Photoreduction of pheophytin in reaction centers of the photosystem II in intact cells of green algae and cyanobacteria under anaerobic conditions

Reversible photoreduction of pheophytin (Pheo) accompanied by a decrease in the chlorophyll fluorescence yield is observed in Photosystem 2 of the intact cells of green algae and cyanobacteria under anaerobic conditions. The photoreaction is inhibited by DCMU and reactivated upon subsequent addition of either ascorbate of dithionite. It is suggested that as a result of electron donation from the water splitting system being in the state S(3), to the reaction centre of Photosystem 2 in the state [P(+)(680)Pheo(-)] Q(-) after the primary photoreaction there occurs formation of the long-living state [P(680)Pheo(-)] Q(-). It was found that oxidized NADP, benzyl viologen and methyl viologen accelerate oxidation of Pheo reduced int he Photosystem 2 in the light indicating that these electron acceptors (typical for Photosystem 1) can accept an election from Pheo in Photosystem 2.     

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     

The nature of photoinhibition in isolated thylakoids

Isolated broken chloroplasts were exposed to irradiances up to 200 W · m⁻² at temperatures between 0°C and 20°C. The degradation of their photochemical apparatus resembles that, induced in photosynthesizing cells by extremely high irradiances (photoinhibition) or by moderate irradiances in presence of chloramphenicol. Electron transport through Photosystem II and variable fluorescence decline in parallel, displaying irradiance-dependent biphasic kinetics (typical half-times of hours). The decay of Photosystem II activities is followed by a delay of several hours by disappearance of the pigment-protein complex CPa (the core of Photosystem II). Photosystem I activity and the corresponding pigment-protein complexes disappear much slower than those of Photosystem II, particularly in thylakoids exposed to light at lower temperatures (below 10°C). Exposures were made in the presence and absence of: electron acceptors, oxygen (vs. nitrogen flushing), ascorbate, catalase and DCMU; none of these agents caused a substantial difference in the rate of degradation. Bovine serum albumin increases nonspecifically the stability of all chloroplast activities both in light and dark. Our results agree with the proposed central role in the inhibition of the Q_B protein. The cause of its inactivation remains obscure. Hypotheses assuming PQ^⨪, PQ⁼ or activated oxygen as the noxious species do not conform with some of our data. The primary step in Q_B protein inactivation need not be a damage to it; its modification serving regulatory purposes is an alternative possibility.     

Site of salicylaldoxime interaction with photosystem II

The reversible inhibition of Photosystem II by salicylaldoxime was studied in spinach D-10 particles by fluorescence, optical absorption, and electron spin resonance spectroscopy. In the presence of 15 mM salicylaldoxime, the initial fluorescence yield was raised to the level of the maximum fluorescence, indicating efficient charge recombination between reduced pheophytin (Ph) and P680⁺. In agreement with the rapid (ns) backreaction expected between Ph^– and P680⁺, the optical absorption transient at 820 mm was not observed. When the particles were washed free of salicylaldoxime, the optical absorption transient resulting from the rereduction of P680⁺ was restored to the µs timescale. These results, along with the previously observed inhibition of electron transport reactions and diminution of the 515-nm absorption change in chloroplasts [Golbeck, J.H. (1980) Arch Biochem Biophys 202, 458–466], are consistent with a site of inhibition between Ph and QA in Photosystem II. ESR Signal IIf and Signal Its were abolished in the presence of 25 mM salicylaldoxime, but both signals could be recovered by washing the D-10 particles free of the inhibitor. The loss of Signal Ilf is most likely a consequence of the inhibition between Ph and Q_A; the rapid charge recombination between Ph^– and P680⁺ would preclude electron transfer from an electron donor on the oxidizing side of Photosystem II. The loss of Signal Its may be due to a change in the environment of the donor complex such that the semiquinone radical giving rise to Signal Its interacts with a nearby reductant.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited chloroplasts - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F₀ prompt chlorophyll a fluorescence yield - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_var variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino) ethanesulfonic acid - P680 reaction center chlorophyll a of photosystem II - Ph pheophytin intermediate electron acceptor - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - Tris tris(hydroxymethyl)aminomethane - Z electron donor to P680⁺     

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     

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     

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.     

