Redesigning the QA binding site of Photosystem II allows reduction of exogenous quinones

Abstract

Strategies to harness photosynthesis from living organisms to generate electrical power have long been considered, yet efficiency remains low. Here, we aimed to reroute photosynthetic electron flow in photosynthetic organisms without compromising their phototrophic properties. We show that 2,6-dimethyl-p-benzoquinone (DMBQ) can be used as an electron mediator to assess the efficiency of mutations designed to engineer a novel electron donation pathway downstream of the primary electron acceptor Q_A of Photosystem (PS) II in the green alga Chlamydomonas reinhardtii. Through the use of structural prediction studies and a screen of site-directed PSII mutants we show that modifying the environment of the Q_A site increases the reduction rate of DMBQ. Truncating the C-terminus of the PsbT subunit protruding in the stroma provides evidence that shortening the distance between Q_A and DMBQ leads to sustained electron transfer to DMBQ, as confirmed by chronoamperometry, consistent with a bypass of the natural Q_A°^- to Q_B pathway.

Figure 1. Efficiency of electrons extraction for three quinone derivatives in the ΔpetA strain lacking the cytochrome b₆f complex.

The steady-state fluorescence yield (F_S) was measured after ∼2.6 s of illumination at 26 μmol photons m⁻² s⁻¹, while the maximum fluorescence (F_M) was measured after a saturating light pulse (250 ms). Variations in (F_M−F_S)/F_M values, related to the PSII photochemical efficiency, were used to estimate the efficiency of electrons extraction (mean±s.d.; n=3). The restoration of PSII photochemical efficiency in the ΔpetA strain by the exogenous quinones indicates their ability to act as electron acceptors. The empty circle and the dashed line show the (F_M−F_S)/F_M value in the WT reference strain in the absence of exogenous quinones under the same light condition (mean±s.d.; n=10).

Figure 2. Ability of three quinone derivatives to deplete electrons from Q_B°⁻ and/or PQ²⁻ in the WT strain using chlorophyll fluorescence analysis.

(a) Effect of quinone derivatives on the ratio of initial fluorescence in the presence of 10 μM DCMU (F_i) to that in its absence (F₀) (mean±s.d.; n=3). Addition of DCMU induces a rise of the initial fluorescence level that reflects the residual electron accumulation in the Q_B site or the plastoquinone pool in the dark. (b) Stern–Volmer plot of the maximum fluorescence yield (F_M) showing the fluorescence quenching effect of exogenous quinones (mean±s.d.; n=3). F_M([Q]=0) represents the F_M value in the absence of quinones.

Figure 3. Efficiency of electron extraction for three quinone derivatives in the WT strain using light-induced absorbance changes of P₇₀₀.

(a) P₇₀₀ reduction ratio as a function of exogenous quinone concentration (mean±s.d.; n=4). P₇₀₀ redox states were estimated from the absorbance changes at 705−730 nm. The P₇₀₀ reduction ratio was calculated as 1 minus the oxidation level reached after a 10 s illumination (26 μmol photons m⁻² s⁻¹) over the level of full P₇₀₀⁺ oxidation after a saturating light pulse. Decreases in the P₇₀₀ reduction ratio indicate a reduced electron flux towards P₇₀₀⁺ in the presence of exogenous quinones. (b) P₇₀₀ reduction ratio as a function of exogenous quinone concentration in the presence of 10 μM DCMU (mean±s.d.; n=3). (c,d) P₇₀₀ reduction rate (k_red), normalized to the rate measured in the absence of quinones (k_red(0)), as a function of exogenous quinone concentration, derived from the data of (a,b), respectively. The normalized k_red/k_red(0) ratio is particularly useful to compare strains or conditions that have different basal levels of P₇₀₀ reduction ratio, as seen here after DCMU treatment.

Figure 4. Peptide environment of the Q_A binding pocket in C. reinhardtii highlighting putative targets for site-directed mutagenesis.

