Specificity of the cyanobacterial orange carotenoid protein: influences of orange carotenoid protein and phycobilisome structures

Abstract

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome (PB), the extramembranous light-harvesting antenna. This mechanism is triggered by the photoactive Orange Carotenoid Protein (OCP), which acts both as the photosensor and the energy quencher. The OCP binds the core of the PB. The structure of this core differs in diverse cyanobacterial strains. Here, using two isolated OCPs and four classes of PBs, we demonstrated that differences exist between OCPs related to PB binding, photoactivity, and carotenoid binding. Synechocystis PCC 6803 (hereafter Synechocystis) OCP, but not Arthrospira platensis PCC 7345 (hereafter Arthrospira) OCP, can attach echinenone in addition to hydroxyechinenone. Arthrospira OCP binds more strongly than Synechocystis OCP to all types of PBs. Synechocystis OCP can strongly bind only its own PB in 0.8 m potassium phosphate. However, if the Synechocystis OCP binds to the PB at very high phosphate concentrations (approximately 1.4 m), it is able to quench the fluorescence of any type of PB, even those isolated from strains that lack the OCP-mediated photoprotective mechanism. Thus, the determining step for the induction of photoprotection is the binding of the OCP to PBs. Our results also indicated that the structure of PBs, at least in vitro, significantly influences OCP binding and the stabilization of OCP-PB complexes. Finally, the fact that the OCP induced large fluorescence quenching even in the two-cylinder core of Synechococcus elongatus PBs strongly suggested that OCP binds to one of the basal allophycocyanin cylinders.

Figure 1.

Schematic orthogonal projections of the various PB cores. In the PBs containing three or five cylinders, the top complete cylinder is formed by four αAPC-βAPC trimers emitting at 660 nm. Each of the basal cylinders of three types of PBs contains two αAPC-βAPC trimers emitting at 660 nm and two trimers emitting at 683 nm. In one of them, one αAPC is replaced by ApcD, and in the other one, αAPC-βAPC is replaced by the dimer ApcF-ApcE. In the five cylinder PBs, two additional semicylinders formed by two αAPC-βAPC trimers are present. In all the cylinders, the two external trimers include an 8.7-kD linker protein (ApcC).

Figure 2.

Composition analysis of the various PBs. A, Polypeptide composition of the isolated Anabaena PBs (Ana), Synechococcus PBs (Sus), and Synechocystis PBs (Sis). L, Ladder. B, Polypeptide composition of the isolated Synechocystis PBs, Arthrospira PBs (Art), and Anabaena PBs. C and D, Room-temperature absorption spectra of the PBs isolated from Synechocystis (solid orange line) and Arthrospira (dashed blue line; C) or Synechococcus (solid pink line) and Anabaena (dashed green line; D). Spectra are normalized at the maximum of absorbance around 620 nm. a.u., Absorbance units. [See online article for color version of this figure.]

Figure 3.

Room temperature (A and C) and 77 K (B and D) fluorescence emission spectra of the isolated PBs. A and B, Synechocystis PBs (solid lines) are compared with Arthrospira PBs (dashed lines). C and D, Synechococcus PBs (solid lines) are compared with Anabaena PBs (dashed lines). Spectra are normalized to the maximum of emission. Excitation was at 590 nm. a.u., Absorbance units.

Figure 4.

Fluorescence quenching induced by Synechocystis OCP in vitro. The PBs (0.012 µm) were illuminated with blue-green light (900 µmol m⁻² s⁻¹) in the presence of an excess of preconverted Synechocystis OCP^r (0.48 µm; 40 per PB) at 23°C and 0.8 m potassium phosphate. Fluorescence decrease was measured using a PAM fluorometer for Synechocystis PBs (orange crosses), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), and Anabaena PBs (green diamonds). [See online article for color version of this figure.]

Figure 5.

Isolation of Arthrospira OCP. A, Immunoblot detection using a primary antibody directed against Arthrospira OCP on whole cell extracts of wild-type (lane 1), oApOCPWT (lane 2), ΔCrtR (lane 3), oApOCPΔCrtR (lane 4), and oSynOCPΔCrtR (lane 5) cells. Three micrograms of chlorophyll was deposited per well. B, Absorbance spectra of Arthrospira OCP^o (orange solid line) and OCP^r (red dashed line) isolated from oApOCPWT cells. [See online article for color version of this figure.]

Figure 6.

