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.     

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.     

Advances on photosystem II investigation by measurement of delayed chlorophyll fluorescence by a phosphoroscopic methods

A method for data acquisition based on recording of light signal from a conventional phophoroscope fluorometer with high-speed digitalization is proposed to extract more information from a delayed chlorophyll a fluorescence (DF) signal. During the signal processing from all points registered by the fluorometer, we obtain simultaneously a large number of induction curves of DF decaying at different time ranges. In addition, it is possible to register a series of dark relaxation kinetics of DF, recorded at different moments during the induction period or at different temperatures. This allows the evaluation of the contribution of DF kinetic components during the induction period or at different temperatures and the comparison between DF signals registered with different phophoroscopes. With the phosphoroscope system used in this study, we have shown that the contribution of the millisecond components (with lifetimes 0.6 and 2-4 ms) predominates during the first second of the induction period. After 1 s of illumination, the amplitudes of the 0.6 ms and 2-4 ms components and of the slower one (with lifetime more than 10 ms) become approximately equal. The change in lifetime of the different components during the induction and during gradual heating is also observed. It is shown that all registered DF kinetic components have different temperature dependences.     

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.     

Comparative study of the water oxidizing reactions and the millisecond delayed chlorophyll fluorescence in photosystem II at different pH

Water splitting activity, the multiline EPR signal associated with S(2)-state of the CaMn(4)-cluster and the fast and slow phases of the induction curve of the millisecond delayed chlorophyll fluorescence from photosystem II (PSII) in the pH range of 4.5-8.5 were studied in the thylakoid membranes and purified PSII particles. It has been found that O(2) evolution and the multiline EPR signal were inhibited at acidic (pK approximately 5.3) and alkaline (pK approximately 8.1) pH values, and were maximal at pH 6.0-7.0. Our results indicate that the loss of O(2) evolution and the S(2)-state multiline EPR signal associated with the decrease of the millisecond delayed chlorophyll fluorescence only in alkaline region (pH 7.0-8.5). Possible correlations of the millisecond delayed chlorophyll fluorescence components with the donor side reactions in PSII are discussed.     

Photoelectric effects on chlorophyll fluorescence of photosystem II in vivo. Kinetics in the absence and presence of valinomycin

Fluorescence induction curves (F(t)) in low intensity 1s light pulses have been measured in leaf discs in the presence and absence of valinomycin (VMC). Addition of VMC causes: (i) no effect on the initial fluorescence level Fo and the initial (O-J) phase of F(t) in the 0.01-1 ms time range. (ii) An approximately 10% decrease in the maximal fluorescence Fm in the light reached at the P level in the O-J-I-P induction curve. (iii) Nearly twofold increase in the rate and extent of the F(t) rise in the J-I phase in the 1-50 ms time range. (iv) A 60-70% decrease in the rise (I-P phase) in the 50-1000 ms time range with no appreciable effect, if at all, on the rate. System analysis of F(t) in terms of rate constants of electron transfer at donor and acceptor sides have been done using the Three State Trapping Model (TSTM). This reveals that VMC causes: (i) no, or very little effect on rate constants of e-transfer reactions powered by PSII. (ii) A manifold lower rate constant of radical pair recombination (k(-1)) in the light as compared to that in the control. The low rate constant of radical pair recombination in the reaction center (RC) in the presence of VMC is reflected by a substantial increase in the nonzero trapping efficiency in RCs in which the primary quinone acceptor (Q(A)) is reduced (semi-open centers). This causes an increase in their rate of closure and in the overall trapping efficiency. Data suggest evidence that membrane chaotropic agents like VMC abolish the stimulation of the rate constant of radical pair recombination by light. This light stimulation that becomes apparent as an increase in Fo has been documented before [Biophys. J. 79 (2000) 26]. It has been ascribed to effects of (changes in) local electric fields in the vicinity of the RC. The decrease of the I-P phase is attributed to a decrease in the photoelectric trans-thylakoid potential in the presence of VMC. Such effects have been hypothesized and illustrated.     

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.     

