Different kinetics of photoinactivation of photosystem I-mediated electron transport and P700 in isolated thylakoid membranes

Photoinactivation kinetics of photosystem I (PSI)-mediated electron transport rate was compared to that of P700 content at room (22 degrees C) and low (4 degrees C) temperatures in isolated spinach thylakoid membranes. The high light treatment was carried out under aerobic and anaerobic conditions. At 22 degrees C the decrease of electron transport rate showed first order exponential kinetics. The amount of P700 decreased linearly, being less affected in the first hours of illumination. During photoinhibition at 4 degrees C in the presence of oxygen, the kinetics of inactivation of PSI photochemical activity and the content of P700 were different. It was found that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had different protective effect on the electron transport rate and on P700 content at both temperatures. Treatment with high light intensity under N(2) atmosphere had no effect on the electron transport rate or P700 content. The possible degradation of PSI reaction centre proteins was determined using immunoblot methods. In the presence of linear electron transport at 22 degrees C correlation between formation of toxic hydroxyl radicals and inhibition of oxygen uptake was observed.     

Investigation of the electron transfer site of p-benzoquinone in isolated photosystem II particles and thylakoid membranes using alpha- and beta-cyclodextrins

The electron transfer sites of p-benzoquinone (pBQ) and 2,6-dichloro-p-benzoquinone (DCBQ) were investigated in thylakoid membranes and isolated photosystem II (PSII) particles from barley (Hordeum vulgare) using alpha- and beta-cyclodextrins (CD) at concentrations up to 16 mM. In CD-treated thylakoid membranes incubated with DCBQ the electron transport through PSII, estimated as oxygen evolution (OE), is largely enhanced according to a S-shaped (sigmoidal) dose-response curve displaying a sharp inflection point, or transition. The maxima percent OE enhancement at cyclodextrin concentrations above 14 mM are about 100% (alpha-CD) and 190% (beta-CD). On the contrary, in thylakoid membrane preparations incubated with pBQ as electron acceptor one observes an OE inhibition of about 30% which might result from the depletion of the thylakoid membrane of its plastoquinone content. It was also found that in isolated PSII particles incubated with either pBQ or DCBQ the cyclodextrins induce only a small OE enhancement. Moreover, the observation in CD-treated thylakoid membranes incubated with pBQ of a residual, non-inhibited oxygen-evolving activity of about 70% puts a twofold question. That is, either the plastoquinone depletion was not complete, or, pBQ binds to electron acceptor sites of different nature. From this and data published in the literature, it is concluded that in the thylakoid membrane (i) DCBQ binds to Q(B), as is generally accepted, and (ii) pBQ binds to the plastoquinol molecules in the PQ pool and most likely also to Q(B), thereby in accord with Satoh et al.'s model [K. Satoh, M. Ohhashi, Y. Kashino, H. Koike, Plant Cell Physiol. 36 (1995) 597-605]. An attractive alternative hypothesis is the direct interaction of pBQ with the non-haem Fe(2+) between Q(A) and Q(B).     

Constitution and energetics of photosystem I and photosystem II in the chlorophyll d-dominated cyanobacterium Acaryochloris marina

This mini review presents current topics of discussion about photosystem (PS) I and PS II of photosynthesis in the Acaryochloris marina. A. marina is a photosynthetic cyanobacterium in which chlorophyll (Chl) d is the major antenna pigment (>95%). However, Chl a is always present in a few percent. Chl d absorbs light with a wavelength up to 30 nm red-shifted from Chl a. Therefore, the chlorophyll species of the special pair in PS II has been a matter of debate because if Chl d was the special pair component, the overall energetics must be different in A. marina. The history of this field indicates that a purified sample is necessary for the reliable identification and characterization of the special pair. In view of the spectroscopic data and the redox potential of pheophytin, we discuss the nature of special pair constituents and the localization of the enigmatic Chl a.     

Modulation of photosystem II chlorophyll fluorescence by electrogenic events generated by photosystem I.

