Photobleaching of Chlorophyll in Light-Harvesting Complex II Increases in Lipid Environment

Abstract

Excess light causes damage to the photosynthetic apparatus of plants and algae primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular environments - protein aggregates, embedded in detergent micelles or in reconstituted membranes (proteoliposomes). The effects of intense light treatment were analyzed by absorption and circular dichroism spectroscopy, steady-state and time-resolved fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching was estimated from the light-induced Chl absorption changes. Photobleaching occurred mainly in Chl a and was accompanied by strong fluorescence quenching of the remaining unbleached Chls. The rate of photobleaching increased by 140% when LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII. Removing oxygen from the medium or adding antioxidants largely suppressed the bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly and indirectly, respectively. The quantum yield of Chl a photobleaching in membranes and detergent was found to be 3.4 × 10^-5 and 1.4 × 10^-5, respectively. These values compare well with the yields of ROS production estimated from spin-trap EPR spectroscopy (around 4 × 10^-5 and 2 × 10^-5). A kinetic model is proposed, quantifying the generation of Chl and carotenoid triplet states and singlet oxygen. The high quantum yield of photobleaching, especially in the lipid membrane, suggest that direct photodamage of the antenna occurs with rates relevant to photoinhibition in vivo. The results represent further evidence that the molecular environment of LHCII has profound impact on its functional characteristics, including, among others, the susceptibility to photodamage.

Keywords: electron paramagnetic resonance; non-photochemical quenching; photoinhibition; photosystem II; reconstituted membranes; singlet oxygen.

FIGURE 1

Photobleaching in LHCII. (A) Absorption spectra of LHCII solubilized in detergent (β-DDM) and reconstituted membranes before (solid lines) and after 30 min of irradiation (dotted lines). (B) Absorption difference spectra (dark–minus–irradiated sample).

FIGURE 2

Time course of LHCII photobleaching in detergent (β-DDM), reconstituted membranes and aggregates during 30 min of irradiation (2000 μmol photons m^–2 s^–1). (A) Absorbance changes at 675 nm and (B) at 652 nm. Circles and lines represent experimental data points and monoexponential fits, respectively.

FIGURE 3

CD spectra of LHCII before, and after 15 and 30 min of irradiation. (A) LHCII in detergent (β-DDM) and (B) in reconstituted membranes. The spectra correspond to absorbance 0.4 at 675 nm.

FIGURE 4

Fluorescence emission spectra of LHCII before and after 30 min irradiation recorded with 436 nm excitation light. (A) LHCII in detergent (β-DDM) and (B) in reconstituted membranes. Note the separate intensity axes (right side) for irradiated samples.

FIGURE 5

EPR spectra of 4-oxo-TEMPO, generated during illumination of LHCII membranes in the presence of 100 mM 4-oxo-TEMP (TEMPD × H₂O). (A) Spectra recorded before and after 7 and 30 min of irradiation of reconstituted membranes. (B) Time course of singlet oxygen trapping by 4-oxo-TEMP (TEMPD × H₂O) during 30 min irradiation. Circles and lines represent experimental data points and monoexponential fits, respectively.

FIGURE 6

(A) EPR spectra of 5-SASL in detergent micelles and lipid membranes. (B) Time course of the 5-SASL concentration estimated from the EPR signal intensity during 30 min of irradiation of the reaction mixture containing LHCII with 50 μM 5-SASL. Circles and lines represent experimental data points and monoexponential fits, respectively.

FIGURE 1

Kinetic scheme of the photosensitization of singlet oxygen in LHCII.