Biochimica et Biophysica Acta, 1142(1993) 155-164 155 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.0(} BBABIO 43803 Two coupled/3-carotene molecules protect P680 from photodamage in isolated Photosystem II reaction centres Javier De Las Rivas, Alison Teller and James Barber AFRC Photosynthesis Research Group, Wolfson Laboratories, Department of Biochemistry. bnperial College of Science, Technology and Medicine, London (UK) (Received 3 August 1992) Key words: PhotosystemI1; Reaction center; Photoinhibition; 0-Carotene; Primary donor; P680 The illumination of isolated Photosystem II (PS II) reaction ccntres 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 /3-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/3-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 /3-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/3-carotene degradation there is also, under the same conditions, an irreversible bleaching of chlorophyll absorbing at 670 nm. It is proposed therefore that 0-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 2/3-carotene and a 1 /3-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 0-carotene to P680 ÷, than is seen in the preparation with only one carotenoid. Introduction Two important roles have been attributed to the function of carotenoids in photosynthesis; as light har- vesting pigments that transfer energy to reaction cen- tres and as protective agents, able to quench singlet oxygen and triplet states of chlorophyll [1]. The protec- tive role is particularly important when the incident light intensity is in excess of the saturation level for photosynthesis and photoinhibitory damage is likely to occur [2]. In addition to these two major functions, it has also been shown that /3-carotene within the Photo- system II (PS II) complex can undergo light-induced Correspondence to: J. Barber, AFRC Photosynthesis Research Group, Wolfson Laboratories, Department of Biochemistry, Imperial College of Science, Technologyand Medicine, London SW7 2AY, UK. Abbreviations: Car, carotenoid; Chl, chlorophyll; Cyt, cytochrome; D1 and D2, products of the psbA and psbD genes, respectively; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; HPLC, high performance liquid chromatography; P680, primary electron donor to PS II; Pheo, pheophytin; PS II, Photosystem II; RC, reaction centre; SiMo, silicomolybdate (SiMo120~o-); Tyrz, redox active tyrosine in PS II. redox reactions [3-5]. It was found that this carotenoid could, under certain circumstances, be oxidised by the primary donor of PS II (P680) to form a cation radical [4,5]. Recently, we have shown that the formation of this cation radical can be detected in the isolated PS II reaction centre which normally contains two /t-caro- tenes and that the oxidant was P680 + [6]. We also showed that the accumulation of photooxidised /3- carotene led to an irreversible loss of the pigment and that the process of degradation did not require the presence of oxygen. The only requirement for the photoinduced oxidation and irreversible bleaching of /3-carotene in the isolated PS II reaction centre com- plex was the presence of a suitable electron acceptor to compete with radical pair recombination and allow a lengthening of the P680 + lifetime. In our previous paper [6] we discussed the possible physiological meaning of the oxidation and degradation of this carotenoid suggesting that this activity func- tioned to protect the reaction centre against other deleterious damage which might occur if the P680 + state was allowed to photoaccumulate. Since P680 + has an oxidising potential in excess of I V [7] it has the unique capacity to oxidise, not only water via Dl-tyro- sine 161 (Tyr z) and the Mn cluster [81, but also pig-