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680⁺ Phe^-) and charge stabilization (formation of P-680⁺ Q_A ^-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced Q_A was about 30% of that measured with oxidized Q_A. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNB m-dinitrobenzene - PPBQ phenyl-p-benzoquinone - PS I photosystem I of green plants - PS II photosystem II of green plants - PSU photosynthetic unit - P-680 primary donor of PS II - Phe intermediary pheophytin acceptor of PS II - Q_A primary quinone acceptor of PS II - RC reaction center     

Chemical modification of photosystem II core complex pigments with sodium borohydride

The reaction of the irreversible chemical reduction of the 13¹-keto C=O group of pheophytin a (Pheo a) with sodium borohydride in reaction centers (RCs) of functionally active spinach photosystem II (PS II) core complexes was studied. Stable, chromatographically purified PS II core complex preparations with altered chromophore composition are obtained in which ～25% of Pheo a molecules are modified to 13¹-deoxo-13¹-hydroxy-Pheo a. Some of the chlorophyll a molecules in the complexes were also irreversibly reduced with borohydride to 13¹-deoxo-13¹-hydroxy-chlorophyll a. Based on the results of comparative study of spectral, biochemical, and photochemical properties of NaBH₄-treated and control preparations, it was concluded that: (i) the borohydride treatment did not result in significant dissociation of the PS II core complex protein ensemble; (ii) the modified complexes retained the ability to photoaccumulate the radical anion of the pheophytin electron acceptor in the presence of exogenous electron donor; (iii) only the photochemically inactive pheo-phytin Pheo_D2 is subjected to the borohydride treatment; (iv) the Q_x optical transition of the Pheo_D2 molecule in the RC of PS II core complexes is located at 543 nm; (v) in the Q_y spectral region, Pheo_D2 probably absorbs at ～680 nm.     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Comparative EPR and thermoluminescence study of anoxic photoinhibition in Photosystem II particles

Photosystem II particles were exposed to 800 W m^–2 white light at 20 °C under anoxic conditions. The F_o level of fluorescence was considerably enhanced indicating formation of stable-reduced forms of the primary quinone electron acceptor, Q_A. The F_m level of fluorescence declined only a little. The g=1.9 and g=1.82 EPR forms characteristic of the bicarbonate-bound and bicarbonate-depleted semiquinone-iron complex, Q_A ^–Fe²⁺, respectively, exhibited differential sensitivity against photoinhibition. The large g=1.9 signal was rapidly diminished but the small g=1.82 signal decreased more slowly. The S₂-state multiline signal, the oxygen evolution and photooxidation of the high potential form of cytochrome b-559 were inhibited approximately with the same kinetics as the g=1.9 signal. The low potential form of oxidized cytochrome b-559 and Signal II_slow arising from Tyr_D ⁺ decreased considerably slower than the g=1.9 semiquinone-iron signal. The high potential form of oxidized cytochrome b-559 was diminished faster than the low potential form. Photoinhibition of the g=1.9 and g=1.82 forms of Q_A was accompanied with the appearance and gradual saturation of the spin-polarized triplet signal of P 680. The amplitude of the radical signal from photoreducible pheophytin remained constant during the 3 hour illumination period. In the thermoluminescence glow curves of particles the Q band (S₂Q_A ^– charge recombination) was almost completely abolished. To the contrary, the C band (Tyr_D ⁺Q_A ^– charge recombination) increased a little upon illumination. The EPR and thermoluminescence observations suggest that the Photosystem II reaction centers can be classified into two groups with different susceptibility against photoinhibition.Abbreviations C band thermoluminescence band associated with Tyr-D⁺Q _a ^– charge recombination - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - F_o initial fluorescence - F_m maximum fluorescence - Q band thermoluminescence band originating from S₂Q _a -charge recombination - Q _a the primary quinone electron acceptor of PS II - P 680 the primary electron donor chlorophyll of PS II - S₂ oxidation state of the water-splitting system - Phe pheophytin - TL thermoluminescence - Tyr _d redox active tyrosine-160 of the D₂ protein     

