Alternating electron and proton transfer steps in photosynthetic water oxidation

Abstract

Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel-production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese-calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S(2) → S(3) transition of the classical S-state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant (Y Z OX). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein-water interface is characterized by a high activation energy (E(a) = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S(0) → S(1) transition are similar (τ, approximately 100 µs; E(a) = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.

Fig. 1.

Photosystem II (A) and reaction cycle of water oxidation (B). In A, crucial redox cofactors and dimensions of the PSII complex are shown (15). Red arrows connect redox cofactors of the ET chain, including the primary electron donor (P₆₈₀), the primary pheophytin acceptor (Phe), the primary (Q_A) and secondary (Q_B) quinone acceptors, and, at the electron donor side, a redox-active tyrosine (Y_Z) and the Mn complex. Water molecules resolved in the crystallographic model (Protein Data Bank entry 3ARC; ref. 15) are shown as red dots; the indicated distances illustrate relevant dimensions. In B, the classical Kok model (16) (inner circle, including states S₄ and S₄′; ref. 22) is extended to describe both oxidation of the Mn complex by ET to the Y_Z radical and proton removal from the Mn complex or its ligand environment by long-distance proton transfer. Coupling of the ET step to local proton shifts is not covered by the shown framework model. The subscripts indicate the number of oxidation equivalents accumulated at the Mn complex; the superscripts indicate the charge relative to the dark-stable S₁-state (+, positive; n, neutral). The proton release steps in the S₀ → S₁ and S₂ → S₃ transitions have not been tracked in time-resolved experiments before, but now these steps are detected in the PBD experiments; the indicated time constants result from the present study.

Fig. 2.

Flash-induced PBD signals and volume changes: S₁ → S₂ (A, A^′), S₂ → S₃ (B, B^′), (C, C^′), and S₀ → S₁ (D, D^′). Thin lines, experimental data; thick lines, simulations using a step-shaped function for the rapid jump caused by formation and single-exponential functions for the slower signal contributions. (Right) Schematic illustration of volume changes deduced from the analysis of the temperature dependence of the PBD signals (time constants for about 20 °C; see Fig. 3).

Fig. 3.

Temperature dependence of the PBD signals of the four resolved transitions. (A) Temperature dependence of the amplitudes as obtained by an exponential simulation (symbols, experimental data; lines obtained by a fit). The dotted lines show the 1σ error ranges of the fit curves. The bars represent the nonthermal part of the PBD signal (volume change ΔV) that corresponds to the PBD amplitude at -14 °C (T₀ = -14 ± 1 °C; SI Text). (B) Arrhenius plots of the rate constants (k = τ^-1, left y axis; time constants, τ, on right y axis). The symbols indicate the experimentally determined values; the lines are fit curves used for determination of the respective activation energy shown in Table 2.

Fig. 4.

Comparison of PBD signals on the four S-transitions measured for PSII membranes in H₂O (black) and D₂O (red) at 20 °C. Thin lines, experimental data; thick lines, simulations with single-exponential functions plus offset, except for the S₀ → S₁ transition, for which a double-exponential function plus offset was used (Fig. S7 and Table S2). The respective rate constant ratio is indicated (KIE = k_H2O/k_D2O). The PBD amplitudes differ because of different thermoelastic properties of H₂O and D₂O.

Fig. 5.

Sequence of events in the classical S₂ → S₃ transition of photosynthetic water oxidation. The Mn₄CaO₅ cluster, the redox-active tyrosine (Tyr₁₆₁), and the key groups of the surrounding hydrogen-bonded network (15) are shown. All indicated amino acid residues are from the D1 subunit of PSII, with exception of CP43–Arg₃₅₇. (Water molecules, H_xO, are indicated as red spheres; putative H-bonds as broken lines that connect H-bond donor and acceptor. Of all the protons, only the phenolic proton is shown as a grey sphere.) The grey mesh outlines a water cluster that includes 4 H_xO in the first coordination sphere of manganese (Mn4), as well as the calcium (Ca) and three second-sphere water molecules. Within less than 100 ns after absorption of a photon and oxidation of the primary chlorophyll donor of PSII (P680), Tyr₁₆₁ (Y_Z) is oxidized by P680⁺ (“1st”). formation results in a rearrangement of the shown H-bonded network (completed within less than 1 µs), likely involving a shift of the phenolic proton to His₁₉₀ and lowering of pK values for deprotonation of the water molecules in the outlined cluster (grey mesh). A proton is removed from the Mn complex/Y_Z environment within about 30 µs, as evidenced by the PBD results presented herein, and a proton vacancy is supposedly created within the outlined water cluster (“2nd”). In the ET to (about 300 µs), Mn oxidation is directly coupled to a proton transfer step involving the previously created proton vacancy of the water cluster (concerted electron–proton transfer) (“3rd”).