A New Pathway of Photoinactivation of Photosystem II. Irreversible Photoreduction of Pheophytin Causes Loss of Photochemical Activity of Isolated D1–D2–Cytochrome b559 Complex

A new pathway of photoinactivation of photosystem II (PS II) connected with irreversible photoaccumulation of reduced pheophytin (Ph) in isolated D1–D2–cytochrome b ₅₅₉ complexes of reaction center (RC) of PS II was discovered. The inhibitory effects of white light illumination on photochemical activity of D1–D2–cytochrome b ₅₅₉ complexes of RCs of photosystem II, isolated from pea chloroplasts, have been compared under anaerobic conditions in the absence and in the presence of sodium dithionite, electron transfer from which to the oxidized primary electron donor P680⁺ results in the photoaccumulation of anion-radical of the primary electron acceptor, PH. In both cases, prolonged illumination (1-5 min, 120 W/m²) led to a pronounced loss of the photochemical activity as it was monitored by measuring the amplitude of the reversible photoinduced absorbance changes at 682 nm related to the photoreduction of Ph. The extent of the photoinactivation depended on the illumination time and pH of the medium. At pH 8.0, the presence of dithionite during photoinactivation brought about a protective effect compared to that in a control sample. In contrast, lowering pH to 6.0 increased the sensitivity to photoinactivation in the dithionite containing samples. For 5 min irradiation, the photochemical activity in the absence and in the presence of dithionite decreased by 35 and 72%, respectively (this was accompanied by an irreversible bleaching of the pheophytin Q_x absorption band at 542 nm). Degradation of the D1 and D2 proteins was not observed under these conditions. A subsequent addition of an electron acceptor, potassium ferricyanide, to the illuminated samples restored neither the amplitude of the signal at 682 nm nor absorption at 542 nm. It is suggested that at pH < 7.0 the photoaccumulated PH is irreversibly converted into a secondary, most probably protonated form, that does not lead to destruction of the RCs but prevents the photoformation of the primary radical pair [P680⁺PH]. A possible application of this effect to photoinactivation of PS II in vivo is discussed.     

Binding of novel inhibitors of electron transfer in photosystem 2, derivatives of perfluoroisopropyldinitrobenzene,with polypeptide D2 of the reaction center

A binding site for novel inhibitors of K15 type (derivatives of perfluoroisopropyldinitrobenzene) with the components of reaction center (RC) of photosystem 2 (PS-2) of higher plants has been investigated. It has been shown that multiple washing the PS-2 submembrane chloroplast fragments (BBY-particles) treated with the K15 inhibitor, including multiple dilution in buffer in the presence of high concentrations of mono- and divalent ions, prolonged (up to 2-5 h) incubation, centrifugation, and subsequent resuspension in buffer deprived of the inhibitor, does not lead to restoration of functional activity of the PS-2. After addition of dithionite, inducing reduction and consequent decomposition of the inhibitor, and subsequent removal of dithionite by washing, the functional activity of PS-2 was completely restored. Incubation in the presence of sodium dodecyl sulfate (SDS), leading to solubilization of the sample to the level of protein components, induced the appearance of a fraction of free K15 retaining the initial inhibitory efficiency. To create a covalent binding of the inhibitor with protein, retained under the conditions of denaturing SDS polyacrylamide gel electrophoresis, the azido-containing analog of K15 (K15-N₃) was used. The need for radioactive label for identification of K15 was avoided by the revealed ability of K15-type inhibitors to emit fluorescence, which retained its features under the experimental conditions. With the technique of photoaffinity binding and denaturing SDS-PAGE in the presence of 6 M urea of submembrane chloroplast fragments enriched in PS-2 the D2-polypeptide, an integral component of the reaction center of PS-2, has been shown to be a binding site for K15-type inhibitors. This conclusion is in agreement with a suggestion (put forward in our earlier publications) that K15-type inhibitors are bound to PS-2 reaction center, replacing Q_A in its binding site. Hence, an agent specifically binding to polypeptide D2 has been found for the first time. The data are compared with information about inhibitory action and binding sites of the known inhibitors of electron transfer in PS-2.     