Rapid chlorophyll a fluorescence transients of Lemna minor leaves as indication of light and exogenous electron carriers effect on photosystem II activity

By using saturating flash, we investigated the change in the rapid fluorescence rise when Lemna minor leaf was exposed to different light conditions and treated with exogenous electron acceptors (methyl viologen and duroquinone) and electron donor (hydroxylamine). Investigation was carried out by using combined pulse amplitude modulated fluorometer and plant efficiency analyzer system, which were employed simultaneously to provide different light conditions and to induce rapid fluorescence rise respectively. We have shown that when leaf of L. minor was exposed to different conditions of illumination, rapid fluorescence rise was greatly influenced by the electron transport functions beyond quinone A-plastoquinone reduction. This was indicated by the change in both fluorescence yield and appearance time of the different transients. When exogenous electron donor (hydroxylamine) and acceptors (methyl viologen and duroquinone) were applied in in vivo condition, we showed that rapid fluorescence rise represented a reliable indicator of PSII-PSI electron transport state and energy dissipation process.     

Light absorption by the chlorophyll a-b complexes of photosystem II in a leaf with special reference to LHCII

To investigate the light-harvesting properties of the Photosystem II chlorophyll (chl) a-b complexes (major light-harvesting complex of Photosystem II [LHCII], CP24, CP26, CP29) in a mature leaf under natural "daylight" illumination, the absorption spectra of the isolated complexes were converted into the photon absorption spectrum (1-T) within a leaf, using the approach of Rivadossi et al. ([1999] Photosynth. Res. 60, 209-215). In the Qy region, significant enhancement of light harvesting by the chl b electronic transitions, with respect to the absorption spectra (optical density [OD]), as well as a large and generalized increase (between two- and four-fold) associated with the vibrational bands of both chl a and b, was observed, which acquires an important light-harvesting role (approximately 30-40% of total). In the Soret region, a small increase in light harvesting by chl b was indicated. To gain more detailed information on these aspects the light harvesting of LHCII in a leaf was investigated. This required describing the pigment absorption (chl a and b, carotenoids) in the LHCII OD spectrum in terms of spectral subbands, which were subsequently used to estimate the relative light harvesting of each pigment type in LHCII of a leaf. When the entire visible spectral interval between 400 and 730 nm is considered, the chl a light harvesting is essentially unchanged with respect to the absorption spectrum (OD) of isolated LHCII, whereas the chl b contribution is 20% higher and the carotenoids are 33% lower. The relative enhancement of the chl b absorption is principally associated with the Qy electronic transition region, the light-harvesting contribution of which becomes prominent in the leaf.     

Carotenoid triplet states associated with the long-wavelength-emitting chlorophyll forms of photosystem I in isolated thylakoid membranes

The carotenoid triplet populations associated with the long-wavelength-emitting chlorophyll forms of photosystem I (PS I)(dagger) have been investigated in isolated spinach thylakoids by means of fluorescence-detected magnetic resonance in zero field. The spectra collected in the 730-800 nm emission range can be globally fitted assuming the presence of four different carotenoid triplet states coupled to long-wavelength-emitting forms of PS I, having zero-field-splitting parameters /D/ = 0.0359 cm(-1) and /E/ = 0.00371 cm(-1), /D/ = 0.0382 cm(-1) and /E/ = 0.00388 cm(-1), /D/ = 0.0395 cm(-1) and /E/ = 0.00397 cm(-1), and /D/ = 0.0405 cm(-1) and /E/ = 0.00411 cm(-1). On the basis of the triplet-associated fluorescence emission profile, it is suggested that those triplets are associated with light-harvesting complex I, the peripheral antenna complex of PS I.     

