Structural evidence for intermediates during O2 formation in photosystem II

Abstract

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn₄CaO₅ cluster first stores four oxidizing equivalents, the S₀ to S₄ intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry^1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S₃→[S₄]→S₀ transition where O₂ is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn₄CaO₅ cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom O_x, which was introduced as a bridging ligand between Ca and Mn1 during the S₂→S₃ transition^4-6, disappears or relocates in parallel with Y_z reduction starting at approximately 700 μs after the third flash. The onset of O₂ evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Fig. 1. An overview of PS II and the electron donor site where water oxidation takes place.

a, The structure of PS II with the membrane-embedded helices and the membrane extrinsic regions on the lumenal side of PS II shown in gray. The main electron transfer components are shown in colour, which include the reaction center chlorophylls (P680), pheophytins, acceptor quinones Q_A and Q_B, redox-active tyrosine Y_z and the catalytic Mn₄CaO₅ cluster. The Y_z and Mn₄CaO₅ cluster are the cofactors of the electron donor site. b, Kok cycle of the water oxidation reaction taking place at the donor site that is sequentially driven by charge separations in the reaction center P680 induced by the absorption of photons (nanosecond light flashes, 1F–4F) in the antenna system of PS II. Room temperature X-ray crystallography data were collected at the time points indicated during the S₃→S₀ transition. c,d, The structure of the OEC in the S₃ (c) and S₀ (d) states and the sequence of events occurring between them. Mn, purple; Ca²⁺, green; O, red. W1, -2, -3 and -4 are water ligands of Mn4 and Ca. The relevant channels for water and proton transfer (O1, O4 and Cl1) are indicated as red, blue and green shaded areas, respectively. The dotted circles mark structural differences between the S₃ and S₀ states.

Fig. 2. mF_obs− DF_calc electron density omit map of key components of the redox active donor site of PS II at five time points along the S₃→S₀ transition as well as the S₃ and S₀ states.

a, Residues D1-Y161 (Y_z) and D1-H190. The omit map from the S₃ state reference is shown in light brown for comparison with the time point data (blue). b, A simplified representation of the structural changes observed at the Y_z region. c, Omit density of atoms O5 and O_x of the OEC. d, Omit density of atoms O5 and the terminal water ligands W1, W2, W3 and W4 of the OEC. e, Omit density of carboxylate oxygen atoms of D1-E189 and D1-D170. All omit maps shown in a and c–e were generated by omitting the atom or residue of interest individually, and only the primary component (that is, the state that is advancing to S₀) was used. Notable features are highlighted with red arrows and black dashed circle. All omit maps shown are contoured at 2.5σ, 3σ and 4σ using the colour scheme annotated in d for easier visualization. See also Supplementary Information Video 1.

Fig. 3. Distance changes between selected atoms/residues in the OEC during the S₃→S₀ transition.

All distances are taken from the refined component of each time point (that is, the state that is advancing to the S₀ state). Error bars are calculated from the end/rapid approach described in Methods and are an upper limit. Data here are shown as mean values ± standard deviation. The error bars for each time point were obtained from n = 100 independent END/RAPID refinements. More details about the END/RAPID procedure can be found in Methods. Dashed arrows in the schematics of the OEC on the right indicate the location of the individual distances. Mn is shown as purple spheres, and O is shown as red spheres.

Fig. 4. Structural changes in select regions of the water and proton channels of PS II during the S₃→S₀ transition.

a, The terminus of the O1 channel near the OEC that includes the group of five waters (W26–W30) in this region. Overlaid is the 2mF_obs− DF_calc electron density map contoured at 0.8σ, 1.0σ and 1.5σ. b, The O4 and Cl1 channels (branch A) that include the D1-D61 and D1-E65/D2-E312 region that is suggested to function as a proton gate. Overlaid are mF_obs− DF_calc omit maps for W1, W2 and D61 shown at 2.5σ, 3.0σ and 4.0σ. Also shown is the F_obs(time point) − F_obs(2F) difference density map within a 1.5-Å radius of W19/W20/W48 in the O4 channel at 3σ (orange map). The observed rotation angle at the side chain of E65 at particular time points is calculated with respect to the corresponding side chain position at the 2F state. Major changes are highlighted with an arrow or dashed circle. All waters are coloured by their B factors according to the diverging colour scheme shown in the figure. Important hydrogen-bond interactions are shown with a binary colour scheme to indicate strength (distance < 2.8 Å is red and 2.8–3.2 Å is gray).

Fig. 5. Schematic of the S₃→S₀ transition and proposed mechanism for O–O formation.

The sequence of events (i–iv) leading to the first deprotonation event, the molecular oxygen release, the water insertion and the second deprotonation event. The OEC atoms are shown in purple (Mn), green (Ca) and red (O). The O1 channel is shown in red, the O4 channel is in blue and the Cl1 channel in green. The ligands of the OEC and the residues forming the water–proton channels are coloured based on the subunit they belong to (D1, blue; D2, green). Possible pathways for proton (cyan arrow), water (red dashed arrow), oxygen (red solid arrow) and electron (green arrow) transfer are depicted. Notable features are highlighted with black arrows. The right tan box shows the suggested models (model a and model b) for O–O bond formation. Oxygen highlighted with magenta indicates the candidate atoms for O–O formation.

Extended Data Fig. 1. mF_obs − DF_calc electron density omit map of relevant atoms in the OEC for time points along the S₃→S₀ reaction.

For comparison of the peak height level, the omit map of the oxygen atom, O2, is also shown. Omit maps for each atom were generated by individually omitting the atom only in the primary conformer in each dataset. The maps are shown at contour levels of 2.5, 3 and 4σ. (a) Omit maps of O_X, O5 and O2 (b) Omit maps of W1, W2, W3, W4 and O2. A clear reduction in omit map peak height of O_X, O5, W1, W2, W3, W4 is observed with respect to the reference O2 omit map between 730–2000 µs.

Extended Data Fig. 2. mF_obs − DF_calc electron density map near the OEC region in the 3F(500 µs) time point.

The map is shown at a contour level of 2.5σ (in yellow). Electron density is observed at this level in-between W3 and W4 (see red arrow), possibly indicating a transient water motion that was also corroborated by the overlap of the omit map densities. For comparison, the individual omit maps of W3 and W4 are also overlaid at 2.5, 3 and 4σ (color scheme same as in Fig. 2 in main text). Stronger electron density is also observed around W1/D61 possibly related to motion involving a proton transfer as discussed in the main text related to Fig. 2. Electron density around the O1 is possibly related to changes observed in the W26-W30 (referred to as the ‘water wheel’ region) in this time point (discussed in main text related to Fig. 4).

Extended Data Fig. 3. Comparing B-factors of key waters in the water wheel region of the O1 channel in the S₃→S₀ transition (W27, W28, W32) against a reference water (W29) for time points shown in Fig. 4 in the main text.

The B-factor distribution of all the channel waters are overlaid for each time point for comparison. The y-axis values of the marker points for the 4 waters are just placeholders. There is a stark shift in the relative B-factor values of W27, W28 and W32 in the 3F(1200 µs) time point which coincides with the disappearance of O_X.