Effect of low temperatures on photochemical activity of PS1 reaction centers from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. frozen under illumination

After cooling of Synechocystis sp. photosystem 1 (PS1) reaction centers (RC) to 160 K under illumination most of the photoactive pigment is fixed for a long time in the oxidized state. The same effect is observed in purple bacteria RC. The dark reduction kinetics of PS1 P700 chlorophyll, which still retains its photochemical activity, in these samples was similar to that in samples cooled in the dark. We suggest that the photoinduced charge separation in PS1 RC, as well as in purple bacteria RC, is accompanied by conformational changes that can be fixed in samples cooled under illumination. As a result, the electrons photomobilized in RC cooled under illumination are unable to return backward the process of electron transfer to P700(+) after cessation of actinic illumination. Such irreversible trapping of electrons can take place in different parts of the PS1 RC electron acceptor chain.     

Photoinactivation of the reactivation capacity of photosystem II in pea subchloroplast particles after a complete removal of manganese

After a complete removal of Mn from pea subchloroplast photosystem-II (PS II) preparations the electron phototransfer and oxygen evolution are restored upon addition of Mn²⁺ and Ca²⁺. Pre-illumination of the sample in the absence of Mn²⁺ leads to photoinhibition (PI) — irreversible loss of the capability of PS II to be reactivated by Mn²⁺. The effect of PI is considerably decreased in the presence of Mn²⁺ (4 Mn atoms per reaction center of PS II) and it is increased in the presence of ferricyanide or p-benzoquinone revealing the oxidative nature of the photoeffect. PI results in suppression of oxygen evolution, variable fluorescence, photoreduction of 2,6-dichlorophenol indophenol from either water or diphenylcarbazide. However, photooxidation of chlorophyll P₆₈₀, the primary electron donor of PS II as well as dark and photoinduced EPR signal II (ascribed to secondary electron donors D ₁ and Z) are preserved. PI is accompanied by photooxidation of 2–3 carotenoid molecules per PS II reaction center (RC) that is accelerated in the presence of ferricyanide and is inhibited upon addition of Mn²⁺ or diuron. The conclusion is made that PI in the absence of Mn leads to irreversible oxidative inactivation of electron transfer from water to RC of PS II which remains photochemically active. A loss of functional interaction of RC with the electron transport chain as a common feature for different types of PS II photoinhibition is discussed.Abbreviations A photoinduced absorbance changes - DPC diphenylcarbazide - DPIP 2,6-dichlorophenol indophenol - F _o constant fluorescence of chlorophyll - F photoinduced changes of Chl fluorescence yield - Mn manganese - P₆₈₀ the primary electron donor in PS II - PI photoinhibition - PS II photosystem II - Q the primary (quinone) electron acceptor in PS II - RC reaction center     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

Characterization of processes responsible for the distinct effect of herbicides DCMU and BNT on Photosystem II photoinactivation in cells of the cyanobacterium Synechococcus sp. PCC 7942

Light-induced modification of Photosystem II (PS II) complex was characterized in the cyanobacterium Synechococcus sp. PCC 7942 treated with either DCMU (a phenylurea PS II inhibitor) or BNT (a phenolic PS II inhibitor). The irradiance response of photoinactivation of PS II oxygen evolution indicated a BNT-specific photoinhibition that saturated at relatively low intensity of light. This BNT-specific process was slowed down under anaerobiosis, was accompanied by the oxygen-dependent formation of a 39 kDa D1 protein adduct, and was not related to stable Q_A reduction or the ADRY effect. In the BNT-treated cells, the light-induced, oxygen-independent initial drop of PS II electron flow was not affected by formate, an anion modifying properties of the PS II non-heme iron. For DCMU-treated cells, anaerobiosis did not significantly affect PS II photoinactivation, the D1 adduct was not observed and addition of formate induced similar initial decrease of PS II electron flow as in the BNT-treated cells. Our results indicate that reactive oxygen species (most likely singlet oxygen) and modification of the PS II acceptor side are responsible for the fast BNT-induced photoinactivation of PS II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electrogenic Protonation of the Secondary Quinone Acceptor Q<Subscript>B</Subscript> in Spinach Photosystem II Complexes Incorporated into Lipid Vesicles

The generation of transmembrane electric potential difference (delta psi) in quinone acceptor complex of proteoliposomes containing core complexes of photosystem II from spinach was studied using for the measurements a direct electrometric technique. Besides the fast increase in the membrane potential associated with the electron transfer between the redox-active tyrosine 161 residue (Y(Z)) in D1 polypeptide and the primary quinone acceptor Q(A), an additional electrogenic phase with tau approximately 0.85 msec at pH 7.3 and the maximal relative amplitude of approximately 11% of the Y(Z)ox Q(A)- phase was observed after the second light flash. The sensitivity of this phase to diuron (an inhibitor of electron transfer between Q(A) and the secondary quinone acceptor Q(B)), the dependence of its amplitude on the light flash parity, and also a decrease in its rate constant with increase in pH indicated that it was due to dismutation of Q(A)- and Q(B)- with the subsequent protonation of a doubly reduced plastoquinone molecule: Q(A)- Q(B)- + 2H+ --> Q(A)Q(B)H2.     

