Alison Telfer

Characterisation of a PS II reaction centre isolated from the chloroplasts of Pisum sativum

A photosystem II reaction centre has been isolated from peas and found to consist of D1, D2 polypeptides and the apoproteins of cytochrome b‐559, being similar to that reported for spinach by Nanba and Satoh [(1987) Proc. Natl. Acad. Sci. USA 84, 109–112]. The complex binds chlorophyll a, pheophytin and the haem of cytochrome b‐559 in an approximate ratio of 4:2:1 and also contains about one molecule of β‐carotene. It binds no plastoquinone‐9 or manganese but does contain at least one non‐haem iron. In addition to a light‐induced signal due to Pheo− seen under reducing conditions, a light‐induced P680+ signal is seen when the reaction centre is incubated with silicomolybdate. In the presence of diphenylcarbazide, the P680+ signal is partially inhibited and net electron flow to silicomolybdate occurs. This net electron flow is insensitive to o‐phenanthroline, 3‐(3,4‐dichlorophenyl)‐1,1‐dimethyl urea and 2‐(3‐chloro‐4‐trifluoromethyl)anilino‐3,5‐dinitrothiophene but is inhibited by proteolysis with trypsin and by other treatments. Fluorescence, from the complex, peaks at 682 nm at room temperature and at 685 nm at 77 K. This emission is significantly quenched when either the P680+Pheo or P680Pheo− states are established indicating that the fluorescence emanates from the back reaction between P680+ and Pheo−.

Direct detection of singlet oxygen from isolated Photosystem II reaction centres

Abstract Both steady-state and time-resolved luminescence measurements at 1270 nm indicate that, when illuminated, isolated reaction centres of Photosystem II can generate singlet oxygen, O 2 ( 1 Δ g ). The oxygen dependent component of the luminescence signal, which was measured in a D 2 O buffer, was quenched by either azide or H 2 O. Singlet oxygen was detected both when primary charge separation was functioning and after it had been inactivated, suggesting that 1 O 2 can be generated from triplet states formed by radical pair recombination (P680 chlorophyll triplet) and by intersystem crossing (accessory chlorophylls). Neither azide nor H 2 O was found to protect against light-induced oxygen dependent irreversible bleaching of chlorophyll and pheophytin of reaction centres. We suggest that the pool of singlet oxygen detected by steady-state luminescence at 1270 nm and quenched by azide and water is located in the bulk medium rather than in the protein matrix of the reaction centre, where the photodamage to pigments occurs, and the singlet oxygen is initially generated.

β-Carotene within the isolated Photosystem II reaction centre: photooxidation and irreversible bleaching of this chromophore by oxidised P680

Illumination of isolated Photosystem II reaction centres in the presence of the electron acceptors, silicomolybdate (SiMo) or 3,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone (DBMIB), leads to selective photooxidation and irreversible photobleaching of β-carotene. No such effect is observed in the absence of the electron acceptors and it is dependent on the ability of the reaction centres to carry out charge separation. Flash absorption studies indicate that prior to the irreversible photobleaching, β-carotene is photooxidised by electron transfer to P680 + . The rate of photobleaching of β-carotene is faster when SiMo is used as the acceptor and occurs both in the presence and absence of oxygen. However, with DBMIB present photobleaching is more clearly observed when oxygen is present. It is argued that when oxygen is absent, photoreduced DBMIB can rapidly rereduce P680 + by an electron transfer cycle involving cytochrome b -559, while in aerobic conditions the cycle is partially inhibited by oxygen acting as an electron acceptor. When Mn(II) is added as an electron donor to P680 + , no photobleaching of β-carotene occurs. The kinetics of photobleaching shows two phases, with 50% loss of the total β-carotene pool occurring rapidly. Coupled with the loss of β-carotene is a photobleaching of accessory chlorophyll which absorbs at 670 nm. Therefore our results indicate that, when the Photosystem II reaction centre is photoactivated under conditions in which P680 + can photoaccumulate, there is a secondary oxidation of β-carotene and accessory chlorophyll which leads to irreversible photobleaching. No such photobleaching occurs if P680 + is rapidly reduced by an exogenous electron donor or by a quinone dependent cyclic flow of electrons around PSII. We discuss the physiological role of β-carotene oxidation and cyclic electron transport in the function of PSII in vivo.

