Structure of the light-harvesting bacteriochlorophyll c assembly in chlorosomes from Chlorobium limicola determined by solid-state NMR

Abstract

We have determined the atomic structure of the bacteriochlorophyll c (BChl c) assembly in a huge light-harvesting organelle, the chlorosome of green photosynthetic bacteria, by solid-state NMR. Previous electron microscopic and spectroscopic studies indicated that chlorosomes have a cylindrical architecture with a diameter of approximately 10 nm consisting of layered BChl molecules. Assembly structures in huge noncrystalline chlorosomes have been proposed based mainly on structure-dependent chemical shifts and a few distances acquired by solid-state NMR, but those studies did not provide a definite structure. Our approach is based on (13)C dipolar spin-diffusion solid-state NMR of uniformly (13)C-labeled chlorosomes under magic-angle spinning. Approximately 90 intermolecular C C distances were obtained by simultaneous assignment of distance correlations and structure optimization preceded by polarization-transfer matrix analysis. It was determined from the approximately 90 intermolecular distances that BChl c molecules form piggyback-dimer-based parallel layers. This finding rules out the well known monomer-based structures. A molecular model of the cylinder in the chlorosome was built by using this structure. It provided insights into the mechanisms of efficient light harvesting and excitation transfer to the reaction centers. This work constitutes an important advance in the structure determination of huge intact systems that cannot be crystallized.

Fig. 1.

Two-dimensional MAS ¹³C¹³C dipolar correlation NMR spectra of uniformly ¹³C-labeled chlorosomes from C. limicola are shown. (A) Chemical structure of BChl c isomers is shown. Numbers with prefix F are carbon atoms in the farnesyl chain. BChl c has two stereoisomers with chirality at the 3¹ position and homologues with different side chains at C8 and C12 as shown on the right. The head, middle, and tail parts are also indicated. The z-axis is perpendicular to the ring plane. (B) Two-dimensional ¹³C correlation spectrum with an rf-driven recoupling mixing time of 1.28 ms is shown. (C) Two-dimensional ¹³C correlation spectrum with a SPC-5 mixing time of 1.1 ms is shown. The red lines stand for negative contour levels. (D) Displayed are the connectivities of the doublet signals shown in a part of the 2D ¹³C NMR spectrum in B. Two individual connectivities for signals a and b are shown by solid and dashed lines, respectively, from C7¹ to C3¹, and are indicated by assignments with and without parentheses.

Fig. 2.

Two-dimensional proton-driven spin diffusion ¹³C¹³C dipolar correlation NMR spectra of uniformly ¹³C-labeled chlorosomes. (A) τ_mix = 4.8 ms. (B) τ_mix = 57.6 ms. (C) τ_mix = 244.8 ms. Cross-peaks between the two sets of sequentially connected signals are shown in C Inset. Arrows indicate cross-peaks whose intensities are shown in Fig. 3 A–D.

Fig. 3.

Build-up curves for ¹³C¹³C spin diffusion. Experimental (symbols) and simulated (lines) cross-peak intensities are presented. (A) Intensities of diagonal peaks for C7¹ (●) and C7 (▴). (B) Cross-peaks C4/C3¹ (●) and C19/C20 (▴). (C) Cross-peaks C9/C3¹ (●) and C10/C7¹ (▴). (D) Cross-peaks C5/C2¹ (●) and C5/C20 (▴). (E) Experimental ¹³C¹³C distance map for r_j,k^eff calculated from the polarization-transfer matrix on the BChl c assembly. Carbons are arranged in the order of the y coordinate defined in Fig. 1A. The diameters of circles are inversely proportional to the distances as shown by examples.

Fig. 4.

BChl c assembly models satisfying head–head, head–tail, and tail–tail contacts and the structure of the BChl c assembly determined under ¹³C¹³C distance constraints. (A) Parallel-dimer layers. (B) Uneven-dimer columns. (C) Antiparallel-dimer layers. (D) Antiparallel-monomer columns. Arrows in the structure indicate the coordination bonds between C3¹OH and Mg. Open rectangles represent BChl c rings. , ◇, and ▵ stand for the head–head, tail–tail, and head–tail contacts, respectively. The arrows on the right of A and C and those at the top of B and D indicate layers and columns, respectively. (E) A side view of the structure of the BChl c assembly. Solid and broken lines represent coordination and hydrogen bonds, respectively. Oxygen atoms are colored red. (F) A top view of the piggyback dimer. (G) A top view of the interdimer full stacking at the center of E. (H) A top view of the intercolumn contact between neighboring dimers. The coordinates of BChl c molecules are given in the legend to SI Fig. 7.

Fig. 5.

A rod element structure built with the parallel dimer layers shown in Fig. 4 E–H. (A) A top view. Magnesium atoms are colored dark green. Columns perpendicular to the page are arranged on the circumference. (B) A side view. The cylindrical structure consists of ≈1,500 molecules. A single spiral layer and a single column are colored green. A layer consisting of 25 dimers makes one spiral rotation. Twenty-nine full stacking dimers (Fig. 4 F and G) constitute the green column along the cylinder axis. The arrows represent the Q_y transition dipole moments of BChls. The Q_y transition dipolar vectors form an angle of 46° with the cylinder axis and an angle of 236° with the radius vector connecting Mg and the cylinder axis. (C) Schematic representation of the excitation energy transfer in a chlorosome. The green lines with arrows indicate the paths of the excitation transfer along the spiral layers to the baseplate shown in B. The model structure was taken from ref. .