Efficient light harvesting by photosystem II requires an optimized protein packing density in Grana thylakoids

Abstract

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

FIGURE 1.

Electron micrographs of freeze-fracture membranes. A, micrographs of lipid liposomes made of the native thylakoid membrane lipid mixture. Scale bar, 100 nm. B, untreated BBY membranes. Larger particles represent PSII complexes (EF face), smaller particles LHCII (PF face). Arrows, indicate EF-PF transitions, which can occur only in stacked grana membranes. Scale bar, 200 nm. C and D, fused BBY (lipid/chrorophyll 10:1). Note the decreased particle density as compared with B. Semicrystalline protein areas are visible in the upper left part in D. Scale bars, C and D, 200 nm.

FIGURE 2.

Steady state chlorophyll a fluorescence analysis. Black circles, F_o; white triangles, F_v; gray squares, F_v/F_m. The degree of lipid addition from liposomes to BBY membranes is expressed as added lipid/chlorophyll (x axis). The lines serve as a guide to the eye.

FIGURE 3.

Shift in the Qy absorption maximum of chlorophyll of fused and control BBY. The absorption maximum of unfused BBY is at 678.5 nm.

FIGURE 4.

A and B, 77 K fluorescence spectra of unfused (A) and fused (B) BBY (lipid/chlorophyll = 3). The area under the spectra was normalized to the measured F_m value. Open circles represent measured data, and the lines are fitted curves. Note that the overall increase of the amplitude in B is mainly caused by an increase in F₆₈₀. C and D, dependence of the amplitudes of the fitted fluorescence components (see A and B) on the added lipid to chlorophyll ratio.

FIGURE 5.

Chlorophyll fluorescence induction analysis. A, normalized F_v induced by broad green excitation light switched on at 0 ms. Curve 1, fusion control, curve 2, fused BBY (lipid/chlorophyll = 1); curve 3, fused BBY (lipid/chlorophyll = 10). Note the faster rise and the stronger sigmoidicity of curve 2 compared with curve 1. B, connectivity plot calculated from A. The deviation from linearity indicates higher cooperativity between PSII centers. The vertical dashed line indicates the 0.33 QA reduction level used for quantifying the connectivity. C, apparent antenna size of PSII expressed as rate of QA reduction (reciprocal half-time) deduced from F_v kinetics as a function of lipid content of BBY membranes (A). D, dependence of the connectivity of PSII on the lipid content. The connectivity parameter is expressed as the F_v value at 0.33 QA reduction (see B) and normalized to the value of unfused BBY. The value of 0 corresponds to linearity in B, equivalent to no connectivity.

FIGURE 6.

488.0 nm resonance Raman spectroscopy in the ν4 region. A, intact thylakoids and BBY membranes. B, xanthophyll distortion in BBY compared with thylakoids is clearly visible in the difference spectrum. C and D, spectra of unfused BBY and BBY fused with liposomes at a lipid to chlorophyll ratio of 1 and 10. The apparent maxima in B are indicated.

FIGURE 7.

Models of protein densities in a grana disc (50 × 50 nm). Structures of the dimeric LHCII-PSII supercomplex and trimeric LHCII are taken from Ref. . For the models a trimeric LHCII to PSII ratio of 3 was assumed (see in the text). A, PSII dimer density 2057/μm², corresponding to unfused BBY. The purple stars indicate putative overlap of protruding neoxanthins with adjacent proteins. B, PSII dimer density 1800/m² corresponding to fused BBY at lipid/chlorophyll of 075. C, PSII dimer density 1100/m² corresponding to fused BBY at lipid/chlorophyll of 10.