Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures

Abstract

Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 μA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 μA/cm2) from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 μA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants.

Fig 1. Cyt c is reduced by PSII in Syn but not in spinach thylakoids.

A. Schematic illustration of the postulated electron transfer pathway from PSII in Syn thylakoid to cyt c_ox. Photo-oxidized electrons are postulated to be transferred from Q_A to cyt c. The addition of DCMU blocks linear electron flow between the Q_A and Q_B and thus increases the reduction rate. B. Stacked spinach thylakoids were incubated for 3 min with cyt c_ox in the dark (black dotted), light (green) or in the light with the addition of DCMU (red dashed). Following centrifugation of the membranes, the absorption spectra of the cyt c containing supernatant was measured. The concentration of reduced cyt c was calculated using the coefficient Δϵ550nm-542nm. The inset depicts the quantification of cyt c photoreduction from three independent experiments in the absence (green) or presence of DCMU (red). C. Analysis of cyt c photoreduction at different levels of spinach PSII isolation. Unstacked spinach thylakoids (Thylakoid, solid green), PSII enriched membranes (BBY, dashed blue) or purified PSII (PSII, dotted brown) were incubated and analyzed as described for panel A. The inset depicts the quantification of cyt c photoreduction by the unstacked thylakoids (green bar) PSII enriched membranes (blue bar) or purified PSII (brown bar) from three independent experiments. No photoreduction occurred in the presence of DCMU.

Fig 2. Syn thylakoids transfer a direct photocurrent from Q_A while higher plants require the presence of an electron transfer mediator.

A. Schematic illustration of the photocell used for photocurrent measurement. A suspension of thylakoid membranes were allowed to settle onto a graphite anode placed at the bottom of a sealed container in a minimal amount of buffer B. The electrode was illuminated from the top by a xenon lamp calibrated to provide an equivalent spectrum of 1 sun. Currents were measured by a potentiostat connected in 3-electrode mode to an Ag/AgCl reference electrode and to a Pt cathode. B. Direct photocurrent produced by wild-type-Syn (solid lines) or spinach (dashed lines) thylakoids illuminated on the graphite anode in the absence (blue) or presence (red) of 50 μM DCMU under a bias of 0.05V_Ag/AgCl. Up and down facing arrows indicate light on and off, respectively. C. DCBQ mediated photocurrent produced by spinach thylakoids on the graphite anode in the absence (blue), the presence of 50μM (green) and of 500 μM DCMU (red) under a bias of 0.24 V_Ag/AgCl. D. Photocurrent produced by isolated spinach PSII on the graphite anode with (dotted lines) or without (solid lines) 0.3mM DCBQ, in the absence (blue) or presence (red) of 50 μM DCMU as in panel B. The inset schematically depicts the role of DCBQ as electron mediator. DCBQ_OX diffuses into the Q_B pocket, and is reduced by Q_A ^- (DCBQ_red). DCBQ_red then diffuses to the anode where it is re-oxidized to become available for another electron transfer cycle.

Fig 3. Modification of the conserved amino acid at position 238 of the D1 protein increased electron abstraction from Q_A in Syn but not in tobacco.

A. Quantification of cyt c (cc) photoreduction by thylakoids of WT-aadA tobacco, WT-Syn, Syn D1-K238E or tobacco D1-R238E. In the absence (green) or following the addition of the herbicide DCMU (red). DCMU enhanced electron transfer by Syn thylakoids, but completely blocked electron transfer in tobacco. Data was collected in three independent experiments. Bars represent standard error. B. Oxygen evolution rates monitored as a measure of the light-dependent electron transfer from the PSII oxygen evolving center of mutated Syn and tobacco (K238E Syn, R238E tobacco, respectively). The exogenous electron acceptors used were DCBQ in the absence of DCMU (blue bars) or cyt c in the presence of the herbicide DCMU (grey bars).

Fig 4. A mediator is required in order to produce photocurrent with tobacco thylakoids.

A. Syn thylakoids transfer electrons directly to the electrode (black, no mediator added). The addition of DCBQ (red) resulted in inhibition of the photocurrent. Direct and DCBQ-mediated photocurrents produced by thylakoid of WT (dashed lines) or K238E (solid lines) strains of Syn were measured on a graphite-working electrode under external bias of 0.05V_Ag/AgCl and 0.24V_Ag/AgCl, respectively. The inset illustrates the schematic representation of the electron abstraction pathways from the Syn thylakoids. Thin and thick arrows indicate the magnitude of electron transfer, respectively. B. Unmediated (orange) and DCBQ-mediated (blue) photocurrent obtained from WT-tobacco (dashed lines) or R238E-tobacco (full lines) as described in panel A. Inset illustrated the electron abstraction pathways from the tobacco lines.