Using MEPS to Generate Photosynthetic Electron Transport in Cyanobacteria

By Laura ThomsonMar 8 2022

Cyanobacteria have evolved complicated mechanisms to transform light energy into chemical energy during three billion years of existence. Cyanobacteria produce reducing power (NADPH) and chemical energy (ATP) for carbon dioxide fixation using only the most basic of ingredients: light and water.

Image Credit: Alexlky/Shutterstock.com

Photosynthesis can be hampered by a process known as photoinhibition at greater light intensities (LI). Despite decades of research highlighting cyanobacteria’s potential, photoinhibition is one element that reduces their appeal as a source of low-cost organic goods and biofuels.

Photosystem II (PSII) and photosystem I (PSI) are the two primary light-activated complexes that regulate the photosynthetic process (PSI). The photosynthetic linear electron transport chain (PETC) is depicted in Figure 1 along with the electrochemical potential of the chain’s components. According to standard hydrogen potential (SHE), all electrochemical potentials presented are midpoint potentials (Eo′, V) at pH 7.

Figure 1. Schematic of the biological components of the photosynthetic electron transport chain (PETC) juxtaposed with the corresponding midpoint potentials versus standard hydrogen electrode (SHE) potentials with green arrows representing electron flow and blue arrows representing proton movement. As depicted in red, the PSII D1 subunit is subject to damage by reactive oxygen species when charge separation does not move forward fast enough in the presence of oxygen, producing singlet oxygen. PSII may be replaced with an electron delivery from duroquinol to the plastoquinone (PQ) pool. Image Credit: Lewis, et al., 2022

PSII is genetically eliminated from the cells in this study, and researchers employ a cathode-mediator device to shuttle electrons between the cell and the electrode, preventing PSII’s oxidative-photo damage, which is an extension of the research team’s earlier intellectual property.

Since this strategy needs the delivery molecule to be isoelectrically comparable to QB (−0.12 V) or at least have a potential more negative than the cyt b₆f pocket (0.30 V), the yellow arrow in Figure 1 denotes a targeted position for an alternate electron source.

Methodology

Figure 2 shows the MEPS reactor, which was a normal three-electrode H-cell reactor. An Ag/AgCl reference electrode (BASi) and a cathode were on one side.

Figure 2. H-cell reactors used for MEPS where the cathode measured light-dependent current density via chronoamperometry (−0.249 V). The ΔpsbB cells in the cathodic chamber acted as a bioanode. The black device represents the laboratory lamps used for exposing reactors to light. Image Credit: Lewis, et al., 2022

The incident light intensity was changed as needed for the experiment by placing a light source directly in front of the glass reactors/cathode.

A MEPS reactor was cleaned, disinfected via autoclaving or UV exposure, and then installed in a laminar flow hood under strict sterile conditions in preparation for an investigation.

CV was first employed in the MEPS reactors to calculate the cathode potential setting that would be used in chronoamperometry tests in BG-11 with the cells present.

The ability of the mediator to transfer electrons intercellularly to the PETC was confirmed using Joliot-type spectroscopy (JTS) with Biologic JTS-10 equipment.

Results and Discussion

To figure out how to artificially stimulate electron flow in the absence of PSII, MEPS was investigated in the presence and absence of living cells that lacked PSII. These findings show that Synechocystis without PSII can behave as a bioanode by receiving electrons from an external cathode. Figure 3 shows an example of light/dark cycles with a LI of 940 μmol photons m^–2 s^–1.

Figure 3. Electrochemical characterization of MEPS is shown. Chronoamperometry measurements (at a bias of −0.249 V vs SHE) of DQH₂-mediated ΔpsbB cells were recorded for 3 h light/dark cycles, where the current density reached −10 μA cm^–2 for each light cycle and delivered 15 μmol of electrons. The baseline (0.07 μA cm^–2), established with DQH₂ and ΔpsbB cells for a 7 h period in the dark, was subtracted out to reflect a starting value of zero. Mediatorless live ΔpsbB (green), mediatorless heat-killed ΔpsbB (blue), and heat-killed ΔpsbB cells with DQH₂ added (gray) generated negligible response. Image Credit: Lewis, et al., 2022

Under a bias of −0.249 V, findings from 940 mol photons m^–2 s^–1 light exposure (Figure 3) show that consistent results may be achieved during the first two light/dark cycles.

To acquire a better grasp of how PSI can work in the absence of PSII, researchers tested MEPS under light circumstances when photodamage and downregulation are known to occur. Figure 4 illustrates this.

