Studies on P-700 photo-oxidation and reduction in Photosystem I subchloroplast particles

The photochemical oxidation and reduction of P-700 were studied in digitonin- and in sodium dodecyl sulphate (SDS)-Photosystem I (PS I) particles in the presence of ascorbate. In digitonin-PS I particles, reduction of P-700⁺ occurs by the bound iron-sulphur protein (P-430) and by ascorbate. The relative contribution of these back reactions depends on the length of the exposure to light and on the temperature and pH of the reaction medium. Experiments performed under anaerobic conditions demonstrate that some endogenous component may serve as the electron acceptor of P-430^?. The rate of the latter reaction is also dependent upon the temperature and pH of the sample. At pH 9 and lower temperatures the rate of this reaction is so much reduced that the reduction of P-700⁺ by ascorbate, which increases rapidly at high pH, can be observed even during illumination. The effects of secondary electron acceptors and of the presence of SDS on the absorption changes due to P-700 are also reported. Low concentrations of SDS are shown to retard the back reaction of P-700⁺ with P-430^?. Studies with SDS-PS I particles (CP_I) confirm the absence of the iron-sulphur centres in this preparation. Three larger P-700-chlorophylla-protein complexes prepared by mild electrophoresis in the presence of SDS plus Triton X-100, however, still contain P-430.     

Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre.

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.     

Absorption changes of P-700 reversible in milliseconds at low temperature in Triton-solubilized Photosystem I particles

Triton-solubilized Photosystem I particles from spinach chloroplasts exhibit largely reversible P-700 absorption changes over the temperature range from 4.2 K to room temperature. For anaerobic samples treated with dithionite and neutral red at pH 10 and illuminated during cooling, a brief (1 μs) saturating flash produces absorption changes in the long wavelength region that decay in 0.95 ± 0.2 ms from 4.2 to 50 K. Above 80 K a faster (100 ± 30 μs) component dominates in the decay process, but this disappears again above about 180 K. The major decay at temperatures above 200 K occurs in about 1 ms. The difference spectrum of these absorption changes between 500 and 900 nm closely resembles that of P-700. Using ascorbate and 2,6-dichlorophenolindophenol as the reducing system with a sample of Photosystem I particles cooled in darkness to 4.2 K, a fully reversible signal is seen upon both the first and subsequent flashes. The decay time in this case is 0.9 ± 0.3 ms.     

Energy transfers from Photosystem II to Photosystem I in Cryptomonas rufescens (Cryptophyceae)

In Cryptomonas rufescens (Cryptophyceae), phycoerythrin located in the thylakoid lumen is the major accessory pigment. Oxygen action spectra prove phycoerythrin to be efficient in trapping light energy.The fluorescence excitation spectra at ?196°C obtained by the method of Butler and Kitajima (Butler, W.L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85) indicate that like in Rhodophycease, chlorophyll a is the exclusive light-harvesting pigment for Photosystem I.For Photosystem II we can observe two types of antennae: (1) a light-harvesting chlorophyll complex connected to Photosystem II reaction centers, which transfers excitation energy to Photosystem I reaction centers when all the Photosystem II traps are closed. (2) A light-harvesting phycoerythrin complex, which transfers excitation energy exclusively to the Photosystem II reaction complexes responsible for fluorescence at 690 nm.We conclude that in Cryptophyceae, phycoerythrin is an efficient light-harvesting pigment, organized as an antenna connected to Photosystem II centers, antenna situated in the lumen of the thylakoid. However, we cannot afford to exclude that a few parts of phycobilin pigments could be connected to inactive chlorophylls fluorescing at 690 nm.     

Membrane phosphorylation leads to the partial detachment of the chlorophyll a/b protein from Photosystem II

The hypothesis that the chlorophyll fluorescence decline due to membrane phosphorylation is caused principally by the detachment and removal of LHCP from the LHCP-PS II matrix is examined. It is demonstrated that when membranes are phosphorylated in the dark (a) the fluorescence decline is greater when excited by light enriched in wavelengths absorbed mainly by LHCP (475 nm) than when excited by light absorbed to a large extent also by the PS II complex (435 nm), (b) titration with different artificial quenchers of chlorophyll fluorescence is unchanged after the phosphorylation-induced fluorescence decline, and (c) the F_v/F_m ratio does not change after the phosphorylation-induced fluorescence decline. These data indicate that it is indeed principally LHCP that interacts with the quencher (PS I presumably). This interaction involves a small fraction of the total PS II-coupled LHCP, which becomes functionally detached from the LHCP-PS II matrix.     

Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of Photosystem II electron acceptors and fluorescence emission from Photosystems I and II

An analysis of the photo-induced decline in the in vivo chlorophyll a fluorescence emission (Kautsky phenomenon) from the bean leaf is presented. The redox state of PS II electron acceptors and the fluorescence emission from PS I and PS II were monitored during quenching of fluorescence from the maximum level at P to the steady state level at T. Simultaneous measurement of the kinetics of fluorescence emission associated with PS I and PS II indicated that the ratio of PS I/PS II emission changed in an antiparallel fashion to PS II emission throughout the induction curve. Estimation of the redox state of PS II electron acceptors at given points during P to T quenching was made by exposing the leaf to additional excitation irradiation and determining the amount of variable PS II fluorescence generated. An inverse relationship was found between the proportion of PS II electron acceptors in the oxidised state and PS II fluorescence emission. The interrelationships between the redox state of PS II electron acceptors and fluorescence emission from PS I and PS II remained similar when the shape of the induction curve from P to T was modified by increasing the excitation photon flux density. The contributions of photochemical and non-photochemical quenching to the in vivo fluorescence decline from P to T are discussed.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.     

Sub-microsecond chlorophyll A delayed fluorescence from Photosystem I Magnetic field-induced increase of the emission yield

(1) In photosystem I (PS I) particles in the presence of dithionite and intense background illumination at 290 K, an external magnetic field (0–0.22 T) induced an increase, ΔF, of the low chlorophyll a emission yield, F ($\text{ΔF}\text{F}\text{? 1–1.5\%}$). Half the effect was obtained at about 35–60 mT and saturation occurred for magnetic fields higher than about 0.15 T. In the absence of dithionite, no field-induced increase was observed. Cooling to 77 K decreased ΔF at 685 nm, but not at 735 nm, to zero. Measuring the emission spectra of F and ΔF, using continuous excitation light, at 82, 167 and 278 K indicated that the spectra of F and ΔF have about the same maximum at about 730, 725 and 700 nm, respectively. However, the spectra of ΔF show more long-wavelength emission than the corresponding spectra of F. (2) Only in the presence of dithionite and with (or after) background illumination, was a luminescence (delayed fluorescence) component observed at 735 nm, after a 15 ns laser flash (530 nm), that decayed in about 0.1 μs at room temperature and in approx. 0.2 μs at 77 K. A magnetic field of 0.22 T caused an appreciable increase in luminescence intensity after 250 ns, probably mainly caused by an increase in decay time. The emission spectra of the magnetic field-induced increase of luminescence, ΔL, at 82, 167 and 278 K coincided within experimental error with those of ΔF mentioned above. The temperature dependence of ΔF and ΔL was found to be nearly the same, both at 685 and at 735 nm. (3) Analogously to the proposal concerning the 0.15 μs luminescence in photosystem II (Sonneveld, A., Duysens, L.N.M. and Moerdijk, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5889–5893), we propose that recombination of the oxidized primary donor P-700⁺ and the reduced acceptor A^?, probably A^?₁, of PS I causes the observed fast luminescence. The effect of an external magnetic field on this emission may be explained by the radical pair mechanism. The field-induced increase of the 0.1–0.2 μs luminescence seems to be at least in large part responsible for the observed increase of the total (prompt + delayed) emission measured during continuous illumination in the presence of a magnetic field.     

Electron acceptors associated with P-700 in Triton solubilized Photosystem I particles from spinach chloroplasts

Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700⁺ with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 μs back reaction of P-700⁺, which we associate with a more primary electron acceptor A₂. In turn, when A₂ is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 μs) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A₁, which functions very close to P-700. The organization of these components probably corresponds to the sequence $\text{P-700-}\text{A}{}_1\text{-}\text{A}{}_2\text{-P-430[}\text{A}\text{B}\text{]}$. The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed.Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.     

Regulation of photosystem stoichiometry,chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure

The structural-functional organization of higher plant chloroplasts has been investigated in relation to the particular light conditions during plant growth. (1) Light intensity variations during growth caused changes in the $\text{Chl}\text{a}\text{Chl}\text{b}$ ratio, in the light-saturated uncoupled rates of electron transport to a Hill oxidant and in the distribution of the chloroplast volume between the membrane and stroma phases. (2) Light quality differences during growth had an effect on the PS II/PS I reaction center ratio and on the chloroplast membrane phase differentiation into grana and stroma thylakoids. Plants grown under far-red-enriched (680–710 nm) illumination contained higher (20–25%) amounts of PS II and simultaneously lower (20–25%) amounts of PS I reaction centers. They also showed a higher grana density along with thicker grana stacks in their chloroplasts. (3) The size of the light-harvesting antenna pool of PS II centers was estimated from the fluorescence time course of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts and was found to be fairly constant (±10%) in spite of the variable PS II/PS I reaction center ratio. The results are compatible with the hypothesis that the structural entities of grana facilitated the centralization and relative concentration increase of a certain group of PS II reaction centers.     