In an attempt to uncover electric field interactions between PS I and PS II during their functioning, fluorescence induction curves were measured on hydroxylamine-treated thylakoids of Chenopodium album under conditions ensuring low and high levels of photogenerated membrane potentials. In parallel experiments with Peperomia metallica chloroplasts, the photocurrents were measured with patch-clamp electrodes and served as indicator of electrogenic activity of thylakoid membranes in continuous light. Inhibition of linear electron flow at PS II donor side by hydroxylamine (0.1 mM) eliminated a slow rise of chlorophyll fluorescence to a peak level and suppressed photoelectrogenesis. Activation of PS I-dependent electron transport using cofactors of either cyclic (phenazine methosulfate) or noncyclic electron transport (reduced TMPD or DCPIP in combination with methyl viologen) restored photoelectrogenesis in hydroxylamine-treated chloroplasts and led to reappearance of slow components in the fluorescence induction curve. Exposure of thylakoids to valinomycin reduced the peak fluorescence in the presence of KCl but not in the absence of KCl. Combined application of valinomycin and nigericin in the presence of KCl exerted stronger suppression of fluorescence than valinomycin alone but was ineffective in the absence of KCl. In samples treated with hydroxylamine and PS I cofactors (DCPIP/ascorbate and methyl viologen), preillumination with a single-turnover flash or a multiturnover pulse shifted the induction curves of both membrane potential and chlorophyll fluorescence to shorter times, which confirms the supposed influence of PS I-generated electrical field on PS II fluorescence. A model is presented that describes modulating effect of the membrane potential on chlorophyll fluorescence and roughly simulates the fluorescence induction curves measured at low and high membrane potentials.     

Application of molecular orbital calculations to interpret the chlorophyll spectral forms in pea photosystem II

The energy and oscillator strength of electronic transitions of chlorophyll (Chl)-amino acid complexes were calculated by using molecular orbital methods. The energies varied widely with coordinated amino acids and the difference between the maximum and minimum energy was about 830 cm-1. This energy difference was comparable with the spreading of absorption bands for light-harvesting Chl-protein complexes of photosystem II (LHC II) of green plants. The feature of the Qy band for pea LHC II was interpreted with the aid of the calculated energies and oscillator strengths. Four spectral components of the band were assigned to individual Chl-amino acid complexes.     

Influence of substituted 1,4-anthraquinones on the chlorophyll fluorescence and photochemical activity of pea thylakoid membranes

The effect of substituted 1,4-anthraquinones on the photochemical activity and chlorophyll fluorescence of thylakoid membranes was examined. Both the fluorescence and the photochemical activity depend on the 1,4-anthraquinone substituent. Stronger quinone-induced quenching of the chlorophyll fluorescence than quinone-induced changes in the activity of photosystem II is observed. The type (Cl or Br) and the position (Cl) of the chalogen atom strongly influence the degree of inhibition of PSII electron transport and the quenching of chlorophyll fluorescence. The data suggest that the quenching of chlorophyll fluorescence is due rather to the interaction of the 1,4-anthraquinones and chlorophyll molecules than to an indirect effect caused by stimulation of the photochemistry.     

On the dimerization of chlorophyll in photosystem II

In photosystem II, absorbed light energy is transferred to a reaction centre consisting of chlorophyll units. Release of an electron from the reaction centre is the starting point for the charge separation and electron transport chain in PSII. Crystal structures of the reaction centre have identified two chlorophyll monomers forming a dimer with a partial structural overlap, thus being stabilized by van der Waals interactions. However, the magnitude of this interaction is not accurately known. In this work, the structure of the chlorophyll dimer has been optimized for the first time using dispersion-corrected density functional theory (B3LYP-DCP) revealing the magnitude of dimerization to be approximately -17 kcal mol(-1). The dispersion interaction is shown to be of great significance for the chlorophyll dimer stabilization. In addition, the redox potential of the chlorophyll dimer is calculated to be 1283 mV in good agreement with recent experimental data.     

