Photoinhibition of photosystem II in vitro: Spectral and kinetic analyses

Two different preparations of photosystem II (PSII) (BBY-type membrane fragments and PSII core complexes) were isolated from 14-day-old pea seedlings (Pisum sativum L.) and used for spectral and kinetic study of photobleaching of chlorophyll (Chl) and amino acids under photoinhibitory conditions. A short-term (2–4 min) illumination of PSII preparations with high-intensity red light (λ > 610 nm, 800 W/m²) resulted in irreversible photobleaching of Chl at 672 and 682 nm under conditions of both acceptor- and donor-side photoinhibition. At longer illumination exposures (> 10 min) the photobleaching maximum at 682 nm was predominant. The calculated kinetic constants for Chl photobleaching in both absorption bands at temperatures of 20 and 4°C had similar values under different photoinhibitory conditions. The shape of action spectrum for Chl photooxidation indicates that photoinhibition of PSII was sensitized by two spectral forms of Chl with absorption maxima at 670 and 680 nm. The photobleaching of amino acids in PSII membrane fragments was only observed during acceptor-side photoinhibition and displayed the photobleaching peaks at 220 and 274 nm. The photogeneration of superoxide anion radical during donor-side photoinhibition was 4–6 times larger than during acceptor-side photoinhibition. Nevertheless, the kinetics of Chl and amino acid photobleaching in PSII preparations showed no appreciable differences. The activation energies for Chl photooxidation were estimated around 3.5 and 9 kcal/mol during acceptor- and donor-side photoinhibition, respectively, providing evidence for the involvement of biochemical stages in PSII photoinhibition. Based on the data obtained, it is proposed that the antenna Chl, rather than Chl of the reaction center, is the sensitizer for both acceptor- and donor-side photoinhibition of PSII in vitro.