Light-driven photoconversion and dark recovery of Arthrospira OCP. Arthrospira OCP^o (1.8 µm) was illuminated using strong white light (5,000 µmol m⁻² s⁻¹), and its A₅₅₀ was recorded over time. A, In 40 mm Tris-HCl, pH 8, Synechocystis OCP (closed symbols) and Arthrospira OCP (open symbols) were compared at 23°C (squares) or 9°C (circles). Data were normalized to the final percentage of the red form in each condition. B, Arthrospira OCP in 40 mm Tris-HCl (blue circles), 0.8 m potassium phosphate (red triangles), or 1.4 m potassium phosphate (green squares) during its photoconversion at 23°C. Data were normalized to the final percentage of the red form at 9°C. C, After Arthrospira OCP photoconversion, the light source was turned off, and A₅₅₀ evolution was followed at 9°C with (blue squares) or without (red circles) FRP addition (one per two OCPs). Recovery was also followed at 23°C (black triangles). Data were normalized to the initial A₅₅₀. [See online article for color version of this figure.]

Figure 7.

Fluorescence quenching induced by Arthrospira OCP in vitro and in vivo. A, Fluorescence quenching triggered by strong blue-green light (1,400 µmol photons m⁻² s⁻¹) in Synechocystis wild-type (orange open circles), oSynOCPΔCrtR (cyan open triangles), oApOCPWT (brown closed squares), and oApOCPΔCrtR cells (purple closed circles) cells at 33°C. F_m′, Maximum PSII fluorescence in the light-adapted state. B, Isolated Synechocystis PBs were illuminated with blue-green light (900 µmol m⁻² s⁻¹) in the presence of an excess of preconverted Synechocystis OCP^r (orange triangles) or Arthrospira OCP^r (blue squares; 0.48 µm; 40 per PB) at 0.8 m potassium phosphate, 23°C. C, Fluorescence quenching induced by strong blue-green light (900 µmol m⁻² s⁻¹) at 0.8 m potassium phosphate in the absence (open symbols) or the presence (closed symbols) of an excess of Arthrospira OCP^r in Synechocystis PBs (orange circles), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), and Anabaena PBs (green diamonds). D, Fluorescence recovery in darkness of “quenched” PBs. The light was turned off after 300 s of illumination. Symbols are as in C. [See online article for color version of this figure.]

Figure 8.

Effect of potassium (K) phosphate concentration on PB fluorescence quenching. PBs (0.012 µm) were illuminated with blue-green light (900 µmol m⁻² s⁻¹) in the presence of preconverted OCP^r (0.48 µm; 40 per PB) at 23°C. A, Percentage of Arthrospira PB fluorescence quenching reached after 5 min of illumination in the presence of Synechocystis OCP^r (black bars) or Arthrospira OCP^r (white bars) in increasing potassium phosphate concentrations from 0.8 to 1.6 m. B, Fluorescence decrease induced by Synechocystis OCP^r in Synechocystis PBs (orange crosses), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), and Anabaena PBs (green diamonds) at 1.4 m potassium phosphate. C, Fluorescence decrease induced by Arthrospira OCP^r in Synechocystis PBs (orange crosses), Arthrospira PBs (blue squares), Synechococcus PBs (red triangles), or Anabaena PBs (green diamonds) at 1.4 m potassium phosphate. [See online article for color version of this figure.]

Figure 9.

Comparison of OCP-related fluorescence quenching in Arthrospira and Synechocystis cells. A, Fluorescence quenching triggered by strong blue-green light (1,400 µmol photons m⁻² s⁻¹) in Synechocystis wild-type (red closed squares) and Arthrospira (black open circles) cells at 33°C. F_m′, Maximum PSII fluorescence in the light-adapted state. B, Coomassie blue-stained gel electrophoresis (top) and immunoblot detection (bottom) of the OCP protein in Synechocystis wild-type cells (lane 1), Arthrospira cells (lane 2), and membrane-PB fractions (prepared as described by Wilson et al. [2006]) obtained from Arthrospira (lane 3) or Synechocystis (lane 4). Each lane contains 2 µg of chlorophyll. C, Room temperature absorbance spectra of Synechocystis wild-type (solid red line) and Arthrospira (dashed black line) cells. Cells were diluted to 3 µg chlorophyll mL⁻¹. a.u., Absorbance units. [See online article for color version of this figure.]

Figure 10.

Structural differences between Arthrospira OCP and Synechocystis OCP. A, Overview of the A. maxima OCP structure. Teal, N-terminal domain; green, linker region; red, C-terminal domain. 3′ hECN is shown in orange. The amino acids changing between Arthrospira OCP and Synechocystis OCP are represented using purple sticks, and blue sticks are used for the ones bearing charges (and for Tyr-171). B, Electrostatic surface maps of Arthrospira and Synechocystis OCPs. For details, see “Materials and Methods.” SB, Salt bridge between Arg-155 and Glu-244(246).