Mechanisms of photosystem I activity stimulation in trypsin-treated pea chloroplast membranes

Trypsin-induced effects on the PSI-photochemical activity of pea chloroplast membranes have been measured under saturating light conditions. It was found that incubation of isolated chloroplasts with trypsin in concentration range of 20–60 /smg trypsin per mg Chl leads to a linear increase of the rate of PSI-mediated electron transport, measured by O₂-uptake with methyl viologen as an electron acceptor and DCPIP·H₂ as electron donor. Low temperature (77 K) chlorophyll fluorescence measurements have indicated that a remarkable redistribution of excitation light energy in favor of PSI occurs under these conditions. The results are interpreted in terms of trypsin-induced lateral reorganization of the chlorophyll-protein complexes within the thylakoid membranes and specific alteration at the level of LHC a/b-PSII supramolecular organization.     

Spin polarization in the lowest triplet state of chlorophyll

Chlorophyll-b in glassy solution has a spin-polarized lowest triplet state at and above 77 K. The magnitude of the effect is different for MTHF and ethanol as solvents, in contrast to what is found for the porphin free base. Chlorophyll-a does not exhibit spin-polarization under identical conditions as for chlorophyll-b. Zero-field parameters are found to be:chlorophyll-a (MTHF) D = (281 ± 6) × 10^?4 cm^?1; E = (39 ± 3) × 10^?4 cm^?1;chlorophyll-b (MTHF) D = (289 ± 4) × 10^?4 cm^?1; E = (49 ± 3) × 10^?4 cm^?1,From ESR signal kinetics it follows that for chlorophyll-b, population and depopulation mainly involve the spin level y?, describing a spin moving in a plane perpendicular to the molecular plane:P_y ? P_x ? P_z; k_x = 240 ± 40 s^?1; k_y = 600 ± 120 s^?1; k_z ? 75 s^?1,where P_i and k_i denote populating and decay rates. Thus, the kinetic scheme for the chlorophyll triplet is different from that of porphyrins with heavier metal ions, but very similar to that of the porphin free base. The spin-lattice relaxation time is found to be anisotropic and shorter than the decay rates of individual spin levels. Nevertheless, spin polarization can be observed, essentially because the ESR signal amplitude depends on population differences.     

The action of oxygen on chlorophyll fluorescence quenching and absorption spectra in pea thylakoid membranes under the steady-state conditions

The effect of oxygen concentration on both absorption and chlorophyll fluorescence spectra was investigated in isolated pea thylakoids at weak actinic light under the steady-state conditions. Upon the rise of oxygen concentration from anaerobiosis up to 412 microM a gradual absorbance increase around both 437 and 670 nm was observed, suggesting the disaggregation of LHCII and destacking of thylakoids. Simultaneously, an increase in oxygen concentration resulted in a decline in the Chl fluorescence at 680 nm to about 60% of the initial value. The plot of normalized Chl fluorescence quenching, F(-O(2))/F(+O(2)), showed discontinuity above 275 microM O(2), revealing two phases of quenching, at both lower and higher oxygen concentrations. The inhibition of photosystem II by DCMU or atrazine as well as that of cyt b(6)f by myxothiazol attenuated the oxygen-induced quenching events observed above 275 microM O(2), but did not modify the first phase of oxygen action. These data imply that the oxygen mediated Chl fluorescence quenching is partially independent on non-cyclic electron flow. The second phase of oxygen-induced decline in Chl fluorescence is diminished in thylakoids with poisoned PSII and cyt b(6)f activities and treated with rotenone or N-ethylmaleimide to inhibit NAD(P)H-plastoquinone dehydrogenase. The data suggest that under weak light and high oxygen concentration the Chl fluorescence quenching results from interactions between oxygen and PSI, cyt b(6)f and Ndh. On the contrary, inhibition of non-cyclic electron flow by antimycin A or uncoupling of thylakoids by carbonyl cyanide m-chlorophenyl hydrazone did not modify the steady-state oxygen effect on Chl fluorescence quenching. The addition of NADH protected thylakoids against oxygen-induced Chl fluorescence quenching, whereas in the presence of exogenic duroquinone the decrease in Chl fluorescence to one half of the initial level did not result from the oxygen effect, probably due to oxygen action as a weak electron acceptor from PQ pool and an insufficient non-photochemical quencher. The data indicate that mechanism of oxygen-induced Chl fluorescence quenching depends significantly on oxygen concentration and is related to both structural rearrangement of thylakoids and the direct oxygen reduction by photosynthetic complexes.     

