Competing charge transfer pathways at the photosystem II-electrode interface

Abstract

The integration of the water-oxidation enzyme photosystem II (PSII) into electrodes allows the electrons extracted from water oxidation to be harnessed for enzyme characterization and to drive novel endergonic reactions. However, PSII continues to underperform in integrated photoelectrochemical systems despite extensive optimization efforts. Here we carried out protein-film photoelectrochemistry using spinach and Thermosynechococcus elongatus PSII, and we identified a competing charge transfer pathway at the enzyme-electrode interface that short-circuits the known water-oxidation pathway. This undesirable pathway occurs as a result of photo-induced O₂ reduction occurring at the chlorophyll pigments and is promoted by the embedment of PSII in an electron-conducting fullerene matrix, a common strategy for enzyme immobilization. Anaerobicity helps to recover the PSII photoresponse and unmasks the onset potentials relating to the Q_A/Q_B charge transfer process. These findings impart a fuller understanding of the charge transfer pathways within PSII and at photosystem-electrode interfaces, which will lead to more rational design of pigment-containing photoelectrodes in general.

Figure 1. Schematic representation of the energy/charge transfer pathways within (A) PSII in the thylakoid membrane of a cyanobacterium; and (B) PSII adsorbed on a mesoporous indium-tin oxide (ITO) electrode connected to a photoelectrochemical cell.

The sequence of the water-oxidation pathway is given by the numbers in bold. The orange arrows represent energy transfer pathways resulting from light (hυ) absorption. The blue arrows represent electron transfer pathways taking place during oxygenic photosynthesis that give rise to the photoanodic currents detected by PF-PEC. The red arrows represent the competing O₂ reduction pathways that give rise to the photocathodic pathways detected by PF-PEC. Electron acceptors such as fullerenes (C₆₀) can enhance both the photoanodic and photocathodic pathways. The solid arrows represent known pathways including: energy transfer at the chlorophyll a (Chl a), charge generation/separation at the reaction center (RC), electron transfer via the pheophytin (Pheo), mediated Q_B/chemical mimic charge transfer,, direct electron transfer from Q_A to the electrode, hole transfer via the tyrosin (Tyr) and water oxidation at the Mn₄Ca cluster. The dashed arrows represent new pathways identified using protein-film photoelectrochemistry under a three-electrode configuration comprising of a working (W), counter (C) and reference (R) electrode. Note that the depiction of the photocathodic pathway at the antenna CP43 subunit is arbitrary.

Figure 2. PSII photocathodic currents stem from protein-bound and isolated Chl a pigments.

Stepped chronoamperometry scans of PSII core complexes isolated from T. elongatus (dark blue) and spinach (light blue); and spinach CP43 antenna subunits (orange), spinach RC complexes with the Mn₄Ca cluster depleted (red), Chl a molecules (green), and β-carotene (β-car) molecules (black) adsorbed on hierarchically structured ITO electrodes. Potentials steps of 0.1 V were scanned in the anodic direction. All experiments were performed in a MES buffer electrolyte solution (pH 6.5) under aerobic conditions at 25 °C with chopped light illumination (679 nm, 5 mW cm^–2). Representative photo-responses are shown and the applied potentials are referenced against the standard hydrogen electrode (SHE); dark current spikes caused by the potential changes were removed for clarity. The photoresponses were normalized to the concentration of Chl a deposited on the electrode (5 μg Chl a cm^–2 electrode surface area).

Figure 3. O₂ and the fullerene derivative, C60-DMePyI, are electron acceptors for photoexcited Chl a.

Photoresponse of (A) Chl a, (B) spinach PSII core complexes, and (C) T. elongatus PSII core complexes adsorbed on ITO electrodes in the presence (black traces) and absence (red traces) of a C₆₀-DMePyl matrix. Stepped chronoamperometry was performed under both aerobic (air, solid traces) and anaerobic (Ar purged, dashed traces) conditions in MES electrolyte buffer solution (pH 6.5) at 25°C with chopped red light illumination (679 nm, 5 mW cm^–2). Potentials steps of 0.1 V were scanned in the anodic direction and representative photoresponses are shown. (D) Proposed mechanisms of photocathodic current generation by Chl a* in the presence of O₂, with enhanced photocathodic currents in the presence of a C₆₀-DMePyl matrix.

Figure 4. Onset potential determination of (A) spinach PSII and (B) T. elongatus PSII adsorbed on ITO electrodes.

Photoresponse plots from stepped chronoamperometry scans performed in the anodic direction. Experiments were conducted in aerobic (air, grey trace), anaerobic (argon (Ar) purged, black trace) conditions; and in the presence of the Q_B inhibitor, 3’-(3,4-dichlorophenyl)-1’1’-dimethylurea (DCMU, blue trace) and the Q_B mimic, 2,6-dichloro-1,4-benzoquinone (DCBQ, red trace). All experiments were conducted in MES electrolyte buffer solution (pH 6.5) at 25°C with light illumination (679 nm, 5 mW cm^–2), and the photoresponses were normalized such that the photocurrent density at 0.6 V is equivalent to 100%. The error bars represent the standard error of the mean (n = 3).

Figure 5. Summary of the photoanodic (blue) and photocathodic (red) charge transfer pathways within PSII and at the PSII-electrode interface.

The applied potential (E_app) range used in this study is shown. Energy levels of materials and cofactors are based on numbers reported within references.,, The P_D1/RC is also commonly referred to as the P680. Grey bars represent uncertain energy levels. The dashed arrows represent pathways and energy levels identified by this study.