Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the energy "red limit" of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.

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−.

During photosynthesis carotenoids normally serve as antenna pigments, transferring singlet excitation energy to chlorophyll, and preventing singlet oxygen production from chlorophyll triplet states, by rapid spin exchange and decay of the carotenoid triplet to the ground state. The presence of two beta-carotene molecules in the photosystem II reaction centre (RC) now seems well established, but they do not quench the triplet state of the primary electron-donor chlorophylls, which are known as P(680). The beta-carotenes cannot be close enough to P(680) for triplet quenching because that would also allow extremely fast electron transfer from beta-carotene to P(+)(680), preventing the oxidation of water. Their transfer of excitation energy to chlorophyll, though not very efficient, indicates close proximity to the chlorophylls ligated by histidine 118 towards the periphery of the two main RC polypeptides. The primary function of the beta-carotenes is probably the quenching of singlet oxygen produced after charge recombination to the triplet state of P(680). Only when electron donation from water is disturbed does beta-carotene become oxidized. One beta-carotene can mediate cyclic electron transfer via cytochrome b559. The other is probably destroyed upon oxidation, which might trigger a breakdown of the polypeptide that binds the cofactors that carry out charge separation.

A reaction center of photosystem II was isolated from Pisum sativum by using immobilized metal affinity chromatography. This reaction center is photochemically active and has a room temperature Qy chlorophyll (Chl) absorption band peaking at 677.5 nm. From HPLC analysis, the pigment stoichiometry was suggested to be 5 Chls per 1 (3-carotene per 2 pheophytins. Low-temperature absorption measurements at 77 K were consistent with the removal of one of the Chls associated with the usual form of the reaction center isolated by using ion-exchange chromatography. Transient absorption spectroscopy on the picosecond time scale indicated that the Chl removed belongs to a pool of Chl absorbing at -670 nm (C6701I) that transfers energy relatively slowly to the primary donor P680 in support of our recently proposed model. The results also support the previous conclusion that radical pair formation is largely associated with a 21-ps time constant when P680 is directly excited and that the identity of C6701 is likely to be peripherally bound ChIs possibly ligated to conserved His residues at positions 118 on the Dl and D2 proteins.that have been assigned to energy transfer from accessory Phs and Chls to the primary donor P680 (17,(19)(20)(21)(22)(23)(24). It has been further suggested that the slow energy transfer processes are associated with two additional Chls bound to the periphery of the reaction center complex (21, 24). Moreover, the presence of these slow energy transfer processes has made the determination of the time constants for primary charge separation more difficult, leading to controversy (16,21,(24)(25)(26)(27)(28). It is therefore particularly interesting to compare the primary photochemistry of the 6-Chl-containing PSII reaction center with those in which 1 or 2 of the peripheral Chl molecules have been removed. In this paper, we report a procedure for isolating the reaction center of PSII by using immobilized metal affinity chromatography. HPLC analysis indicated that the complex contained 5 Chls per 2 Phs and, relative to that of the 6-Chl-containing preparation, showed a spectral shift indicative of the removal of an accessory Chl absorbing on the blue side of the main Qy band. The role of this Chl as an accessory pigment was confirmed by time-resolved picosecond absorption spectroscopy.

The photosystem II reaction centre of all oxygenic organisms is subject to photodamage by high light i.e. photoinhibition. In this review I discuss the reasons for the inevitable and unpreventable oxidative damage that occurs in photosystem II and the way in which beta-carotene bound to the reaction centre significantly mitigates this damage. Recent X-ray structures of the photosystem II core complex (reaction centre plus the inner antenna complexes) have revealed the binding sites of some of the carotenoids known to be bound to the complex. In the light of these X-ray structures and their known biophysical properties it is thus possible to identify the two beta-carotenes present in the photosystem II reaction centre. The two carotenes are both bound to the D2 protein and this positioning is discussed in relation to their ability to act as quenchers of singlet oxygen, generated via the triplet state of the primary electron donor. It is proposed that their location on the D2 polypeptide means there is more oxidative damage to the D1 protein and that this underlies the fact that this latter protein is continuously re-synthesised, at a far greater rate than any other protein involved in photosynthesis. The relevance of a cycle of electrons around photosystem II, via cytochrome b(559), in order to re-reduce the beta-carotenes when they are oxidised and hence restore their ability to quench singlet oxygen, is also discussed.

By measuring time-resolved luminescence emission at 1270 nm, we have detected singlet oxygen formation by illuminated, reaction centers of photosystem II isolated from Pisum sativum, which is in agreement with earlier work (Macpherson, A. N., Telfer, A., Barber, J., & Truscott, T. G. (1993) Biochim. Biophys. Acta 1143, 301-309). In this paper we show that the yield of singlet oxygen is significantly increased if the number of beta-carotene molecules bound per isolated complex is reduced from two to one. We conclude, therefore, that beta-carotene can act as an effective quencher of singlet oxygen in the photosystem II reaction center. This conclusion is supported by the finding that the rate of light-induced irreversible bleaching of chlorins in the reaction center is increased with decreasing beta-carotene levels. The results demonstrate the direct intermediacy of singlet oxygen in causing photooxidative damage within a biological environment and are discussed, specifically, in terms of the role of beta-carotene in protecting photosystem II against photoinhibition.

No abstract

No abstract