Phycobilisomes supply excitations to both photosystems in a megacomplex in cyanobacteria

Abstract

In photosynthetic organisms, photons are captured by light-harvesting antenna complexes, and energy is transferred to reaction centers where photochemical reactions take place. We describe here the isolation and characterization of a fully functional megacomplex composed of a phycobilisome antenna complex and photosystems I and II from the cyanobacterium Synechocystis PCC 6803. A combination of in vivo protein cross-linking, mass spectrometry, and time-resolved spectroscopy indicates that the megacomplex is organized to facilitate energy transfer but not intercomplex electron transfer, which requires diffusible intermediates and the cytochrome b6f complex. The organization provides a basis for understanding how phycobilisomes transfer excitation energy to reaction centers and how the energy balance of two photosystems is achieved, allowing the organism to adapt to varying ecophysiological conditions.

Fig. 1. Schematic outline of the experimental work-flow established for the genetic modification, isolation, and preliminary characterization of the MCL

(A) Genetic modification of PsbO protein at C terminus [PsbOH strain (5)]. (B) Luminal side of the PSII monomer, indicating the solvent-accessible PsbO C terminus with His₆ tag (purple) introduction. PsbO is colored yellow; PsbU, orange; PsbV, light blue; and loop E of CP43, lime. (C) In vivo model of PBS and photosystems. NTA, nitrilotriacetic acid. (D) BN-PAGE (blue native polyacrylamide gel electrophoresis) analysis of isolated PSII (PsbOH), dimer (D), and monomer (M). (E) Ultracentrifugation isolation of MCL after affinity chromatography. (F and G) LC-MS/MS and TRF spectroscopy of the MCL.

Fig. 2. Identification of interprotein cross-links between PBS and two photosystems

(A) ApcE-PSII cross-links. The N-terminal domain of ApcE is the only phycobilin-attached region in linker proteins (Cys¹⁹⁰). Five cross-links were found between ApcE and PSII. PsbB, sky blue; PsbC, wheat; PsbD, light gray; PsbI, orange. All lysines (²²⁷K:PsbB, ⁴⁵⁷K:PsbC, ³⁵K:PsbI, and ²³K:PsbD) are represented as spheres. (B) Docking model of ApcD to PSI trimmer (cytoplasmic view) based on the identified cross-links presented in (C). PsaA, marine; PsaB, wheat; PsaC, lime; PsaD, light pink; PsaE, yellow; PsaF, gray; PsaL, cyan; ApcD, green; ApcA, light blue; ApcB, teal. (D and E) Close-up views of the model shown in (C) and PCB (red sticks). Cross-links were found between ⁴⁸K:ApcD (orange) and ¹¹K:PsaA, ¹⁷K:ApcB and ³⁰K:PsaA, ⁵⁸K:ApcB and ¹⁰K:PsaD, and ⁴⁹K:ApcD and ⁷⁶K:PsaD. Lysine residues from PSI, ApcD, and ApcB are presented as red, orange, and chocolate spheres, respectively.

Fig. 3. Time-resolved fluorescence of the MCL at 77 K, fluorescence decay analysis, and model of the PBS-PSII-PSI association in the MCL

(A) Two-dimensional profile of TRF recorded in 1-ns time windows after excitation at 550 nm. (B) Representative TRF spectra taken at various delay times after excitation. a.u., arbitrary unit. (C) Representative fluorescence decay traces taken at three distinct bands (PC, PSII, and PSI) accompanied with fits. IRF, instrument response function. (D) Rise and decay of fluorescence at 720 nm (PSI) recorded for the MCL and the isolated PSI. (E) The MCL model of the PBS-PSII-PSI association, showing that PSII is fully covered by close association with the PBS core, whereas PSI is associated with ApcD through a side-on orientation.