Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29 - Effects of zeaxanthin, pH and phosphorylation

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosys tem II has been shown to correlate with the onset of nonphotochemical quenc hing in vivo. We have used recombinant CP29 protein, over-expressed in Esch erichia coli and refolded in vitro with purified pigments, to obtain a prot ein indistinguishable from the native complex extracted from thylakoids, bi nding either violaxanthin or zeaxanthin together with lutein. These recombi nant proteins and the native CP29 were used to measure steady-state chlorop hyll fluorescence emission and fluorescence decay kinetics. We found that t he presence of zeaxanthin bound to CP29 induces a approximate to 35% decrea se in fluorescence yield with respect to the control proteins (the native a nd zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics sho wed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived component s (tau (3) and tau (4)) are modified by the presence of zeaxanthin. The mos t relevant changes are observed in the rfqy of tau3 and in the average life time (approximate to 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control p roteins). When studied in vitro, no significant effect of acidic pH (5.2-5. 3) is observed on chlorophyll a fluorescence yield or kinetics. The data pr esented show that recombinant CP29 is able to bind zeaxanthin and this prot ein-bound zeaxanthin induces a significant quenching effect.