Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells

Abstract

Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min^-1 Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

Keywords: artificial cells; giant lipid vesicles; light transduction; photosynthetic reaction center; proton gradient.

Fig. 1.

Preparation of GUVs by the droplet transfer method (48). (A) Water in oil (w/o) droplets, prepared by the emulsification of an aqueous solution (I-solution) in a lipid-rich oil phase, are transferred to an aqueous solution (O- solution) by centrifugation. (B) For preparing RC@GUVs, a detergent-stabilized RC solution (RC-micelles) is emulsified in oil, giving the w/o droplets. Owing to asymmetric RC-micelle structure a preferential “physiological” RC orientation is expected, namely, with the H subunit (in orange) facing toward the aqueous core of the droplets (the cytoplasm-like GUV lumen), and the photoactive dimer (SI Appendix, Fig. S2 A and B) facing the GUV exteriors (in white). (C) RC@GUVs (POPC:POPG, 9:1) as imaged by confocal microscopy. Red-fluorescent AE-RC was reconstituted in calcein-containing GUVs. (C1) Green fluorescence channel (calcein). (C2) Red fluorescence channel (AE-RC). (C3) Bright field. (C4) Overlay of the C1, C2, and C3 channels.

Fig. 2.

Charge recombination of RCs reconstituted in giant vesicles after a saturating light flash. The points represent the experimental data, and the lines are the biexponential best fit curves. Data refer to charge recombination in the absence (blue points) and in the presence (dark green points) of excess of reducing agent (cyt²⁺). In a control experiment (red points), a full recovery of RC photoactivity is measured after the addition of an electron acceptor, the dQ, and the exhaustion of cyt²⁺. Note that values in the y axis represent the absolute values of ΔA₈₆₅. (Inset) Theoretical charge recombination curves in the absence (blue) and in the presence (green) of cyt²⁺, corresponding to different RC orientation (100, 50, and 0% of physiological orientation). The histogram represents the initial amplitude of the curves ΔA₈₆₅(0). The green bar marked with the asterisk refers to the experimental trace reported in the main plot.

Fig. 3.

Scheme of RC@GUVs function under red-light illumination. (A) RC is reconstituted in a highly oriented manner (90%) in the membrane of GUVs, whose average diameter is 20 μm. The asterisk marks a nonphysiologically oriented RC. (B) Detail of the photochemical mechanism generating the pH gradient. hv, light energy.

Fig. 4.

Generation of a pH gradient by RC@GUVs. (A) Bulk fluorescence measurements of pyranine-containing RC@GUVs, which have been suspended in a fluorescence cuvette and illuminated from the top (SI Appendix, Fig. S9). Blue and red points refer to RC@GUVs with final RC concentration of 10 nM and 20 nM, respectively. Black lines represent the best-fit straight line, whose slopes are (2.64 ± 0.03) × 10⁻⁴ a.u.⋅min⁻¹ and (5.57 ± 0.03) × 10⁻⁴ a.u.⋅min⁻¹, respectively, for the blue and red datasets. (B) Confocal images of three pyranine-containing RC@GUVs illuminated with red light. (C) Quantitative image analysis reveals the increase of intravesicle pH in time (fluorescence values converted by means of a calibration; SI Appendix, section S2h). The best-fit slope is 0.061 ± 0.004 pH units per min. (D) Comparison between the experimentally observed pH increase in the aqueous core of giant vesicles: circles with error bars (as in C) and the theoretical outcomes (colored bands). a.u., arbitrary units.