ISOLATED PHOTOSYNTHETIC REACTION-CENTER OF PHOTOSYSTEM-II AS A SENSITIZER FOR THE FORMATION OF SINGLET OXYGEN - DETECTION AND QUANTUM YIELDDETERMINATION USING A CHEMICAL TRAPPING TECHNIQUE

Singlet oxygen formation by photosystem II reaction centers isolated f rom Pisum sativum has been detected by two chemical trapping technique s: histidine dependent oxygen uptake and bleaching of p-nitrosodimethy laniline by the intermediary endoperoxide of histidine. The quantum yi eld of singlet oxygen formation determined by these methods was estima ted to be 0.16 by comparison with the known quantum yields of standard singlet oxygen sensitizers. Singlet oxygen was formed on illumination of reaction centers under conditions that lead to formation of the tr iplet state of the primary electron donor, P680. Experiments with deut erated buffer and active oxygen scavengers indicated that singlet oxyg en was the only active oxygen species produced by this reaction. Neith er azide nor histidine, which are scavengers of singlet oxygen, protec ted against photobleaching of the chlorophyll of reaction centers that occurs concomitantly with singlet oxygen formation, suggesting that b leaching involves singlet oxygen generated within the protein matrix o f the complex. Singlet oxygen sensitized exogenously by rose bengal (w hen excited specifically at 550 nm) was also found to bleach reaction center chlorophyll in a manner similar to the intrinsic mechanism. We conclude that singlet oxygen formed within the hydrophobic interior of the reaction center attacks the chlorophylls of P680, and presumably also amino acids in the vicinity, and that only the singlet oxygen tha t escapes to the medium is affected by added scavengers or deuterated medium. These experiments extend our earlier report of the detection o f singlet oxygen by its luminescence at 1270 nm when isolated photosys tem II reaction centers are illuminated (Macpherson, A. N., Telfer, A. , Barber, J., and Truscott, T. G. (1993) Biochim. Biophys. Acta 1143, 301-309). Moreover, our results support the hypothesis that production of singlet oxygen underlies the vulnerability of photosystem II to ph otodamage and hence necessitates the rapid turnover of the D1 protein of the reaction center.