Investigations on the electron transfer processes in Photosystem II: New insights by chlorophyll fluorescence decay measurements with additional saturating light pulses

Electron transfer processes in leaves were investigated by chlorophyll fluorescence decay measurements. A fast chlorophyll fluorescence decay was observed in the intact state, reflecting normal electron transfer in Photosystem II. After treatment with DCMU a slow chlorophyll fluorescence decay was measured due to blocked electron transfer after the primary quinone Q_A. Additional saturating light pulses, one between each two measuring pulses, were used to completely reduce Q_A of the intact leaf: the chlorophyll fluorescence decay became similar to that of a DCMU treated leaf. A decreased electron donation rate to the reaction centre P680 was obtained after treatment with hydroxylamine. The intensity of the additional saturating light pulses was not sufficient to reduce all Q_A under this condition and only a small increase of the average chlorophyll fluorescence decay time occurred. Following our previous paper [Berg et al. (1997) Photosynthetica 34, in press], we investigated the effects of water stress with the additional saturating light pulses. An almost complete reduction of Q_A was possible after water stress started. A small, but systematic shortening of the slow chlorophyll fluorescence decay followed, up to a relative loss of leaf mass of 80%. At this time a rapid shortening of the chlorophyll fluorescence decay occurred, caused by an electron deficiency at the donor site of PS II. Additional saturating light pulses had no effects on the chlorophyll fluorescence decay any more, revealing a radiationless recombination between the reduced primary quinone Q and the oxidized reaction centre P680⁺.     

The donor side of Photosystem II as the copper-inhibitory binding site

We have measured, under Cu (II) toxicity conditions, the oxygen-evolving capacity of spinach PS II particles in the Hill reactions H₂OSiMo (in the presence and absence of DCMU) and H₂OPPBQ, as well as the fluorescence induction curve of Tris-washed spinach PS II particles. Cu (II) inhibits both Hill reactions and, in the first case, the DCMU-insensitive H₂O SiMo activity. In addition, the variable fluorescence is lowered by Cu (II). We have interpreted our results in terms of a donor side inhibition close to the reaction center. The same polarographic and fluorescence measurements carried out at different pHs indicate that Cu (II) could bind to amino acid residues that can be protonated and deprotonated. In order to reverse the Cu (II) inhibition by a posterior EDTA treatment, in experiments of preincubation of PS II particles with Cu (II) in light we have demonstrated that light is essential for the damage due to Cu (II) and that this furthermore is irreversible.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenilcarbazide - F_o initial non-variable fluorescence - F_I intermediate fluorescence yield - F_m maximum fluorescence yield - F_v variable fluorescence yield - Mes 2,-(N-morpholino)ethanosulfonic acid - OEC oxygen-evolving complex - P680 Primary electron donor chlorophyll - Pheo pheophytin - PPBQ phenyl-p-benzo-quinone - PS II Photosystem II - SiMo Silicomolybdate - Q_B secondary quinone acceptor - Q_A primary quinone aceptor - Tris N-tris(hydroxymethyl)amino ethane - Tyr_z electron carrier functioning between P680 and the Mn cluster This article is dedicated to Prof. Dr. Harmut Lichtenthaler on the occasion of his 60th birthday.     