Chemical environment heterogeneity around the two chlorophyll a′ molecules in photosystem I

Treatment of whole and fractionated plant tissues with hydrophilic and hydrophobic solvent mixtures of varied volume ratio liberates two chlorophyll (Chl) a′ molecules from the photosystem (PS)I core when the solvent hydrophilicity exceeds a critical level, whereas only one molecule is extracted in hydrophobic media. The PSI core proteins, PSI-A and PSI-B, which form a heterodimer, appear to bind one Chl a′ molecule each, in local environments significantly different regarding their hydrophilicity or hydrophobicity.     

Time-resolution of the antheraxanthin- and delta pH-dependent chlorophyll a fluorescence components associated with photosystem II energy dissipation in Mantoniella squamata

The electronic excited-state behavior of photosystem II (PSII) in Mantoniella squamata, as influenced by the xanthophyll cycle and the transthylakoid pH gradient (delta pH), was examined in vivo. Mantoniella is distinguished from other photosynthetic organisms by two main features namely (1) a unique light-harvesting complex that serves both photosystems I (PSI) and II (PSII); and (2) a violaxanthin (V) cycle that undergoes only one de-epoxidation step in excess light to accumulate the monoepoxide antheraxanthin (A) as opposed to the epoxide-free zeaxanthin (Z). The cells were treated first with high light to induce the delta pH and A accumulation, followed by herbicide-induced closure of PSII traps and a chilling treatment, to sustain and stabilize the delta pH and nigericin-sensitive fluorescence level in the dark. De-epoxidation was controlled with subsaturating concentrations of dithiothreitol (DTT) and was 5-10 times more sensitive to DTT than higher plant thylakoids. The PSII energy dissipation involved two steps: (1) the pH activation of the xanthophyll binding site that was associated with a narrowing and slight attenuation of the main 2 ns (ns = 10(-9) s) fluorescence lifetime distribution; and (2) the concentration-dependent binding of A to the activated binding site yielding a second distribution centered around 0.9 ns. Consistent with the model of Gilmore et al. (1998) (Biochemistry 37, 13,582-13,593), the fractional intensity of the 0.9 ns component depended almost entirely on the A concentration and correlated linearly with the decrease of the steady-state chlorophyll alpha fluorescence intensity.     

Reversed-phase HPLC determination of chlorophyll a' and naphthoquinones in photosystem I of red algae: existence of two menaquinone-4 molecules in photosystem I of Cyanidium caldarium.

Chlorophyll (Chl) a', the C13(2)-epimer of Chl a, is one of the two Chl molecules constituting the primary electron donor (P700) of photosystem (PS) I of a thermophilic cyanobacterium Synechococcus elongatus. To examine whether PS I of other oxygenic photosynthetic organisms in general contain one Chl a' molecule in P700, the pigment composition of thylakoid membranes and PS I preparations isolated from red algae Porphyridium purpureum and Cyanidium caldarium was examined by reversed-phase HPLC with particular attention to Chl a' and phylloquinone (PhQ), the secondary electron acceptor of PS I. The two red algae contained one Chl a' molecule at the core part of PS I. In PS I of C. caldarium, two menaquinone-4 (MQ-4) molecules were detected in place of PhQ used by higher plants and cyanobacteria. The 1:2:1 stoichiometry among Chl a', PhQ (MQ-4) and P700 in PS I of the red algae indicates that one Chl a' molecule universally exists in PS I of oxygenic photosynthetic organisms, and two MQ-4 molecules are associated with PS I of C. caldarium.     