The primary electron donor of photosystem II of the cyanobacterium Acaryochloris marina is a chlorophyll d and the water oxidation is driven by a chlorophyll a/chlorophyll d heterodimer

We present a theoretical analysis of the flash-induced absorbance difference spectrum assigned to the formation of the secondary radical pair P(+)QA(-) in photosystem II of the chlorophyll d-containing cyanobacterium Acaryochloris marina. An exciton Hamiltonian determined previously for chlorophyll a-containing photosystem II complexes is modified to take into account the occupancy of certain binding sites by chlorophyll d instead of chlorophyll a. Different assignments of the reaction center pigments to chlorophyll a or d from the literature are investigated in the calculation of the absorbance difference spectrum. A quantitative explanation of the experimental data requires one chlorophyll a molecule per reaction center, located at the site of P(D1). The remaining sites are occupied by chlorophyll d and pheophytin a. By far, the lowest site energy is found for the accessory chlorophyll of the D1 branch, Chl(D1), which represents the sink of excitation energy and therefore the primary electron donor. The cationic state is stabilized at the chlorophyll a, which drives the oxidation of water.     

Pulsed high-frequency EPR study on the location of carotenoid and chlorophyll cation radicals in photosystem II

When the primary electron-donation pathway from the water-oxidation complex in photosystem II (PS II) is inhibited, chlorophyll (Chl(Z) and Chl(D)), beta-carotene (Car) and cytochrome b(559) are alternate electron donors that are believed to function in a photoprotection mechanism. Previous studies have demonstrated that high-frequency EPR spectroscopy (at 130 GHz), together with deuteration of PS II, yields resolved Car(+) and Chl(+) EPR signals (Lakshmi et al. J. Phys. Chem. B 2000, 104, 10 445-10 448). The present study describes the use of pulsed high-frequency EPR spectroscopy to measure the location of the carotenoid and chlorophyll radicals relative to other paramagnetic cofactors in Synechococcus lividus PS II. The spin-lattice relaxation rates of the Car(+) and Chl(+) radicals are measured in manganese-depleted and manganese-depleted, cyanide-treated PS II; in these samples, the non-heme Fe(II) is high-spin (S = 2) and low-spin (S = 0), respectively. The Car(+) and Chl(+) radicals exhibit dipolar-enhanced relaxation rates in the presence of high-spin (S = 2) Fe(II) that are eliminated when the Fe(II) is low-spin (S = 0). The relaxation enhancements of the Car(+) and Chl(+) by the non-heme Fe(II) are smaller than the relaxation enhancement of Tyr(D)(*) and P(865)(+) by the non-heme Fe(II) in PS II and in the reaction center from Rhodobactersphaeroides, respectively, indicating that the Car(+)-Fe(II) and Chl(+)-Fe(II) distances are greater than the known Tyr(D)(*)-Fe(II) and P(865)(+)-Fe(II) distances. The Car(+) radical exhibits a greater relaxation enhancement by Fe(II) than the Chl(+) radical, consistent with Car being an earlier electron donor to P(680)(+) than Chl. On the basis of the distance estimates obtained in the present study and by analogy to carotenoid-binding sites in other pigment-protein complexes, possible binding sites are discussed for the Car cofactors in PS II. The relative location of Car(+) and Chl(+) radicals determined in this study provides valuable insight into the sequence of electron transfers in the alternate electron-donation pathways of PS II.     

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.     

How energy funnels from the phycoerythrin antenna complex to photosystem I and photosystem II in cryptophyte Rhodomonas CS24 cells

We report an investigation of energy migration dynamics in intact cells of the photosynthetic cryptophyte Rhodomonas CS24 using analyses of steady-state and time-resolved fluorescence anisotropy measurements. By fitting a specific model to the fluorescence data, we obtain three time scales (17, 58, and 113 ps) by which the energy is transferred from phycoerythrin 545 (PE545) to the membrane-associated chlorophylls (Chls). We propose that these time scales reflect both an angular distribution of PE545 around the photosystems and the relative orientations of the donor dihydrobiliverdin (DBV) bilin and the acceptor Chl. Contrary to investigations of the isolated antenna complex, it is demonstrated that energy transfer from PE545 does not occur from a single-emitting bilin, but rather both the peripheral dihydrobiliverdin (DBV) chromophores in PE545 appear to be viable donors of excitation energy to the membrane-bound proteins. The model shows an almost equal distribution of excitation energy from PE545 to both photosystem I (PSI) and photosystem II (PSII), whose trap times correspond well to those obtained from experiments on isolated photosystems.     