State 1-state 2 transition in leaves and its association with ATP-induced chlorophyll fluorescence quenching

By using chlorophyll fluorescence, a study has been made of changes in spillover of excitation energy from Photosystem (PS) II to PS I associated with the State 1–State 2 transition in intact pea and barley leaves and in isolated envelope-free chloroplasts treated with ATP. (1) In pea leaves, illumination with light preferentially absorbed by PS II (Light 2) led to a condition of maximum spillover (state 2) while light preferentially absorbed by PS I induced minimum spillover condition (State 1) as judged from the redox state of Q and low-temperature emission spectra. The State 1–State 2 transitions took several minutes to occur, with the time increasing when the temperature was lowered from 19 to 6°C. (2) In contrast to the wild type, leaves of a chlorophyll b-less mutant barley did not exhibit a State 1–State 2 transition, suggesting the involvement of the light-harvesting chlorophyll ab-protein complex in spillover changes in higher plants. (3) Spillover in isolated pea chloroplasts was increased by treatment with ATP either (a) in Light 2 in the absence of an electron acceptor or (b) in the dark in the presence of NADPH and ferredoxin. These observations can be interpreted in terms of the model that a more reduced state of plastoquinone activates the protein kinase which catalyzes phosphorylation of the light-harvesting chlorophyll ab-protein complex (Allen, J.F., Bennett, J., Steinback, K.E. and Arntzen, C.J. (1981). Nature 291, 25–29). This process was found to be very temperature sensitive. (4) Pea chloroplasts illuminated in the presence of ATP seemed to exhibit a slight decrease in the degree of thylakoid stacking, and an increased intermixing of the two photosystems. (5) The possible mechanism by which protein phosphorylation regulates the State 1–State 2 changes in intact leaves is presented in terms of changes in the spatial relationship of two photosystems resulting from alteration in membrane organization.

Two coupled β-carotene molecules protect P680 from photodamage in isolated Photosystem II reaction centres

Abstract The illumination of isolated Photosystem II (PS II) reaction centres in the presence of the artificial electron acceptors, silicomolybdate (SiMo) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) leads to the irreversible bleaching of β-carotene. The bleaching is biphasic with approx. 50% of the carotenoid being bleached rapidly and the remainder more slowly. This is attributed to the sequential bleaching of the two β-carotenes contained in the isolated complex, due to oxidation by the primary oxidant, P680+. Using SiMo as electron acceptor we found that only when all the β-carotene is irreversibly bleached does further illumination induce a loss of electron transfer activity. This rate of loss is exacerbated by the presence of oxygen. In addition to β-carotene degradation there is also, under the same conditions, an irreversible bleaching of chlorophyll absorbing at 670 nm. It is proposed therefore that β-carotene and chlorophyll 670 contained in the reaction centre protect against deleterious effects due to the photoaccumulation of the highly oxidising P680+ state by acting as electron donors. Comparison of the spectra of the normal 2 β-carotene and a 1 β-carotene containing preparation of the PS II reaction centre indicates that in the former these two carotenoids are excitonically coupled. The excitonic coupling allows a more rapid initial rate of electron donation by the β-carotene to P680+, than is seen in the preparation with only one carotenoid.

Spectral resolution of more than one chlorophyll electron donor in the isolated Photosystem II reaction centre complex

Abstract Prolonged illumination of the isolated Photosystem II reaction centre under aerobic conditions causes a selective photodestruction of chlorophyll which absorbs maximally at 680 nm. Concomitant with this effect is a loss of photochemical activity. When oxygen is absent, the reaction centre is no longer damaged by illumination. Protection of the 680 nm absorbing chlorophyll against photodamage and maintenance of photochemical activity can also be achieved if silicomolybdate (SiMo) is present as an electron acceptor, although in this case there is an irreversible bleaching at 670 nm, whether oxygen is present or not. We therefore suggest that there are two chlorophyll species in the reaction centre absorbing at 670 nm and 680 nm, respectively. The latter we attribute to the primary donor P680 and the former to an accessory chlorophyll. It seems highly likely that in the absence of SiMo but under aerobic conditions, the photodestruction of P680 involves singlet oxygen generated from the P680 triplet. When SiMo is present the yield of the triplet is significantly reduced (Nugent, J.H.A., Telfer, A., Demetriou, C. and Barber, J. (1989) FEBS Lett. 255, 53–58) due to electron transfer to the acceptor and thus this mode of photodegradation is reduced. However, the accumulation of P680+ when SiMo is present facilitates the oxidation of the accessory 670 nm chlorophyll which seems to result in its photodestruction by a mechanism not involving oxygen.

