The origin of chlorophyll fluorescence In vivo and its quenching by the photosystem II reaction centre

Isolated chlorophyll a, in contrast to when it is dissolved in organic solvents, shows a lower and variable yield of fluorescence when bound to protein and embedded in the thylakoid membrane of photosynthetic organisms. There are two current theories that attempt to explain the origin of this variable yield of fluorescence. (i) It may be emitted directly from the photosystem II (PSII) antenna system and therefore in competition with photochemical trapping (prompt fluorescence). (ii) It may be derived from a recombination reaction between oxidized P$_{680}$ and reduced pheophytin within the PSII reaction centre (delayed fluorescence). We have isolated a PSII reaction centre complex that binds only four chlorophyll a molecules and can carry out primary charge separation. The complex contains no plastoquinone and therefore is devoid of the secondary electron acceptor Q$_A$. It does, however, contain two pheophytin $a$ molecules, and one of these acts as a primary electron acceptor. The electron donor is P$_{680}$, which is either a monomeric or dimeric form of chlorophyll a. The isolated PSII reaction centre fluoresces at room temperature with a maximum at 683 nm, and the intensity of this emission is almost totally quenched when reduced pheophytin (bright light plus sodium dithionite) or oxidized P$_{680}$ (bright light plus silicomolybdate) is photoaccumulated. The photoinduced quenching of chlorophyll fluorescence when sodium dithionite is present is also observed in intact PSII preparations containing plastoquinone Q$_A$. In the latter case Q$_A$ is chemically reduced in the dark by dithionite. Bearing in mind the above two postulates for the origin of variable chlorophyll fluorescence it has been possible to investigate the relative quantum yields for the photoproduction of the P$_{680}$Pheo$^-$ state either in the absence (with isolated PSII reaction centres) or presence (with PSII-enriched membranes) of reduced Q$_A$. It has been shown that in the absence of Q$^-_A$ the quantum efficiency for production of the P$_{680}$Pheo$^-$ is several orders of magnitude greater than when Q$_A$ is present. This difference probably partly reflects the coulombic restraints on primary charge separation when Q$_A$ is reduced and would suggest that under these conditions the PSII reaction centre is a less efficient trap. Such a conclusion is therefore consistent with postulate (i) that the increase in yield of chlorophyll fluorescence as Q$_A$ becomes reduced is not due to a back reaction between P$^+_{680}$ and Pheo$^-$ but rather to a decrease in competition between emission and trapping. The results do emphasize however, that the P$_{680}$Pheo$^-$ and P$^+_{680}$Pheo states are quenchers of chlorophyll fluorescence. In addition to the above, it has been noted that at 77 K fluorescence from the isolated PSII reaction centre reaches a maximum at 685 nm and does not have a peak at 695 nm. This observation appears to invalidate the postulate that the 695 nm emission is from the pheophytin of the PSII reaction centre.