Effect of detergents on the reliability of a chemical assay for P-700

A chemical assay for P-700 was developed using 0.36 mM potassium ferricyanide as oxidant and 1.6 mM sodium ascorbate as reductant. The major difference from other chemical assays for P-700 is procedural. The method is designed to take advantage of the availability of microprocessor-linked spectrophotometers to obtain greater accuracy by minimizing the spectral changes due to irreversibly oxidized antenna chlorophyll molecules. The value measured for the P-700 concentration in a sample of chloroplasts was not changed by the presence of EDTA, Mg²⁺ or sucrose in the assayed solution. Similarly, half of the detergents tested (Triton X-100, Nonidet P-40, digitonin, Deriphat 160, Miranol S2M-SF and Miranol M2M) did not alter the value when added to the chloroplasts. The remainder of the detergents examined caused a significant decrease or increase in the value for P-700 content. Sodium dodecyl sulfate, of particular interest due to its widespread use, caused a doubling in the amount of apparent P-700. This effect may be due to this detergent and some others enabling an additional long wavelength form of chlorophyll, possibly an intermediary electron acceptor in Photosystem I, to be chemically oxidized and reduced under the assay conditions.     

Evidence that the low-potential (−700 mV) electron acceptor (X) in Photosystem I has two iron-sulphur centres

The Photosystem I reaction centre contains two groups of iron-sulphur centres: Fe-S_A and Fe-S_B with redox potentials between ?510 and ?590 mV, and Fe-S_X with redox potential about ?700 mV. Spin quantitation (Heathcote, P., Williams-Smith, D.L. and Evans, M.C.W. (1978) Biochem. J. 170, 373–378) and Mössbauer spectroscopy (Evans, E.H., Dickson, D.P.E., Johnson, C.E., Rush, J.D. and Evans, M.C.W. (1981) Eur. J. Biochem. 118, 81–84) did not show unequivocally whether Fe-S_X has one or two centres. Experiments are described which support the proposal that Fe-S_X has two centres. Fe-S_X can be photoreduced irreversibly by 210 K illumination of dithionite-reduced samples or reversibly by 7.5 K illumination of these samples. The amplitude of the Fe-S_X signal reversibly induced by illumination at 7.5 K is never more than 50% of the amplitude of the signal when Fe-S_X is prereduced by room temperature illumination or by 210 K illumination. Approx. half of the Fe-S_X is rapidly reduced by 210 K illumination, the remainder more slowly. The extent of reversible Fe-S_X reduction and P-700 photooxidation is little affected by the fast reduction of about half of the Fe-S_X. Subsequent reduction of the remaining Fe-S_X is paralleled by loss of the reversible photoreaction.     

Bicarbonate effects on chlorophyll a fluorescence transients in the presence and the absence of diuron

We investigated the effect of HCO^?₃ addition to CO₂-depleted thylakoids by means of fluorescence techniques. (1) In the presence of diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), the net reduction of the primary quinone-type electron acceptor (Q) of Photosystem (PS) II is about 2-times faster in the absence of HCO^?₃ than in its presence, whether normal, heat-treated or NH₂OH-treated samples are used. This effect of HCO^?₃ is, therefore, not on the O₂-evolving apparatus. It is, however, interpreted to be due to an influence of HCO^?₃ on the kinetics of the reduction of Q, perhaps combined with an effect on the back reaction of Q^? with P-680⁺, the oxidized form of the PS II reaction center chlorophyll a. (2) Fluorescence experiments in the absence of diuron indicate that the absence of HCO^?₃ results in a complete block at the quinone level; the area over the fluorescence induction curve in the absence of HCO^?₃ was found to be 2.2-times higher in the absence than in the presence of diuron, pointing to a complete block of BH₂ oxidation in the absence of HCO^?₃. (3) No change in the midpoint potential of Q is observed when HCO^?₃ is added to CO₂-depleted membranes. HCO^?₃ not only has a large (on/off) effect on the reoxidation of BH₂, but also a smaller effect between P-680 and Q. We propose that HCO^?₃ binding to its specific site in the thylakoid membrane results in a conformational change, allowing normal electron transport between the two photosystems.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

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.     

Photosystem II reaction center with altered pigment-composition: reconstitution of a complex containing five chlorophyll a per two pheophytin a with modified chlorophylls

Pigment-depleted Photosystem II reaction centers (PS II-RCs) from a higher plant (pea) containing five chlorophyll a (Chl) per two pheophytin a (Phe), were treated with Chl and several derivatives under exchange conditions [FEBS Lett. 434 (1998) 88]. The resulting reconstituted complexes were compared to those obtained by pigment exchange of “conventional” PS II-RCs containing six Chl per two Phe. (1) The extraction of one Chl is fully reversible. (2) The site of extraction is the same as the one into which previously extraneous pigments have been exchanged, most likely the peripheral D1-H118. (3) Introducing an efficient quencher (Ni-Chl) into this site results in only 25% reduction of fluorescence, indicating incomplete energy equilibration among the “core” and peripheral chlorophylls.     

Association of chlorophyll a/c2 complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c₂ proteins were found. The most common complexes have Chl a/c₂ complexes at both sides of the PSI core monomer and have dimensions of about 17 × 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c₂ light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c₂ proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c₂ proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.