_1/2 > 50 ms) Q_A⁻ yield was ∼ 95% in D1:3, and ∼ 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q_x band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13¹-keto of Pheo_D1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P_D1, in particular Ser153Ala. Photo-induced Q_A⁻ formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P⁺.     

Effect of Photosystem II inhibitor K-15 on photochemical reactions of the isolated D1/D2 cytochrome b559 complex

Effect of a highly efficient inhibitor of Photosystem II (PS II), K-15 (4-[methoxy-bis-(trifluoromethyl)methyl)-2,6-dinitrophenyl hydrazone methyl ketone), was investigated using the D1/D2/cytochrome b559 reaction centre (RC) complex. A novel approach for photoaccumulating reduced pheophytin (Pheo^–) in the absence of the strong reducing agent, sodium dithionite, was demonstrated which involved illumination in the presence of TMPD (from 5 to 100 M) under anaerobic conditions. The addition of K-15 at concentrations of 0.5 M and 2 M resulted in approx. 50% and near 100%, respectively, inhibition of this photoreaction, while subsequent additions of dithionite eliminated the inhibitory effect of K-15. Methyl viologen induced similar inhibition at much higher concentrations (>1 mM). Moreover, K-15 efficiently quenched the variable part of chlorophyll fluorescence (which is the recombination luminescence of the pair P₆₈₀ ⁺ Pheo^–). A 50% inhibition was induced by 5 M K-15 and the effect was maximal in the range 20 to 200 M. Photooxidation of P₆₈₀ in the presence of 0.1 mM silicomolybdate was also efficiently inhibited by K-15 (50% inhibition at 15 M). The data are consistent with the idea put forward earlier (Klimov et al. 1992) that the inhibitory effect of K-15 is based on facilitating a rapid recombination between Pheo^– and P₆₈₀ ⁺ (or Z⁺) via its redox properties. The inhibitor can be useful for suppressing PS II reactions in isolated RCs of PS II which are resistant to all traditional inhibitors, like diuron, and probably functions by substituting for Q_A missing in the preparation.At a concentration of 0.5–50 M K-15 considerably increased both the rate and extent of cytochrome b559 photoreduction in the presence, as well as in the absence, of 5 mM MnCl₂. Consequently it is suggested that K-15 also serves as a mediator for electron transfer from Pheo^– to cytochrome b559.Abbreviations K-15 4-[methoxy-bis-(trifluoromethyl)methyl]-2,6-dinitrophenyl hydrazone methyl ketone - P₆₈₀ the primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A and Q_B the primary and the secondary electron acceptor of PS II - RC reaction centre - SiMo silicomolybdate - TMPD N,N,N,,N,-tetramethyl-p-phenylenediamine - Z secondary electron donor of PS II     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

Effects of detergent on the excited state structure and relaxation dynamics of the photosystem II reaction center: A high resolution hole burning study

Low temperature (4.2 K) absorption and hole burned spectra are reported for a stabilized preparation (no excess detergent) of the photosystem II reaction center complex. The complex was studied in glasses to which detergent had and had not been added. Triton X-100 (but not dodecyl maltoside) detergent was found to significantly affect the absorption and persistent hole spectra and to disrupt energy transfer from the accessory chlorophyll a to the active pheophytin a. However, Triton X-100 does not significantly affect the transient hole spectrum and lifetime (1.9 ps at 4.2 K) of the primary donor state, P680^*. Data are presented which indicate that the disruptive effects of Triton X-100 are not due to extraction of pigments from the reaction center, leaving structural perturbations as the most plausible explanation. In the absence of detergent the high resolution persistent hole spectra yield an energy transfer decay time for the accessory Chl a Q_Y-state at 1.6 K of 12 ps, which is about three orders of magnitude longer than the corresponding time for the bacterial RC. In the presence of Triton X-100 the Chl a Q_Y-state decay time is increased by at least a factor of 50.Abbreviations PS I photosystem I - PS II photosystem II - RC reaction center - P680, P870, P960 the primary electron donor absorption bands of photosystem II, Rhodobacter sphaeroides, Rhodopseudomonas viridis - NPHB nonphotochemical hole burning - TX Triton X-100 - DM Dodecyl Maltoside - Chl chlorophyll - Pheo pheophytin - ZPH ero phonon hole     

Thermoluminescence properties of the isolated photosystem two reaction centre

Formation of thermoluminescence signals is characteristics of energy- and charge storage in Photosystem II. In isolated D1/D2/cytochrome b-559 Photosystem II reaction centre preparation four thermoluminescence components were found. These appear at -180 (Z band), between -80 and -50 (Z_v band), at -30 and at +35°C. The Z band arises from pigment molecules but not correlated with photosynthetic activity. The Z_v and -30°C bands arise from the recombination of charge pairs stabilized in the Photosystem II reaction centre complex. The +35°C band probably corresponds to the artefact glow peak resulting from a pigment-protein-detergent interaction in subchloroplast preparations (Rózsa Zs, Droppa M and Horváth G (1989) Biochim Biophys Acta 973, 350–353).Abbreviations Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - D1 psbA gene product - D2 psbD gene product - P680 primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - RC reaction centre of PS II - TL thermoluminescence     

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.     

Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

On the mechanism of linolenic acid inhibition in photosystem II

Recent studies in our laboratory have reexamined the interaction of the unsaturated fatty acid, linolenic acid, with Photosystem II and have documented two principal regions of inhibition: one associated with the donor complex (Signal 2_f or D₁) to the reaction center, and the other located on the reducing side between pheophytin and Q_a (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263–271). A further characterization of fatty acid inhibition of secondary electron transport in Photosystem II at room and cryogenic temperatures is presented in this paper. These studies demonstrate that linolenic acid, and related fatty acid analogs, (1) eliminate the transient absorption increase at 320 nm, attributed to Q⁻_a; (2) abolish the production, either chemically or photochemically, of the ESR signal (Q⁻Fe) associated with the bound quinone acceptor, Q⁻_a; and (3) prevent the photooxidation of Signal 2_1t(D₁) at cryogenic temperature. Linolenic-acid-treated samples are characterized by a high initial fluorescence yield (F_i) equivalent to the maximum level of fluorescence (F_max); however, the spin-polarized triplet, associated with the reactioncenter electron donor, P-680, is observed only in inhibited samples that have been prereduced with sodium dithionite. These results suggest the presence of an additional acceptor intermediate between pheophytin and Q_a. The donor-assisted photoaccumulation of pheophytin anion in Photosystem II particles, as monitored by the decline of fluorescence yield, is inhibited by linolenic acid. Redox titrations of the fluorescence yield in control and inhibited preparations demonstrate that the midpoint potential for the primary acceptor for Photosystem II is insensitive to the fatty acid (E_m ≈ −583 mV) and thus indicate that primary photochemistry is functional during linolenic-acid inhibition. These data are consistent with the hypothesis that unsaturated fatty acids inhibit secondary electron transport in Photosystem II via displacement of endogenous quinone from quinone-binding peptides.     

Oxidized Amino Acid Residues in the Vicinity of QA and PheoD1 of the Photosystem II Reaction Center: Putative Generation Sites of Reducing-Side Reactive Oxygen Species

Under a variety of stress conditions, Photosystem II produces reactive oxygen species on both the reducing and oxidizing sides of the photosystem. A number of different sites including the Mn₄O₅Ca cluster, P₆₈₀, Pheo_D1, Q_A, Q_B and cytochrome b₅₅₉ have been hypothesized to produce reactive oxygen species in the photosystem. In this communication using Fourier-transform ion cyclotron resonance mass spectrometry we have identified several residues on the D1 and D2 proteins from spinach which are oxidatively modified and in close proximity to Q_A (D1 residues ²³⁹F, ²⁴¹Q, ²⁴²E and the D2 residues ²³⁸P, ²³⁹T, ²⁴²E and ²⁴⁷M) and Pheo_D1 (D1 residues ¹³⁰E, ¹³³L and ¹³⁵F). These residues may be associated with reactive oxygen species exit pathways located on the reducing side of the photosystem, and their modification may indicate that both Q_A and Pheo_D1 are sources of reactive oxygen species on the reducing side of Photosystem II.     

Increases in the fluorescence Fo level and reversible inhibition of Photosystem II reaction center by high-temperature treatments in higher plants

Effects of high temperatures on the fluorescence F_m (maximum fluorescence) and F_o (dark level fluorescence) levels were studied and compared with those of the photochemical reactions of PS II. These comparisons were performed during and after the high temperature treatments. The following results were obtained; (1) increases in the F_o level at high temperatures were partly reversible, (2) the F_m level in the presence of dithionite in spinach chloroplasts decreased at high temperatures and also showed a partial reversibility, (3) photoreductions of pheophytin a and Q_a were reversibly inhibited at high temperatures parallel to the decrease in the difference between the F_m and F_o levels, and (4) the decrease in the fluorescence F_m level seemed to be related to denaturation of chlorophyll-proteins. All the data suggested that, as well as the separation of light-harvesting chlorophyll a/t b protein complexes of PS II from the PS II core complexes, partly reversible inactivation of the PS II reaction center at high temperatures is the cause of the increase in the F_o level.