Superoxide,Hydrogen Peroxide and Hydroxyl Radical in D1/D2/cytochrome b-559 Photosystem II Reaction Center Complex

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.     

An evaluation of the potential triggers of photoinactivation of photosystem II in the context of a Stern-Volmer model for downregulation and the reversible radical pair equilibrium model

Photoinactivation of photosystem II (PS II) is a light-dependent process that frequently leads to break-down and replacement of the D1 polypeptide. Photoinhibition occurs when the rate of photoinactivation is greater than the rate at which D1 is replaced and results in a decrease in the maximum efficiency of PS II photochemistry. Downregulation, which increases non-radiative decay within PS II, also decreases the maximum efficiency of PS II photochemistry and plays an important role in protecting against photoinhibition by reducing the yield of photoinactivation. The yield of photoinactivation has been shown to be relatively insensitive to photosynthetically active photon flux density (PPFD). Formation of the P680 radical (P680+), through charge separation at PS II, generation of triplet-state P680 (3P680*), through intersystem crossing and charge recombination, and double reduction of the primary stable electron acceptor of PS II (the plastoquinone, Q(A)) are all potentially critical steps in the triggering of photoinactivation. In this paper, these processes are assessed using fluorescence data from attached leaves of higher plant species, in the context of a Stern-Volmer model for downregulation and the reversible radical pair equilibrium model. It is shown that the yield of P680+ is very sensitive to PPFD and that downregulation has very little effect on its production. Consequently, it is unlikely to be the trigger for photoinactivation. The yields of 3P680* generated through charge recombination or intersystem crossing are both less sensitive to PPFD than the yield of P680+ and are both decreased by down regulation. The yield of doubly reduced Q(A) increases with incident photon flux density at low levels, but is relatively insensitive at moderate to high levels, and is greatly decreased by downregulation. Consequently, 3P680* and doubly reduced Q(A) are both viable as triggers of photoinactivation.     

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     

Evidence for localisation of accumulated chlorophyll cation on the D1-accessory chlorophyll in the reaction centre of Photosystem II

Absorption and circular dichroism spectra of Photosystem II (PS II) reaction centres (RC) were studied and compared with spectra calculated on the basis of point-dipole approximation. Chlorophyll cation was accumulated during a light treatment of PS II RC in the presence of artificial electron acceptor silicomolybdate. Light-induced difference spectra and their calculated counterparts revealed the location of accumulated cation at the accessory chlorophyll of the D1 protein subunit.     