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     

Inactivation of photosynthetic oxygen evolution by UV-B irradiation: A thermoluminescence study

The influence of UV-B irradiation on photosynthetic oxygen evolution by isolated spinach thylakoids has been investigated using thermoluminescence measurements. The thermoluminescence bands arising from the S₂Q_B ^- (B band) and S₂Q_A ^– (Q band) charge recombination disappeared with increasing UV-B irradiation time. In contrast, the C band at 50°C, arising from the recombination of Q_A ^- with an accessory donor of Photosystem II, was transiently enhanced by the UV-B irradiation. The efficiency of DCMU to block Q_A to Q_B electron transfer decreased after irradiation as detected by the incomplete suppression of the B band by DCMU. The flash-induced oscillatory pattern of the B band was modified in the UV-B irradiated samples, indicating a decrease in the number of centers with reduced Q_B. Based on the results of this study, UV-B irradiation is suggested to damage both the donor and acceptor sides of Photosystem II. The damage of the water-oxidizing complex does not affect a specific S-state transition. Instead, charge stabilization is enhanced on an accessory donor. The acceptor-side modifications decrease the affinity of DCMU binding. This effect is assumed to reflect a structural change in the Q_B/DCMU binding site. The preferential loss of dark stable Q_B ^- may be related to the same structural change or could be caused by the specific destruction of reduced quinones by the UV-B light.Abbreviations Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A first quinone electron acceptor of PS II - Q_B second quinone electron acceptor of PS II - Tyr-D accessory electron donor of PS II - S₀-S₄ charge storage states of the water-oxidizing complex     

Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level F_j are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_i initial fluorescence level immediately after shutter open - F_pt intermediate plateau fluorescence level - F_m maximum fluorescence level emitted when all PS II centers are closed - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Electron transfer to P-680+ in active and inhibited Photosystem II fractions from higher plants

We have studied the kinetics of P-680⁺ reduction in PS II fractions which were prepared with high rates of oxygen evolution from pea thylakoids. In noninhibited fractions, about two-thirds of the photooxidized P-680 was reduced in less than 2 μs (the minimum time resolution of the instrument), and phases with lifetimes of 5–40 μs may be linked with electron donation in PS II. After inhibition of electron donation to P-680⁺, rereduction kinetics were biphasic with lifetimes of 90–150 μs and 600–900 μs. We argue that the faster component may represent a back-reaction from the state [D⁺₁ P-680⁺ ... Q⁻B⁻], and that the slower component may represent a back-reaction from the state (D₁//P680⁺Q⁻B]. From experiments with low concentrations of water analogues we propose that the complex multi-phasic kinetics of electron donation from D₁ to P-680⁺ could be a reflection of the dynamic properties of the water binding site.     

Modulation of herbicide-binding by the redox state of Q400, an endogenous component of Photosystem II

Preincubation of isolated chloroplasts with ferricyanide, prior to addition of DCMU, unmasks a high-potential electron acceptor (Q₄₀₀) in Photosystem II that acts as an additional quencher and prolongs the fluorescence induction curve in the presence of DCMU (Ikegami, I. and Katoh, S. (1973) Plant Cell Physiol. 14, 829–836). This study confirms that Q₄₀₀ is endogenous to Photosystem II and is not a bound ferricyanide, and several new characteristics of this high potential acceptor are established. (a) It is accessible to ferricyanide even in the presence of DCMU. The rate of oxidation, however, is very slow, consistent with access only via Q_A. Accessibility may be enhanced by magnesium, reminiscent of the oxidation of Q⁻_A by ferricyanide. (b) Oxidation of Q₄₀₀ drastically suppresses the binding of DCMU at neutral and alkaline pH. Below pH 6, however, DCMU binding is essentially normal. The pH dependence of DCMU binding is consistent with the known pH dependence of the redox midpoint potential of Q₄₀₀. (c) Binding of many other inhibitors of Q_A-to-Q_B electron transfer is much less affected or even completely unaffected. These results have implications for current notions of herbicide binding and may also bear on the origin of slow phases of fluorescence induction in the presence of DCMU.     

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.