Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function?

Abstract

In nature, an oxo-bridged Mn₄ CaO₅ cluster embedded in photosystem II (PSII), a membrane-bound multi-subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light-induced charge separations in the reaction center of PSII. The Mn₄ CaO₅ cluster accumulates four oxidizing equivalents to enable the four-electron four-proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S-states, S₀ - S₄ in the Kok cycle. One important question related to the catalytic mechanism of the oxygen-evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S-states and if such equilibria are essential for the mechanism of the O-O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S₂ state of PSII with structural data collected of the S₁ , S₂ and S₃ states by serial crystallography at neutral pH (∼6.5) using an X-ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S₂ state, the room temperature crystallography data can be well-described by just one S₂ structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.

Fig. 1.

S₂-g2 and S₂-g4 transition.(A) Transition scheme from S₁ to the S₂-g2 and S₂-g4 states in PSII from plants and the oxidation states of the Mn and the total spin of S-states. (B) Proposed HS and LS S₂ state structures by Pantazis et al. 2012 and Isobe et al. 2014 C, Formation and the conversion between the S₂-g2 and S₂-g4 state and the difference in their physical properties. A detailed discussion can be found in several reviews and papers (Haddy 2007, Pokhrel and Brudvig 2014, Boussac et al. 2015, Boussac et al. 2018).

Fig. 2.

EPR and XAS spectra of spinach PSII. (A) EPR spectra of spinach PSII samples illuminated at (b) 195 K (blue) and (c) 140K with NIR (red) along with corresponding (a) dark (black) EPR spectra. The difference spectra are between the spectra after illumination and the spectra of the same dark-adapted sample. (B) Mn XANES spectra (top) and their second derivative spectra (bottom) of HS (red) and LS (blue) S₂ states, in comparison with S₁ (black) states. (C) Mn EXAFS spectra of HS (red) and LS (blue) S₂ states. The spectrum of the 140K illuminated sample (HS-rich) after 200K annealing is also shown (cyan) (D) EPR spectrum of the sample annealed at 200K (cyan) after 140K with illumination compared to the 140K with NIR (black) spectrum. The figure is adapted from Chatterjee et al. 2016.

Fig. 3.

Comparison of the MnCaO₅ cluster of the OEC between the S₁ and S₂ states. (A) 2mF_o-DF_c density (blue mesh) of the OEC in the S₂ state shown at 0.7σ contour level. In addition, the mF_o-DF_c difference density between the data and the model was plotted at 3.0σ contour level but no peaks are visible as the differences have low intensity, indicating a good fit of the model to the experimental data (B) O5 omit map density shown of the OEC in the S₂ state, contoured at 3.0σ (green mesh). (C, D) Atomic distances in the OEC averaged across both monomers in S₁ and S₂ states, respectively. Ca remains 8-coordinate upon insertion of Ox by way of movement of D1-Glu189 away from the OEC. E, mF_o-DF_c difference density between the data and the model shown at +3.0σ (green) and −3.0σ (red) contour. This density was calculated using the 1:1 mixture of the open cubane (right open) and closed cubane (left open) models (Fig. 1B). The figure is adapted from Kern et al. (2018).

Fig. 4.

Comparison of the water environment of the OEC between the S₁ and S₂ states. (A, B) Water environment of the OEC at the O4 water chain (A, B) and next to O1(C) in the S₁ (0F, brown) and S₂ (1F, blue) states. We note that Water 20 is highly unstable in position and there is not sufficient density in the S₂ state data to model the water 20 position. (C) the overlaid 2mF_o-DF_c density maps at 1.5σ contour level of the S₁ (0F, brown) and S₂ (1F, blue) states show the changes in water positions. The O1-W26 distance changes from 3.09 to 3.01 Å upon transition from S₁ to S₂. The figures are adapted from Kern et al. (2018).