Tryptophan at position 181 of the D2 protein of photosystem II confers quenching of variable fluorescence of chlorophyll: Implications for the mechanism of energy-dependent quenching

The lumenal CD loop region of the D2 protein of photosystem II contains res idues that interact with a reaction center chlorophyll and the redox-active Tyro. Using combinatorial mutagenesis, photoautotrophic mutants of Synecho cystis sp. PCC 6803 have been generated with multiple amino acid changes in this region. The CD loop mutations were transferred into a photosystem I-l ess Synechocystis strain to facilitate characterization of photosystem II p roperties in the mutants. Most of the combinatorial photosystem I-less muta nts obtained had a high yield of variable fluorescence, F-V. However, in th ree mutants, which shared a replacement of Phe181 by Trp, the F-V yield was dramatically reduced although a high rate of oxygen evolution was maintain ed. A site-directed F181W D2 mutant shared similar properties. Picosecond t ime-resolved fluorescence measurements revealed that in the combinatorial F 181W mutants the fluorescence lifetimes in closed and open photosystem II c enters were essentially identical and were similar to the fluorescence life time in open centers of the control strain. These results are explained by quenching of variable fluorescence in the mutants by charge separation betw een Trp181 and excited reaction center chlorophyll. This reaction competes efficiently with fluorescence and nonradiative decay in closed photosystem II centers, where the Lifetime of the excitation in the chlorophyll antenna is long. Thermodynamic considerations favor the formation of oxidized tryp tophan and reduced chlorophyll in the quenching reaction, presumably follow ed by charge recombination. A possible role of tryptophan-chlorophyll charg e separation in the mechanism of energy-dependent quenching of excitations in photosynthesis is discussed.