Short-term responses of Photosystem I to heat stress

When 23°C-grown potato leaves (Solanum tuberosum L.) were exposed for 15 min to elevated temperatures in weak light, a dramatic and preferential inactivation of Photosystem (PS) II was observed at temperatures higher than about 38°C. In vivo photoacoustic measurements indicated that, concomitantly with the loss of PS II activity, heat stress induced a marked gas-uptake activity both in far-red light (>715 nm) exciting only PS I and in broadband light (350–600 nm) exciting PS I and PS II. In view of its suppression by nitrogen gas and oxygen and its stimulation by high carbon-dioxide concentrations, the bulk of the photoacoustically measured gas uptake by heat-stressed leaves was ascribed to rapid carbon-dioxide solubilization in response to light-modulated stroma alkalization coupled to PS I-driven electron transport. Heat-induced gas uptake was observed to be insensitive to the PS II inhibitor diuron, sensitive to the plastocyanin inhibitor HgCl₂ and saturated at a rather high photon flux density of around 1200 E m^–2 s^–1. Upon transition from far-red light to darkness, the oxidized reaction center P700⁺ of PS I was re-reduced very slowly in control leaves (with a half time t_1/2 higher than 500 ms), as measured by leaf absorbance changes at around 820 nm. Heat stress caused a spectacular acceleration of the postillumination P700⁺ reduction, with t_1/2 falling to a value lower than 50 ms (after leaf exposure to 48°C). The decreased t_1/2 was sensitive to HgCl₂ and insensitive to diuron, methyl viologen (an electron acceptor of PS I competing with the endogenous acceptor ferredoxin) and anaerobiosis. This acceleration of the P700⁺ reduction was very rapidly induced by heat treatment (within less than 5 min) and persisted even after prolonged irradiation of the leaves with far-red light. After heat stress, the plastoquinone pool exhibited reduction in darkness as indicated by the increase in the apparent Fo level of chlorophyll fluorescence which could be quenched by far-red light. Application (for 1 min) of far-red light to heat-pretreated leaves also induced a reversible quenching of the maximal fluorescence level Fm, suggesting formation of a pH gradient in far-red light. Taken together, the presented data indicate that PS I responded to the heat-induced loss of PS II photochemical activity by catalyzing an electron flow from stromal reductants. Heat-stress-induced PS I electron transport independent of PS II seems to constitute a protective mechanism since block of this electron pathway in anaerobiosis was observed to result in a dramatic photoinactivation of PS I.Abbreviations PFD photon flux density - PS Photosystem - Apt and Aox amplitude of the photothermal and photobaric components of the photoacoustic signal, respectively - P700 reaction center pigment of PS I - Fo and Fm initial and maximal levels of chlorophyll fluorescence, respectively - Fv=Fm Fo-variable chlorophyll fluorescence - Q_A primary (stable) electron acceptor of PS II - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Cyt cytochrome     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.     

Donor function of phosphate ions in photosystem 2

Evidence was obtained for the interaction between the photosystem 2 (PS2) reaction centre (RC) chlorophyll (Chl) P680 and inorganic phosphate, Pi. The light-induced endogenous basal electron transport to ferricyanide in PS2 depended on endogenous Pi. The electron transport in phosphate deficient chloroplasts was absent, and could be resumed upon the addition of exogenous Pi or of the exogenous electron donor, diphenylcarbazide. Some chloroplast Chl molecules were apparently bound with Pi to a complex via the magnesium atom that was detected by the increase in absorbance in the Chl a absorption maximum at 435 nm observed after the consumption of endogenous Pi in the photophosphorylation reactions. The electron paramagnetic resonance (EPR) Signal I, found in the spectra at 77 K after irradiation of frozen samples in chloroplasts poor in endogenous Pi, was the sum of P700+ and P680+ signals. The P680+ signal disappeared after addition of Pi, diphenylcarbazide or diuron to the chloroplasts before freezing. In addition, the EPR doublet signal of the phosphate anion radicals was recorded at 77 K after irradiation in the ethanol solutions of Chl a containing potassium phosphate. The same doublet signal was discovered in the difference EPR spectrum "chloroplasts minus chloroplasts with diuron" at 77 K after irradation. The results are a possible evidence of the participation of phosphate ions in the primary light reactions of PS2. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the complex interaction between the electron acceptor silicomolybdate and Photosystem II

Silicomolybdate (SiMo) and its effects on thylakoids have been characterized to evaluate its use as a probe for Photosystem II (PS II). It can accept electrons at two places in the electron transport chain: one at PS II and the other at PS I. In the presence of 1 M 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) only the site at PS II is available. It is suggested that SiMo must disp;ace bicarbonate from its binding site to be able to function as an electron acceptor. This displacement is non-competitive. The binding of SiMo is inhibited differentially by PS II inhibitors: dinoseb>ioxynil> diuron. This difference is determined by the different positions of the inhibitors within the Q_B binding niche and their interaction with bicarbonate. The experimental results show that the SiMo-binding niche is located between the parallel helices of the D1 and D2 proteins of PS II, close to the non-heme iron. We conclude that SiMo is an electron acceptor with unique characteristics useful as a probe of the acceptor side of PS II.     