The structure of PSII and its mutated variants was based on the PDB entry 3ARC (ref. 26) and built with the SWISS-MODEL workspace. (a) Three dimensional structure of the Q_A binding pocket of PSII. The targets for point mutation or truncation in D2 are marked in orange rectangles, and the C-terminal ends of CP43 and PsbT are highlighted in blue and pink rounded rectangles, respectively. (b) Surface structure of PSII upon truncation of CP43 or PsbT. Red outlines highlight the truncated region of CP43 (upper panel) or PsbT (lower panel). On each view, the number shows the shortest distance measured between the conjugated ring of Q_A and the accessible surface of PSII. It is of note that the I30 and K31 residues of PsbT are not shown as they are absent in the crystal structure. (c) Illustration of the 24-RDPP-27 peptide of PsbT and its surroundings. Positively and negatively charged amino acids are circled in red and blue, respectively. Arrows represent putative electrostatic interactions between amino acids of opposite charges. (d) Substitution of the 24-RDPP-27 peptide for GGAG results in loss of electrostatic interaction of this part with its neighbouring subunits. The number shows the shortest distance between the conjugated ring of Q_A and the water-excluded surface of PSII, determined from the model structures.

Figure 5. Accumulation of photosynthetic complexes in the mutant strains.

Cells were grown in TAP medium at 25 °C under LED white light (8 μmol photons m⁻² s⁻¹) and collected at the mid-log phase. Two independent lines are shown for each construct. Protein samples were loaded on an equal chlorophyll basis (0.5 μg per lane), and a dilution series of WT samples is shown for semi-quantitative comparison. Antibodies against essential subunits of PSII (D2), PSI (PsaA), cytochrome b₆f (PetA) and ATP synthase (AtpB) probed the accumulation of the respective photosynthetic complexes. Numbers on the left side of the blots are molecular weights in kD. See Supplementary Fig. 9 for the uncropped blot images.

Figure 6. Comparison of the efficiency of electron extraction by DMBQ in the mutant strains.

The efficiency of electron extraction was measured in the psbD (a), psbT deletion (b), psbT-C-terminus (c) and psbC-C-terminus (d) mutant strains by the normalized k_red/k_red(0) ratio determined from the P₇₀₀ absorbance changes shown in Supplementary Fig. 3. Asterisks indicate a statistically significant difference with respect to the corresponding aadA control strain or a significant difference with respect to WT for the ΔpsbT strain using a two-tailed t-test. *: 0.001<P<0.05, **: P<0.001. Error bars are s.d. of at least three independently performed experiments.

Figure 7. Evidence of electron transfer from Q_A°⁻ to DMBQ in the psbT mutant strains.

(a) Direct electron transfer from Q_A°⁻ to DMBQ as revealed by chlorophyll fluorescence analysis under dim light in the psbT mutant strains. The ratio of (F_M−F_S)/F_M normalized to the level in the absence of DMBQ was measured in the presence of 10 μM DCMU at actinic light of 0.6 μmol photons m⁻² s⁻¹. The larger normalized (F_M−F_S)/F_M ratio in these mutant strains relative to the aadA control strain suggests that additional electron transfer from Q_A°⁻ to DMBQ competes with fluorescence. Asterisks indicate a statistically significant difference from the level of the aadA control strain using a two-tailed t-test. *: 0.001<P<0.05, **: P<0.001 (mean±s.d.; n=6). (b) Chronoamperometic measurements to estimate the sustained electron transfer from Q_A°⁻ to DMBQ in the presence of DCMU. The slope of current rise, which relates to the reduction rate of DMBQ, was estimated at 5 and 15 min after the addition of DCMU and normalized to the slope at 2.5 min before the DCMU addition (see Methods for details). Asterisks indicate a statistically significant difference from the level of the aadA control strain using a two-tailed t-test. *: 0.001<P<0.05, **: P<0.001. Error bars are s.d. of at least four independently performed experiments.

Figure 8. Pre-incubation with DCMU supports a direct electron transfer from Q_A°⁻ in the psbT(Δ24–31) mutant strain.

(a) A pre-incubation with DCMU (10 μmol l⁻¹) in the psbT(Δ24–31) mutant strain does not fully inhibit the production of a photo-induced current (illumination is indicated by the red horizontal bar) due to the electron transfer between PSII and DMBQ. (b) The current recorded in the control strain is fully inhibited by a pre-incubation with DCMU.