Transforming Escherichia coli Proteomembranes into Artificial Chloroplasts Using Molecular Photocatalysis

Abstract

During the light-dependent reaction of photosynthesis, green plants couple photoinduced cascades of redox reactions with transmembrane proton translocations to generate reducing equivalents and chemical energy in the form of NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate), respectively. We mimic these basic processes by combining molecular ruthenium polypyridine-based photocatalysts and inverted vesicles derived from Escherichia coli. Upon irradiation with visible light, the interplay of photocatalytic nicotinamide reduction and enzymatic membrane-located respiration leads to the simultaneous formation of two biologically active cofactors, NADH (nicotinamide adenine dinucleotide) and ATP, respectively. This inorganic-biologic hybrid system thus emulates the cofactor delivering function of an active chloroplast.

Keywords: Biocatalysis; Cofactors; Photocatalysis; Photosynthesis; Synthetic Biology.

Scheme 1

Schematic overview of the photobiocatalytic process connecting the photocatalytic NADH formation of 1 with the enzymatic ATP and G6P production using inverted E. coli vesicles as the central cofactor conversion machinery.

Figure 1

A) Ground‐state UV/Vis absorption (solid lines) and steady‐state emission spectrum (dotted lines) of the complexes 1 and 3 in DMSO. B) Photo‐independent NADH production using 1 (5 μM) and NaHCO₂ (100 mM) as chemical reductant. C) Photocatalytic reduction of NAD⁺ in the photobiocatalysis buffer, using complex 1 (20 μM). D) Evaluation of the in vitro ATP‐synthesis with inverted E. coli vesicles (650 μg mL⁻¹ total protein concentration) using a luminol‐luminescence assay. ATP synthesis was monitored at 560 nm in the presence (red curve) or the absence of vesicles (black curve). The black arrow indicates addition of NADH (500 μM final concentration) to initiate the vesicle respiration and the ATP synthesis. E) Emission of AO in the presence of vesicles upon initial addition of the respective substrate (ATP or NADH; 1.0 mM or 0.5 mM final concentration, respectively) or the pure buffer (black triangle) and subsequent addition of NH₄Cl (green triangle). F) Course of the AO fluorescence in the presence of 650 μg mL⁻¹ (total protein concentration) of inverted E. coli vesicles. As indicated with the first arrow, NADH addition (500 μM, final concentration) initiates respiratory activity of the vesicles. Addition of ADP, NH₄Cl or the pure buffer is indicated with the asterisk marked arrow. G) Course of the AO fluorescence after addition of NADH (500 μM) to 650 μg mL⁻¹ (total protein concentration; addition is indicated by the black arrows) of the inverted E. coli vesicles which were either used directly (black curve) or irradiated for 3 days at room temperature (red curve) using one LED‐stick (λ _max=465 nm, 45–50 mW cm⁻²). H) Emission of AO in the presence of an independently irradiated photocatalysis mixture with the subsequent addition of the vesicles (t=50 s). The photocatalysis mixture was irradiated for 2 h in the typical photobiocatalysis buffer (1 mM NAD⁺, 25 μM 1 (black trace) or 0 μM 1 (i.e. “blank”, orange trace)); the inset shows the emission resulting from the photogenerated NADH (λ _max≈460 nm) after these 2 h of irradiation. I) Course of formazan formation, detected by its absorbance at 492 nm. The mixture consisted of glucose (0.1 mM), hexokinase (1 U), G6P‐DH (1 U), NADP⁺ (0.5 mM), diaphorase (0.8 U) and INT (0.1 mM). The addition of ATP (0.5 mM, final concentration) is indicated by the black arrow and induces the enzymatic reaction cascade depicted in Scheme S1. J) Exemplary kinetic analysis of the photogenerated NADH by formazan absorbance at 492 nm after workup of the aliquots taken from the photobiocatalysis at the given irradiation time using 5 μM 1 as catalyst (see also Figures S5 and S10). K) Exemplary kinetic development of ATP concentration during the photobiocatalysis using 5 μM 1 as the catalyst (see also Figure S5). L) Comparison of the ATP output from different catalysis mixtures using 5 μM of catalyst 1.

Figure 2

A) and B) Changes of the luminescence intensity of complexes 3 (A) and 5 (B) (both 10 μM and dissolved in the photobiocatalysis buffer) upon addition of increasing volumes of the vesicle suspension (650 μg mL⁻¹). C) Comparison of the relative emission intensity increase of complexes 3 and 5 upon addition of the vesicles (650 μg mL⁻¹, N=2; the errors are small and are thus mostly overlayed by the data points). D) Evaluation of complex‐vesicle interactions by comparing the ground‐state UV/Vis absorption spectra of the respective complexes prior and after an 1 h long incubation at room temperature (see Figure S13 for detailed spectra, N=2). Bottom: Molecular structures of the complexes 2, 3 and 5. The phosphonic acid groups highlighted by blue color become deprotonated under the utilized buffer conditions.