On the relation between the Kautsky effect (chlorophyll a fluorescence induction) and Photosystem II: basics and applications of the OJIP fluorescence transient

Chlorophyll a fluorescence is a highly sensitive, non-destructive, and reliable tool for measuring, rather quickly, photosynthetic efficiency, particularly of Photosystem II (PSII), the water-plastoquinone oxidoreductase. We briefly review here the connection between the fast (up to 2 s) chlorophyll fluorescence rise and PSII, as well as the empirical use of the fluorescence rise kinetics in understanding photosynthetic reactions, particularly of PSII. When dark-adapted photosynthetic samples are exposed to light, a fluorescence induction is observed, known as the Kautsky effect, after Hans Kautsky, the discoverer of the phenomenon showing the existence of variable fluorescence. The chlorophyll fluorescence intensity rises from a minimum level (the O level), in less than 1 s, to a maximum level (the P-level) via two intermediate steps labeled J and I. This is followed by a decline to a lower semi-steady state level, the S level, which is reached in about one minute. We provide here an educational review on how this phenomenon has been exploited through analysis of the fast OJIP fluorescence transient, by discussing basic assumptions, derivation of equations, as well as application to PSII-related questions.     

Heterogeneity of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) at the level of trimeric subunits.

To study organization of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) from spinach thylakoid membranes at the level of trimeric subcomplexes, we have applied non-denaturing isoelectric focusing (ndIEF) in vertical, slab polyacrylamide gels. When analyzed by two consecutive ndIEF/electroelution runs, spinach BBY membrane preparations (PSII(alpha)-enriched, stacked thylakoid membranes) were resolved into nine fractions of 100% purity, labelled 1-9 in order of decreasing pI values. Seven of these fractions (3-9) were shown by absorption spectroscopy to stand for LHCII subcomplexes. The subcomplexes were established - by monitoring their circular dichroism spectra and comparing them to the spectra of native LHCII trimers and monomers - to be structurally intact trimers. The analysis of polypeptide composition of the subcomplexes in terms of apparent molecular masses and Lhcb genes' products led us to the conclusion that each of the subcomplexes might be a mixed population of closely similar individual trimers, comprising of permutations of Lhcb1 and Lhcb2 (subcomplexes 3-7) or Lhcb1, Lhcb2 and Lhcb3 (subcomplexes 8 and 9).     

Quenching of chlorophyll a singlets and triplets by carotenoids in light-harvesting complex of photosystem II: comparison of aggregates with trimers

Laser-induced changes in the absorption spectra of isolated light-harvesting chlorophyll a/b complex (LHC II) associated with photosystem II of higher plants have been recorded under anaerobic conditions and at ambient temperature by using multichannel detection with sub-microsecond time resolution. Difference spectra (ΔA) of LHC II aggregates have been found to differ from the corresponding spectra of trimers on two counts: (i) in the aggregates, the carotenoid (Car) triplet–triplet absorption band (ΔA>0) is red-shifted and broader; and (ii) the features attributable to the perturbation of the Q_y band of a chlorophyll a (Chla) by a nearby Car triplet are more pronounced, than in trimers. Aggregation, which is known to be accompanied by a reduction in the fluorescence yield of Chla, is shown to cause a parallel decline in the triplet formation yield of Chla; on the other hand, the efficiency (100%) of Chla-to-Car transfer of triplet energy and the lifetime (9.3 μs) of Car triplets are not affected by aggregation. These findings are rationalized by postulating that the antenna Cars transact, besides light-harvesting and photoprotection, a third process: energy dissipation within the antenna. The suggestion is advanced that luteins, which are buried inside the LHC II monomers, as well as the other, peripheral, xanthophylls (neoxanthin and violaxanthin) quench the excited singlet state of Chla by catalyzing internal conversion, a decay channel that competes with fluorescence and intersystem crossing; support for this explanation is presented by recalling reports of similar behaviour in bichromophoric model compounds in which one moiety is a Car and the other a porphyrin or a pyropheophorbide.     

Non-linear absorption and fluorescence of chlorophyll a excited by a ruby laser

Excitation of chlorophyll a in solution by a Q-switched ruby laser permits the determination of the two-photon and excited singlet state cross sections from fluorescence and transmission measurements.