Identification of the chromophores involved in aggregation-dependent energy quenching of the monomeric photosystem II antenna protein Lhcb5

Abstract

Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light-harvesting complex II in ADQ and exclusively monomeric Lhcb proteins in charge transfer quenching. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin, a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than light-harvesting complex II during aggregation and that this is enhanced by binding to zeaxanthin, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll 612 and 613, in close contact with lutein bound at site L1, are important facilitators of ADQ.

FIGURE 1.

Fluorescence decay of Lhb5 and LHCII under “quenching” and “unquenching” conditions. Shown are fluorescence decay traces (solid lines) of LHCII (A) and Lhcb5 (B) proteins recorded at 685 nm, under either quenching (Q) or unquenching (UNQ) conditions (see “Results” for details). Decay traces were then fitted using either two or three exponential functions for unquenching or quenching samples, respectively; resulting fitting curves (fit) are shown as dashed lines.

FIGURE 2.

Average fluorescence lifetime ratios between samples in detergent and under conditions favoring aggregation. A, fluorescence average lifetimes of monomeric Lhcb4 to -6, trimeric LHCII proteins and of a mixture of Lhcb6 and LHCII trimers (3:1 molar ratio) in detergent (τ_AV UNQ) or under aggregation conditions (τ_AV Q). Time-resolved fluorescence analysis is reported in Table 1. Lhcb4 to -6 and LHCII trimer average fluorescence lifetimes were measured either in violaxanthin binding (Vio) or zeaxanthin binding (Zea) samples; calculated fluorescence average lifetimes of the LHCII and Lhcb6 mixture are reported (theor). B, average fluorescence lifetime ratios among samples measured in detergent (τ_AV UNQ) or in aggregation conditions (τ_AV Q), showing actual and calculated fluorescence average lifetimes of the LHCII and Lhc6 mixture under aggregation conditions.

FIGURE 3.

77 K emission spectra of Lhcb proteins in detergent or under conditions favoring aggregation. Fluorescence emission spectra (recorded at 77 K) of Lhcb proteins binding either Vio or Zea. Different samples were diluted with detergent (UNQ) or under aggregation conditions (Q) as described under “Results.” A, LHCII trimers; B, Lhcb4 complexes; C, Lhcb5 complexes; D, Lhcb6 complexes. Black traces, violaxanthin-binding complexes in detergent; red traces, zeaxanthin-binding complexes in detergent; blue traces, violaxanthin-binding complexes under aggregation conditions; cyan traces, zeaxanthin-binding complexes under aggregation conditions.

FIGURE 4.

77 K emission spectra of Lhcb5 WT and mutants in detergent or under conditions favoring aggregation. 77 K fluorescence emission spectra for Lhcb5 wild type and chlorophyll-binding mutants. Different samples, binding either Vio or Zea, were diluted with detergent (UNQ, dashed traces) or under aggregation conditions (Q, solid lines) as described under “Results.” A, wild type (black traces), A2 (red traces), A3 (blue traces), and B3 (cyan traces) violaxanthin-binding complexes; B, wild type (black traces), A4 (red traces), A5 (blue traces), B5 (cyan traces), and B6 (green traces) violaxanthin-binding complexes; C, wild type (black traces), A2 (red traces), A3 (blue traces), and B3 (cyan traces) zeaxanthin-binding complexes; D, wild type (black traces), A4 (red traces), A5 (blue traces), B5 (cyan traces), and B6 (green traces) zeaxanthin-binding complexes.

FIGURE 5.

CD spectra of Lhcb5 WT and mutants. A, CD spectra of Vio binding Lhcb5 wild type in detergent (UNQ, dashed black line) and under aggregation conditions (Q, solid black line) and the difference between them (gray dashed traces). Difference spectrum peaks are indicated. B, CD spectra of Zea binding Lhcb5 wild type in detergent (UNQ, dashed black line) and under aggregation conditions (Q, solid black line) and the difference between them (gray dashed traces). Difference spectrum peaks are indicated. C, CD difference spectra in the 650–750 nm region of Vio binding Lhcb5 wild type (solid black line), A2 (dashed black line), and A3 (dashed gray line) mutants, calculated by subtracting the spectrum measured in detergent (UNQ) from the spectrum measured under aggregation conditions (Q). D, CD difference spectra in the 650–750 nm region of Vio binding Lhcb5 wild type (solid black line), zeaxanthin binding Lhcb5 wild type (dashed black line), and the zeaxanthin binding mutant A3 (dashed gray line), calculated by subtracting the spectrum measured in detergent (UNQ) from the spectrum measured under aggregation conditions (Q).