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 photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). 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 presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.
Crimi
M
M
Dipartimento Scientifico e Tecnologico, Università di Verona, Strada le Grazie, Cà Vignal 1, 37134 Verona, Italy. crimi@sci.univr.net
Dorra
D
D
Bösinger
C S
CS
Giuffra
E
E
Holzwarth
A R
AR
Bassi
R
R
D002338:Carotenoids / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D045342:Light-Harvesting Protein Complexes; D014975:Lutein / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D024341:Xanthophylls; D065146:Zeaxanthins; D019207:beta Carotene / Q000031:analogs & derivatives* / Q000378:metabolism
C069660:CP29 light harvesting complex; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins; D024341:Xanthophylls; D065146:Zeaxanthins; D019207:beta Carotene; D002338:Carotenoids; C005613:violaxanthin; D014975:Lutein