Photo-electrochemical control of photosystem II chlorophyll fluorescence in vivo

The effect of electric field on chlorophyll fluorescence is considered on the basis of the reversible radical pair model. The hypothesis is presented that the electric fields generated by photosynthetic charge separation in reaction centers and propagated laterally through the thylakoid lumen are associated with changes in chlorophyll fluorescence yield.     

Role of LHCII organization in the interaction of substituted 1,4-anthraquinones with thylakoid membranes

The chlorophyll fluorescence, photochemical activity and surface electric properties of thylakoid membranes with different stoichiometry of pigment-protein complexes and organization of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHCII) were studied in the presence of substituted 1,4-anthraquinones. Data show strong dependence of the quenching of the chlorophyll fluorescence on the structural organization of LHCII. The increase of the LHCII oligomerization, which is associated with significant reduction of the transmembrane electric charge asymmetry and electric polarizability of the membrane, correlates with enhanced quenching effect of substituted 1,4-athraquinones. Crucial for the large quinone-induced changes in the membrane electric dipole moments is the structure of the quinone molecule. The strongest reduction in the values of the dipole moments is observed after interaction of thylakoids with 3-chloro-9-hydroxy-1,4-anthraquinone (TF33) which has the highest quenching efficiency. The quinone induced changes in the photochemical activity of photosystem II (PSII) correlate with the total amount of the supramolecular LHCII-PSII complex and depend on the number of substituents in the 1,4-anthraquinone molecule.     

Intramolecular interactions in the triplet excited states of benzophenone-thymine dyads

Time-resolved and product studies on the synthesized dyads 1 and 2 have provided evidence that the benzophenone-to-thymine orientation strongly influences intramolecular photophysical and photochemical processes. The prevailing reaction mechanism has been established as a Paterno-Büchi cycloaddition to give oxetanes 3-6; however, the ability of benzophenone to achieve a formal hydrogen abstraction from the methyl group of thymidine has also been evidenced by the formation of photoproducts 7 and 8. These processes have been observed only in the case of the cisoid dyad 1. Adiabatic photochemical cycloreversion of the oxetane ring is achieved upon direct photolysis to give the starting dyad 1 in its excited triplet state. The photobiological implications of the above results are discussed with respect to benzophenone-photosensitized damage of thymidine.     

Role of triplet states of aryldiazonium cations in the Meerwein reaction

The mechanism of the Meerwein reaction was interpreted on the basis of quantum-chemical calculations of the optimal geometry, electronic spectra, and ground state of a large number of diazonium cations. The relationship of this process with the triplet excited state of aryldiazonium cations and the role of spin catalysis in thermal excitation of these states via ligand-to-metal and metal-to-ligand electron transfer involving organic ligands and complex copper chlorides as catalysts were examined.     

ESR and ENDOR of triplet states in the charge-transfer crystal naphthalene-tetracyanobenzene

The ENDOR spectrum of localized triplet states (X-traps) in napthalene-tetracyanobenzene crystals at 4.2 J has been analyzed. From the symmetry of the spin density distribution on the donor and acceptor, it is concluded that the chargetransfer state is distributed over one donor and two acceptors. Between 130 and 300 K, the ESR spectrum or mobile triplet excitons is measured.     

Rapid cleavage of the naphthylmethyl-oxygen bond in higher triplet excited states

Rapid cleavage of the naphthylmethyl-oxygen bond of 1- and 2-[(4-benzoylphenoxy)methyl]naphthalenes in higher triplet excited states occurred within a laser flash of 5 ns to give 1- and 2-naphthylmethyl radicals with formation quantum yields of 0.042 +/- 0.004 and 0.020 +/- 0.002, respectively, during two-colour two-laser flash photolysis.