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     

The PQ/PQH2 ratio and occupancy of photosystem II-QB site by plastoquinone control the degradation of D1 protein during photoinhibition in vivo

Photoinactivation of photosystem II (PSII) and light-dependent degradation of the reaction center II (RCII) protein D1 have been investigated in Chlamydomonas reinhardtii mutants D6, AC208, and B4 deficient in cytochrome b6/f, plastocyanin, and photosystem I (PSI) activity, respectively. These mutants possess active PSII and reduce plastoquinone (PQ) but cannot oxidize plastoquinol (PQH2) via light-dependent reduction of NADP. In light-exposed cells a high ratio PQH2/PQ and a low turnover of PQ/PQH2 at the RCII-QB site are maintained. In all mutants photoinactivation of RCII was slower as compared to the wild-type (wt) cells, and D1 degradation was drastically decreased. The degradation of D1 was also lower in the wt cells under anaerobic conditions and presence of ascorbate, while raising the concentration of dissolved oxygen increased the degradation of the D1 protein in the AC208 mutant. Photoinactivation and light-dependent degradation of the D1 protein were drastically increased in the Scenedesmus obliquus LF-1 mutant cells altered in its PSII manganese binding and thus unable to reduce PQ using water as an electron donor. Diuron inhibited the light-dependent degradation of D1 protein in both the LF-1 mutant and wt cells. Based on these results we propose that availability of PQ at the QB site is required for (i) the photoinactivation process of the RCII acceptor side followed by inactivation of the donor side leading to the generation of harmful cation radicals (Z+, P680+, chlz+) which damage the D1 protein, and (ii) the accessibility of the cleavage site of the damaged D1 protein to proteolytic degradation.     

Spectral properties of stabilized D1/D2/cytochrome b-559 photosystem II reaction center complex Effects of Triton X-100, the redox state of pheophytin, and β-carotene

Absorption, fluorescence, and CD spectral properties of the isolated D1/D2/cytochrome b-559 photosystem II reaction center complex were examined in stabilized reaction center material at 77 K. Spectral properties were dependent on the presence or absence of 0.05% Triton X-100 in the RC suspension medium, on the redox state of pheophytin, and on the state of inactivation of the complex. The specific spectral properties of the PS II RC complex in the red suggest that the primary donor is not a bacterial-type special pair and could be a monomer. Furthermore, the spectral properties in the PS II RC may be the result of excitonic interactions among all the porphyrin molecules in the complex. Interactions between β-carotene and porphyrins indicate a significant role for β-carotene in the PS II RC.     

Mechanism of proton transfer inhibition by Cd(2+) binding to bacterial reaction centers: determination of the pK(A) of functionally important histidine residues

The bacterial photosynthetic reaction center (RC) uses light energy to catalyze the reduction of a bound quinone molecule Q(B) to quinol Q(B)H(2). In RCs from Rhodobacter sphaeroides the protons involved in this process come from the cytoplasm and travel through pathways that involve His-H126 and His-H128 located near the proton entry point. In this study, we measured the pH dependence from 4.5 to 8.5 of the binding of the proton transfer inhibitor Cd(2+), which ligates to these surface His in the RC and inhibits proton-coupled electron transfer. At pH <6, the negative slope of the logarithm of the dissociation constant, K(D), versus pH approaches 2, indicating that, upon binding of Cd(2+), two protons are displaced; i.e., the binding is electrostatically compensated. At pH >7, K(D) becomes essentially independent of pH. A theoretical fit to the data over the entire pH range required two protons with pK(A) values of 6.8 and 6.3 (+/-0.5). To assess the contribution of His-H126 and His-H128 to the observed pH dependence, K(D) was measured in mutant RCs that lack the imidazole group of His-H126 or His-H128 (His --> Ala). In both mutant RCs, K(D) was approximately pH independent, showing that Cd(2+) does not displace protons upon binding in the mutant RCs, in contrast to the native RC in which His-H126 and His-H128 are the predominant contributors to the observed pH dependence of K(D). Thus, Cd(2+) inhibits RC function by binding to functionally important histidines.     

Stress-induced degradation of D1 protein and its photoprotection by DCPIP in isolated thylakoid membranes of barley leaf

Effects of various stress treatments such as NaCl, hydrogen peroxide, hydroxyl free radical, and high irradiance (HI, 1 000 μmol m⁻² s⁻¹) on the photosystem (PS) 2 mediated electron transport rate and the degradation of D1 protein in the thylakoid membranes of barley were studied. The applied stresses caused significant reduction in the PS 2-mediated electron transport and a degradation of D1 protein that was highest during the HI-treatment. Presence of 2,6-dichlorophenol indophenol (DCPIP), which is an artificial electron acceptor from water, significantly minimizes the HI-induced deleterious effect on the PS 2-mediated electron transport rate, disarrangement of PS machinery, and degradation of the D1 protein. HI in the absence of an acceptor resulted in production of reactive oxygen species due to electron transfer to oxygen.