Mutation-induced shift of the photosystem II active site reveals insight into conserved water channels

Abstract

Photosystem II (PSII) is the water-plastoquinone photo-oxidoreductase central to oxygenic photosynthesis. PSII has been extensively studied for its ability to catalyze light-driven water oxidation at a Mn₄CaO₅ cluster called the oxygen-evolving complex (OEC). Despite these efforts, the complete reaction mechanism for water oxidation by PSII is still heavily debated. Previous mutagenesis studies have investigated the roles of conserved amino acids, but these studies have lacked a direct structural basis that would allow for a more meaningful interpretation. Here, we report a 2.14-Å resolution cryo-EM structure of a PSII complex containing the substitution Asp170Glu on the D1 subunit. This mutation directly perturbs a bridging carboxylate ligand of the OEC, which alters the spectroscopic properties of the OEC without fully abolishing water oxidation. The structure reveals that the mutation shifts the position of the OEC within the active site without markedly distorting the Mn₄CaO₅ cluster metal-metal geometry, instead shifting the OEC as a rigid body. This shift disturbs the hydrogen-bonding network of structured waters near the OEC, causing disorder in the conserved water channels. This mutation-induced disorder appears consistent with previous FTIR spectroscopic data. We further show using quantum mechanics/molecular mechanics methods that the mutation-induced structural changes can affect the magnetic properties of the OEC by altering the axes of the Jahn-Teller distortion of the Mn(III) ion coordinated to D1-170. These results offer new perspectives on the conserved water channels, the rigid body property of the OEC, and the role of D1-Asp170 in the enzymatic water oxidation mechanism.

Keywords: cryo-EM; hydrogen-bond network; mutagenesis; oxygen-evolving complex; photosynthesis; photosystem II; quantum mechanics/molecular mechanics; water channel.

Figure 1

Water channels in photosystem II.A, the structure of D1-D170E PSII, colored according to subunit, is shown with portions of the O1 channel, the Cl-1 channel, and the O4 channel shown in red, green, and blue volumes, respectively. B, a focused view showing the location of relevant structures in relation to the OEC.

Figure 2

Difference map peaks show changes near the OEC. Overlays of D1-D170E PSII, shown in yellow; WT PSII, shown in grey; and the D1-D170E-minus-WT difference map, shown in positive green and negative red volumes; (A) the Asp to Glu mutation shifting the OEC metal positions, (B) the Glu sidechain “buckling,” (C) CP43-R357 pivoting, and (D) individual OEC metals shifting. The rescaled cryo-EM maps and difference maps are shown as isomesh and isosurfaces, respectively. Background map features are excluded for clarity; see Tables S8 and S9 for details.

Figure 3

Disorder is observed among structured waters. Water molecules in the (A) Cl-1 water channel, (B) O4 water channel, (C) water tetramer, and (D and E) O1 water channel are shown for rescaled D1-D170E PSII (left), rescaled WT PSII (middle), and a merge of rescaled D1-D170E PSII and WT PSII (right). C, the stabilized water position in the water tetramer is indicated with a red arrow in D1-D170E PSII and WT PSII. D and E, water molecules that are highly conserved between D1-D170E PSII and WT PSII. Throughout, D1-D170E PSII is shown in yellow and WT PSII is shown in grey. The isomesh shows lower map contour while the isosurface shows a higher map contour. Additional atoms are included for spatial reference but map features are excluded for clarity; see Tables S8 and S9 for details.

Figure 4

QM/MM calculations show an altered energy landscape for the hydrogen-bonding network (HBN). QM/MM optimized structures for (A and B) HBN1 and (C and D) HBN2 with labeled distances (Å). A and C, show low energy configurations for WT PSII. B and D, low-energy configurations for D1-D170E PSII. The relative potential energy landscapes are shown for (E) WT PSII and (F) D1-D170E PSII configurations, where the energies corresponding to structures in (A–D) are annotated.