Time resolved monitoring of flash induced changes of fluorescence quantum yield in spinach thylakoids

Abstract

The anionic and cationic chlorin radicals P680+ and Pheo- of photosystem II (PSII) act as powerful nonphotochemical quenchers of chlorophyll fluorescence. After excitation with short laser flashes the primary radical pair P680+Pheo- is formed and stabilized by rapid electron transfer from Pheo- to QA via a 300 ps kinetics. Accordingly, PSII reaction centers populate the state P680+QA- about 1 ns after a saturating short laser pulse. The subsequent reduction of P680+ by the redox active tyrosine Y within the ns-timescale removes the quencher P680+ and the fluorescence quantum yield rises until the photochemical quencher QA becomes restored by Q A- reoxidation via markedly slower m s-kinetics with QB (Q B-). This offers the possibility to measure the kinetics of P680+ reduction with Yz monitoring flash induced changes of fluorescence quantum yield with sufficiently high time resolution. The present study describes new equipment that satisfies this condition by using a rapidly gated multichannel plate detector in order to eliminate distortions due to fluorescence induced by the strong actinic flash. This method is especially suited for noninvasive analyses of the reaction pattern of P680+ reduction of PSII with intact water oxidation in thylakoids and whole algal cells. Data analyses require a suitable separation of signals that originate from the transient formation of carotenoid triplets. The applicability of the new assay is illustrated.