Structures of Rhodopseudomonas palustris RC-LH1 complexes with open or closed quinone channels

Abstract

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo-electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris A 2.65-Å resolution structure of the RC-LH1₁₄-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC Q_B site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange.

Fig. 1. Overall architecture of RC-LH1₁₄-W and RC-LH1₁₆ complexes.

(A and B) The complexes in surface representation. (C and D) Bound pigments in stick representation. (E and F) The complexes viewed from the cytoplasmic face with peptides in cartoon representation and LH1 subunits numbered clockwise from the protein-W gap [consistent with numbering for the Rba. sphaeroides complex (13)]. Protein subunits are colored in yellow for LH1-α, light blue for LH1-β, red for protein-W, cyan for RC-H, orange for RC-L, and magenta for RC-M. Cofactors are shown in stick representation with BChl and BPh a molecules in green, carotenoids in purple, and UQ₁₀ molecules in yellow. (G and H) Zoomed views of the protein-W gap in the RC-LH1₁₄-W complex (G) and equivalent region of the RC-LH1₁₆ complex (H). Cofactors are shown in space-filling representation with sequestered quinones displayed in blue. The protein-W gap is highlighted with a blue dashed line in (G), and small pores for quinone/quinol diffusion across the LH1₁₆ ring are highlighted with black dashed lines in (H).

Fig. 2. Spectral and biochemical analyses of RC-LH1₁₄-W and RC-LH1₁₆ complexes.

(A) Ultraviolet/Vis/NIR absorption spectra with peaks labeled with their corresponding pigments and normalized to the BPh peak at 775 nm. (B) Circular dichroism spectra normalized to BChl absorbance at 805 nm. (C and D) Selected ΔA spectra from time-resolved absorption spectroscopy of the RC-LH1₁₄-W complex (C) and RC-LH1₁₆ complex (D). For better comparability, all spectra are normalized to a ∆A of −1 at 0.2 ps. (E) The rate of cytochrome c₂ oxidation upon illumination in the presence of various concentrations of UQ₂ (see fig. S8 for raw data). (F) Ratios of protein-W and the RC-L subunit in purified complexes and isolated membranes from cells grown under low, medium or high intensity illumination (10, 30, or 300 μM m⁻² s⁻¹, respectively). Protein levels were determined by SDS–polyacrylamide gel electrophoresis and immunodetection (see fig. S9 for raw data). Ratios were determined relative to the purified RC-LH1₁₄-W complex, which has a 1:1 RC-L to protein-W stoichiometry.

Fig. 3. Structure of protein-W and interactions with LH1.

(A) Protein-W viewed facing the interface with LH1 αβ14 in cartoon representation with side chains as sticks (red) shown within its portion of the electrostatic potential map (transparent gray surface at a contour level of 0.13). (B) Protein-W in surface representation colored by hydrophobicity. Polar and charged regions are shown in cyan, hydrophobic regions are shown in white, and strongly hydrophobic regions are in orange. (C and D) Protein-W in cartoon representation in the same orientation as in (A) (C) and rotated 180° (D). Resolved residues are in a rainbow color scheme according to position in the sequence with the N terminus in blue and C terminus in red. (E) Protein-W in the same view as in (A) with residues at the protein-W:LH1 interface in stick representation with accompanying labels. (F) Protein-W rotated 90° relative to (E) with LH1 αβ14 in cartoon representation and interface residues in stick representation. Highlighted residues from the β polypeptide are labeled. Cofactors are shown as sticks with coloring matching Fig. 1, and the resolved β-DDM is shown in gray with oxygens in red. (G) The view in (F) rotated 180° with highlighted residues from the α polypeptide labeled.

Fig. 4. Density maps colored by local resolution as determined with the Relion local resolution tool.

The maps for RC-LH1₁₄-W (A and B) and RC-LH1₁₆ (C and D) are shown from the same top/side view in Fig. 1 (A and B) (A and C) and from the lumenal surface of the complex (B and D). The color key is shown on the right.

Fig. 5. Structurally defined lipids and quinones.

(A and B) RC-LH1₁₄-W (A) and RC-LH1₁₆ (B) polypeptides are shown in cartoon representation and pigments as sticks, using the color scheme in Fig. 1. Lipids are shown in red, and detergents are shown in gray. UQs bound to the RC Q_A and Q_B sites are in yellow, and sequestered UQs are in blue. (C and D) The same view as in (A) and (B) with the lipids omitted. (E to G) Zoomed views of Q1 (E), Q2 (F), and Q3 (G) from RC-LH1₁₆ with interacting side chains in stick representation. Hydrogen bonds are shown with a black dashed line.

Fig. 6. Conformational changes upon quinone binding to the RC Q_B site.

(A) Overlaid cartoons of holo (chain L, orange/chain M, magenta) and apo (gray) structures with key residues displayed in stick representation. UQ₁₀ is shown in stick representation in yellow. Dashed lines show hydrogen bonds formed in the holo structure. (B and C) Surface representations of apo and holo structures, respectively, with L-Phe²¹⁷ highlighted in blue and the side chain oxygen of L-Tyr²²³ in red. Subunit L is orange; and subunits M and H are not colored. (D and E) The apo (D) and holo (E) RC Q_B sites [colored as in (A)], respectively, aligned to the Thermosynechococcus vulcanus PSII (green with plastoquinone in blue; PDB ID: 3WU2) (58).