Probing substrate water access through the O1 channel of Photosystem II by single site mutations and membrane inlet mass spectrometry

Abstract

Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO₂ to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn₄CaO₅ cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated from Thermosynechococcus vestitus (T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn₄CaO₅ cluster during its S₂→S₃ and S₃→S₄→S₀ state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacterium Synechocystis sp. PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitus numbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn₄CaO₅ cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn₄CaO₅ cluster that includes the pentameric, near planar 'water wheel' of the O1 channel.

Keywords: CP43-V410; D1-E329; O1 channel; Oxygen evolving complex; Photosystem II; Substrate water exchange; Synechocystis sp. PCC 6803; Water delivery; Water oxidation; Water wheel.

Fig. 1

(a) Kok-cycle showing substrate/products for each light-induced S-state transition. (b) The open cubane structure of the Mn₄CaO₅ cluster in S₂ state and of the Mn₄CaO₅-O_X structure of the S₃ state that is reached by water insertion, shown from left to right. (c) Active site of the water oxidation in Photosystem II in Synechocystis sp. PCC 6803 and its connection to the O1 channel system. Cyan, magenta and green denotes; D1, CP43 and PsbV residues in order. Red spheres show water molecules or oxo-bridges, purple and green spheres show manganese and calcium ions, respectively. Light-green dashed lines are showing potential hydrogen bonds. Grey surface and dots signify the O1 channel network calculated using CAVER 3.0 (Chovancova et al. 2012) on the PDB entry 7N8O. Structure based on PDB 7N8O is prepared using Pymol

Fig. 2

The profile of the calculated O1 channel. (a) The channel model is depicted as the grey surface leading from the O1 bridge (left) to the lumen (right). Red spheres are showing the molecules aligned in the channel cavity. The bottleneck residues are displayed. Structures based on PDB entry 7N8O. (b) The radius of the channel cavity versus the length from the O1 bridge as the starting point. Dashed lines between (a) and (b) are drawn to indicate specific locations; start point, bottleneck, end point from left to right

Fig. 3

Substrate water exchange kinetics of PSII core complexes isolated from Synechocystis. Data (symbols) and fits (solid lines) for the site directed mutants D1-E329F and D1-E329L are shown in blue and green, respectively, while the dashed black lines show the fit obtained for WT PSII data (see SI Figure S2 for the WT data). The titles m/z 34 and m/z 36 denote the single labeled oxygen (^18,16O₂) and double labeled oxygen (^18,18O₂), respectively. The insets in m/z 34 graphs show a detailed view of the fast phase of substrate water exchange reflecting W_f exchange. The data recorded at 10 °C and pH 6.5

Fig. 4

Substrate water exchange kinetics of PSII core complexes isolated from Synechocystis. Results (symbols) and fits (solid lines) for the site directed mutant CP43-V410S is shown in red, while the fit obtained for WT PSII is shown as dashed black lines (see SI Figure S2 for the WT data). The titles m/z 34 and m/z 36 denote the single labeled oxygen (^18,16O₂) and double labeled oxygen (^18,18O₂), respectively. The insets in m/z 34 graphs show an expansion of the fast phase of substrate water exchange reflecting W_f exchange. The data were recorded at 10 °C and pH 6.5. Note the change in the time scale between top and bottom panels

Fig. 5

Normalized variable fluorescence decay kinetics recorded after a single actinic flash applied on whole cells in the presence of 25 µM DCMU. The lines denote the three exponential fit model optimized for the datasets shown with spheres. WT, E329F, E329L and V410S are shown with colors; black, blue, green and red, respectively

Fig. 6

Light-minus-dark S₂ state multiline EPR signals obtained for WT, D1-E329F and CP43-V410S PSIIcc by illumination at 200 K. The spectra are normalized with respect to the chlorophyll concentrations. WT, E329F and V410S are shown with colors; black, blue and red, respectively. EPR conditions: microwave frequency: 9.38 GHz, microwave power: 20 mW, modulation amplitude: 20 G, temperature: 7 K

Scheme 1

Simplified and expanded representation of the substrate exchange mechanism in S₂ and S₃ states proposed by Siegbahn (Siegbahn, 2013). The red and blue oxygen atoms denote W_s and W_f, respectively. The upper row is showing the exchange in the S₂ state, while the lower row is a possible exchange mechanism in the S₃ state. Paths A and B denote protonation steps needed to initiate W_f and W_s exchange, respectively. The association of O5 and O_X with Ca is not shown for simplicity