Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii

Abstract

Photosystem I (PSI) is a large pigment-protein complex mediating light-driven charge separation and generating a highly negative redox potential, which is eventually utilized to produce organic matter. In plants and algae, PSI possesses outer antennae, termed light-harvesting complex I (LHCI), which increase the energy flux to the reaction center. The number of outer antennae for PSI in the green alga Chlamydomonas reinhardtii is known to be larger than that of land plants. However, their exact number and location remain to be elucidated. Here, applying a newly established sample purification procedure, we isolated a highly pure PSI-LHCI supercomplex containing all nine LHCA gene products under state 1 conditions. Single-particle cryo-EM revealed the 3D structure of this supercomplex at 6.9 Å resolution, in which the densities near the PsaF and PsaJ subunits were assigned to two layers of LHCI belts containing eight LHCIs, whereas the densities between the PsaG and PsaH subunits on the opposite side of the LHCI belt were assigned to two extra LHCIs. Using single-particle cryo-EM, we also determined the 2D projection map of the lhca2 mutant, which confirmed the assignment of LHCA2 and LHCA9 to the densities between PsaG and PsaH. Spectroscopic measurements of the PSI-LHCI supercomplex suggested that the bound LHCA2 and LHCA9 proteins have the ability to increase the light-harvesting energy for PSI. We conclude that the PSI in C. reinhardtii has a larger and more distinct outer-antenna organization and higher light-harvesting capability than that in land plants.

Keywords: Chlamydomonas; Chlamydomonas reinhardtii; LHCI; cryo-electron microscopy; cryo-electron microscopy (cryo-EM); green algae; light-harvesting complex (antenna complex); outer antenna; photosynthesis; photosynthetic efficiency; photosystem I; single-particle analysis.

Figure 1.

Purification of the PSI–LHCI supercomplexes from 137c WT. Shown is SDS-PAGE of the PSI–LHCI supercomplex purified only with a sucrose density gradient (before the Fd column) and with a combination of sucrose density gradient and Fd column affinity chromatography (after the Fd column). A total of 0.8 μg of Chl was loaded in each lane.

Figure 2.

Cryo-EM projection map of the PSI–LHCI supercomplex of 137c WT. White arrow, NAPol ring density. Scale bar, 5 nm.

Figure 3.

The 2D projection map of single-particle cryo-EM of the PSI–LHCI supercomplex. A, the 2D average of PSI–LHCI supercomplex from the 137c WT, where these transmembrane helices were used as a marker for the assignment of the orientation of the individual LHCA proteins. The white contours mark a border for each LHCA protein, and the red contours mark LHCA helix C. B, the 2D average of PSI–LHCI supercomplex from the lhca2 mutant. The crystal structure of the plant PSI–LHCI (PDB entry 5L8R) and the model structures of LHCA2 and LHCA9 were overlaid on the projection maps of the PSI–LHCI from the 137c WT (C) and the lhca2 mutant, highlighting the missing density with a dotted line (D). PsaA and PsaB are presented in blue; PsaC, PsaD, and PsaE are in yellow; PsaF and PsaJ are in lilac; PsaG is in cyan; PsaK is in red-purple; PsaI, PsaL, PsaH are in ocher; plant-LHCA1 is in green; plant-LHCA2 is in orange; plant-LHCA3 is in yellow orange; LHCA4 is in yellow green; LHCA9 is in magenta, and LHCA2 is in red. Images are the sums of 3814 (137c WT) and 3511 (lhca2 mutant) aligned projections. Scale bars, 5 nm. In A, the contrast of Fig. 2 has been increased to assist visualization; no contrast change was applied to B–D.

Figure 4.

The 3D single-particle cryo-EM of the PSI–LHCI supercomplex. Surface representations of the PSI–LHCI supercomplex from 137c WT are shown. Densities are illustrated at the different contours (6σ (A) and 12σ (B)) with transmembrane helices rendered as a ribbon diagram using PDB structures (B). Top panels, view from the stromal side; bottom panels, view from the PsaG side (turned 90° along the x axis). PDB models and color codes are same as in Fig. 3. Scale bar, 5 nm.

Figure 5.

Relative abundance of the LHCA proteins and the PSI subunits in the lhca2 mutant. LHCA composition in the PSI–LHCI supercomplex from the lhca2 mutant was determined using stable isotope (¹⁵N)-labeled LC-MS/MS spectrometry. The relative amounts of each subunit for the PSI–LHCI supercomplex were determined based on the ¹⁴N:¹⁵N isotopomer values derived from the chromatograms between the 137c WT and the lhca2 mutant. Data shown are the mean ± S.E. (error bars) (n = 3).

Figure 6.

Absolute fluorescence emission spectra at 77 K. Fluorescence emission spectra were obtained by excitation at 440 nm with an integrating sphere at 77 K with sample concentration of 3 μg of Chl/ml. a.u., arbitrary unit.

Figure 7.

A schematic model of the PSI–LHCI supercomplex of C. reinhardtii. A, the PSI–LHCI supercomplex viewed from the stromal side. The LHCI belt at the PsaF (F, lilac) and PsaJ (J, lilac) side of the PSI core complex is composed of eight LHCA proteins (dark green oval) forming two layers. The LHCA2 and LHCA9 proteins exist between the PsaG (G, cyan) and PsaH (H, ocher), subunits of the PSI core complex. B, the PSI–LHCI supercomplex horizontally viewed from the LHCI belt side. Three extrinsic subunits, PsaC (C, yellow), PsaD (D, yellow), and PsaE (E, yellow), are located on the stromal side. The N-terminal region of PsaF subunit is projected to the luminal side. The color codes for PsaK (K) is red-purple. PsaM (M), which does not exist in C. reinhardtii, is shown by a dotted circle. Four of the LHCI subunits are assigned in the map as LHCA2 (2), LHCA9 (9), LHCA1a (1a), and LHCA1b (1b).