Figure 4. (a) Individual light/dark cycle examples for four Lis: 150 (orange), 320 (brown), 940 (teal), and 2050 (black) μmol photons m^–2 s^–1 normalized to mg-chl (at −0.249 V vs SHE). (b) Triplicate studies determining the MEPS light-activated electron delivery rate in 50 mL reactors at maximum current density levels (which reached those points at different times). The following Lis were tested: 150, 320, 940, and 2050 μmol photons m^–2 s^–1 and resulted in electron delivery rates of 24, 35, 70, and 113 μmol e h^–1 mg-chl^–1, respectively. Image Credit: Lewis, et al., 2022

As illustrated in Figure 4b, triplicate investigations compared the maximal electron delivery rate generated during the first three light cycles.

For the first three light cycles, Figure 5 compares current densities after a 1.5-hour exposure to light (within the first 24 hours). Figure 6 depicts the rates of JTS-based re-reduction.

Figure 5. Bar diagram depicting triplicate results of current density generated in the first 1.5 h of three consecutive light cycles for four light intensities (at −0.249 V vs SHE): 150 (orange), 320 (gray), 940 (teal), and 2050 (black) μmol photons m^–2 s^–1. Image Credit: Lewis, et al., 2022

Figure 6. Joliot-type spectroscopy absorbance kinetics showing the re-reduction rate (absorbance units per s normalized to chlorophyll = a.u. s^–1 mg-chl^–1) of P700⁺ → P700 when exposed to 940 μmol photons m^–2 s^–1 of actinic light. Samples tested: ΔpsbB cells with reduced mediator (burgundy), ΔpsbB cells with oxidized mediator (blue), ΔpsbB cells lacking mediator (violet), PSII-less cells with reduced mediator with DBMIB, a cyt b₆f inhibitor (orange) and wild-type cells (green). Image Credit: Lewis, et al., 2022

Conclusion

MEPS reactors were created with the goal of allowing photosynthetic electron transport to occur without being hampered by PSII photodamage. Figure 7 depicts the interpretation of the system’s operation.

Figure 7. Schematic of the microbial electro-photosynthetic system (MEPS) with green arrows representing electron flow and blue arrows representing proton movement. (1) Molecular mediator (duroquinone to duroquinol) reduction at the cathode −0.249 V vs SHE). (2) Live ΔpsbB cells act as bioanodes where the oxidation of the mediator occurs. (3) Within the cells, at the interface of the thylakoid membrane, duroquinol mediators donate electrons to (4) the cyt b₆f complex, releasing protons into the luminal space and passing electrons further down the chain; (5) cytochrome c₅₅₃ or plastocyanin then transport the electrons to Photosystem I. (6) Upon exposure to light, PSI catalyzes the light-driven electron transfer from P700 to the cytoplasmic site of the membrane where it reduces (7) ferredoxin (FD) that shuttles electrons to FNR for the reduction of NADP⁺ to NADPH. A proton gradient is generated in the process, which drives ATP synthesis. If NADP⁺ is not available electrons will be shuttled back into the PETC via cyclic electron flow (CEF) to the cyt b₆f complex or may be dissipated via Flv1/Flv3 that reduces enzymes or redox regulation pathways in the cell (for clarity not depicted in the figure). Image Credit: Lewis, et al., 2022

The MEPS cathode started photosynthesis in the first stage by electrochemically controlling the reduction of quinone-type redox mediators in the bioreactor’s bulk medium. Exogenous mediators were able to effectively feed electrons to the thylakoid interface at rates comparable to or higher than PSII in wild-type cells and were provided to the PETC prior to cyt b₆f, enabling proton pumping and ATP synthesis, according to JTS data.

These findings suggest that MEPS could be utilized to improve PETC at high light intensities (for example, for biotechnological applications), as well as to investigate additional photoinhibitory processes in the lack of PSII, as well as light-driven electron flow via PSI without PSII confounding effects.

Journal Reference

Lewis, C. M., Flory, J. D., Moore, T. A., Moore, A. L., Rittmann, B. E., Vermaas, W. F. J., Torres, C. I., Fromme, P. (2022) Electrochemically Driven Photosynthetic Electron Transport in Cyanobacteria Lacking Photosystem II. Journal of the American Chemical Society, 144(7), pp. 2933–2942. Available Online: https://pubs.acs.org/doi/10.1021/jacs.1c09291.

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