Identification of psbA and psbD gene products, D1 and D2, as reaction centre proteins of photosystem 2

A recent report (Nanba O, Satoh K: Proc. Natl. Acad. Sci. USA 84: 109–112, 1987) described the isolation from spinach of a putative photosystem 2 reaction centre which contained cytochrome b-559 and three other electrophoretically resolvable polypeptide bands, two of which have molecular weights comparable to the D1 and D2 polypeptides. We have used in vivo labelling with radioactive methionine and probed with D1 and D2 monospecific antibodies (raised against synthetically expressed sequences of the psbA and psbD genes) for specific detection of these proteins in a similarly prepared photosystem 2 reaction centre preparation. These techniques identified a 32 000 dalton D1 band, a 30 000 dalton D2 band and a 55 000 dalton D1/D2 aggregate, the latter apparently arising from the detergent treatments employed. Digestions with a lysine-specific protease further confirmed the identification of the lysine-free D1 polypeptide and also confirmed that the D1 molecules in the 55 000 dalton band were in aggregation with other bands and not in self-aggregates. The D1 and D2 polypeptides (including the aggregate) are considerably enriched in the photosystem two reaction centre preparation compared to the other resolved fractions.

Thylakoid protein phosphorylation during State 1—State 2 transitions in osmotically shocked pea chloroplasts

Abstract In osmotically shocked pea chloroplasts illuminated with modulated blue-green light (light 2), phosphorylation of the light-harvesting chlorophyll a b- protein complex (LHCP) accompanies the slow decrease in modulated fluorescence that indicates adaptation to light absorbed predominantly by Photosystem II (State 2). On subsequent additional illumination with continuous far-red light (absorbed predominantly by Photosystem I; light 1) both effects are reversed: modulated chlorophyll fluorescence emission increases (indicating adaptation towards State 1) and LHCP is dephosphorylated. Net phosphorylation and dephosphorylation of LHCP induced by light 2 and excess light 1, respectively, occur on the same time scale as the ATP-dependent chlorophyll fluorescence changes indicative of State 2 and State 1 transitions. The phosphatase inhibitor NaF (10 mM), stimulates the effect of blue-green light on fluorescence and prevents the effect of far-red light. These results provide a demonstration that light of different wavelengths can control excitation energy distribution between the two photosystems via the plastoquinol-activated LHCP phosphorylation mechanism suggested previously (Allen, J.F., Bennett, J., Steinback, K.E. and Arntzen, C.J. (1981) Nature 291, 25–29; and Horton, P. and Black, M.T. (1980) FEBS Lett. 119, 141–144).

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA820) in the microsecond time range. The extent of ΔA820 induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg2+ free medium (plus 15 mM K+) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll ab-protein complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg2+ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.

Quantitative analysis of State 1–State 2 transitions in intact leaves using modulated fluorimetry — evidence for changes in the absorption cross-section of the two photosystems during state transitions

Abstract Using a combination of modulated and non-modulated light with synchronized detection it has been possible to monitor State 1–State 2 transitions in intact leaves as changes in the yield of modulated chlorophyll fluorescence. In the presence of excess far-red non-modulated light (713 nm) absorbed mainly by Photosystem I (PS I), the modulated fluorescence intensity was taken to represent F o — the emission yield which occurs when the reaction centres of Photosystem II (PS II) are all open. On the other hand, superimposing saturating non-modulated wide-band, blue-green light resulted in a transitory maximum yield of modulated chlorophyll fluorescence, F m , due to the total closure of the PS II reaction centres. In the absence of these additional lights the fluorescence level assumed a steady-state value, F s , between F o and F m . All these parameters changed as the leaf slowly adapted to light of a given spectral composition. It was found that both F o and F m increased reversibly (by about 15–20%) during the transition from State 2 to State 1 such that the ratio of F m to F o remained constant, indicative of changes in absorption cross-section of PS II and PS I rather than alterations in ‘spillover’ which would cause preferential changes in F m . It was also possible to estimate the fractions of light, β and α, channeled to PS II and PS I, respectively, from the values of F o , F m and F s . In one approach, β was estimated in State 1, using the assumption that α + β = 1, and its variation during the subsequent state transition was assumed to follow proportional changes in F o (or F m ). It was found that in State 2 there is a small loss (about 4%) of the total utilization of light in both photosystems. However, if such loss is neglected, assuming α + β is always unity, the calculated β was found to vary in the same direction and almost with the same magnitude as F o (or F m ), indicating independently that a change in absorption cross-section in PS II (and PS I) had occurred. Consistent with these data were the light-saturation curves for the non-modulated far-red light-quenching effect in bringing the fluorescence from F s to F o in States 1 and 2. The ratio of the initial slopes of these curves indicates quantitatively both redistribution of light between PS I and PS II during the State 1–State 2 transitions and a partial loss of excitation energy in State 2.