Life Science Research Frontiers 2024 18 Photosynthetic water oxidation is catalyzed by a Mn 4 CaO 5 cluster, which is bound to photosystem II (PSII), a huge membrane protein complex (Figs. 1(a,b)) [1]. Through this reaction, two molecules of water are split into four electrons, four protons, with the concomitant generation of one dioxygen, using the light energy from the sun. This reaction is important because it converts light energy to biologically useful chemical energy and generates molecular oxygen, both of which are indispensable for sustaining almost all life forms on the earth. This reaction proceeds through the so- called S-state cycle (Fig. 1(c)), in which the Mn 4 CaO 5 cluster undergoes state transitions upon absorption of photons in the sequence of S 1 → S 2 → S 3 → (S 4 ) → S 0 . The structure of the dark-stable S 1 state, as well as those of the meta-stable S 2 , S 3 states, have been solved [2,3], and some time-resolved studies have also been conducted [4]. However, the full time-resolved structural changes of the S 2 and S 3 states have not yet been elucidated. Using the X-ray free electron laser (XFEL) at SACLA BL2 , we conducted pump-probe serial femtosecond X-ray crystallography (SFX) experiments of PSII microcrystals at room temperature at delay times of 20 ns, 200 ns, 1 m s, 30 m s, 200 m s, and 5 ms after either one (1F) or two (2F) flashes, corresponding to the S 1 → S 2 and S 2 → S 3 transitions, respectively [5]. We calculated difference Fourier maps between those after light illumination and before illumination ( F o (after pump) – F o (before pump) ), which sensitively detect maps of areas where structural changes have occurred. In this way, we are able to detect transient structural changes of the catalyst during the formation of the S 2 and S 3 states. While we observed many structural changes following either 1F or 2F illumination, including both the electron acceptor and donor sides of PSII [5], we will focus these changes on the electron donor side and around the Mn 4 CaO 5 cluster here. At 200 ns following 1F, P680, a chlorophyll a molecule of the reaction center of PSII, showed some positive density at its Mg ion, reflecting the charge separation event after light illumination (Fig. 2(a)). Accompanying this, an electron is donated to P680 through Y Z , a Try residue of D1-Y161, from the Mn 4 CaO 5 cluster. D1-F186, a residue lying between Y Z and P680, is shifted toward P680 together with Y Z and D1-Q165 (Fig. 2(a)), reflecting the electron donation from Y Z to P680 at this time range. D1-H190, which forms a short, low-barrier H-bond with Y Z in the S 1 state, moved away from Y Z , making the H-bond between Y Z ‘normal’ ( Figs. 2(b,c)). These structural changes disappeared gradually following time progression, indicating that the structure returned to its original state after electron donation (Fig. 2). The structural changes following 2F are more or less the same as those following 1F in this area, but they appeared later and smaller than those following 1F, indicating some loss of efficiency as well as the time required to donate the second electron than donating one electron. In the Mn 4 CaO 5 cluster and its immediate environment, a number of difference densities appeared after either 1F or 2F (Fig. 3), indicating large structural changes during the S 1 → S 2 and S 2 → S 3 transitions. At 200 ns following 1F, Mn1–Mn3 were found to be unstable (Fig. 3(a)), which corresponds to the electron donation from the Mn 4 CaO 5 cluster to P680. After the meta-stable S 2 is formed (5 ms after 1F), these Mn ions mostly become stable again; instead, Mn4 slightly shifted its position, corresponding to the stable donation of an electron from Mn4 to P680. At this time, W16, a water molecule that participates Time-resolved protein crystallography – Structural changes of the catalyst for water oxidation captured by XFEL Fig. 1. Structure of PSII and the S-state model of water oxidation. (a) Structure of a PSII dimer with a side view of the membrane. The area highlighted in weak yellow in the middle is the trans-membrane area, and a dashed line divides two monomers. The areas encircled with red lines are the site where the Mn 4 CaO 5 cluster is bound. (b) Structure of the Mn 4 CaO 5 cluster, with distances indicated in Å. (c) Kok cycle (S-state model) for water oxidation. (a) (b) (c) CP43 CP47 Cyt b 559 cyt c 550 12 kDa 33 kDa W1 2.2 2.2 2.1 2.1 2.1 2.1 1.9 1.8 2.1 2.3 2.5 2.5 2.7 2.4 2.4 2.4 2.3 2.0 Mn4 Mn3 Mn2 Mn1 W2 W3 W4 O1 O3 O2 O5 O4 Ca Stromal side Lumenal side 40 μ s <1.6 ms 1.6 ms 100 μ s S 1 S 0 O 2 e – H 2O (H 2 O) S 4 S 2 S 3 300 μ s h ν D1 D2 e – e – e – H + H + H + H + h ν h ν h ν Research Frontiers 2024 19 in the H-bond network in the O4-channel, has disappeared, and W10 is largely displaced. In addition, D1-E189, a unique carboxylate ligand that is bound to Mn1 in a monodentate way and thus has the freedom to rotate, rotated its side chain slightly (Fig. 3(a)). Following 2F, there are also a number of structural changes observed at various time points. Among them, the most remarkable one is that a new water molecule named O6*, appeared near Ca and the side chain of D1-E189 at 1 m s following 2F (Fig. 3(b)). The density for O6* increased at 30– 200 m s following 2F, but disappeared completely by 5 ms. Instead, the density for O6 (or Ox), a water molecule previously reported to be inserted into a position close to O5 [2,3], appeared at 200 m s after 2F, and became maximum at 5 ms after 2F (Fig. 3(b)). This indicates that O6* is the precursor for O6, and is translocated to the O6 position during formation of the meta-stable S 3 state. Accompanying these structural changes, Mn1 and Mn4 shifted toward the opposite side, resulting in the elongation of the distance between them which is required for the insertion of O6. D1-E189 also shifted its side chain largely in order for the insertion of O6. Finally, O6 is ligated to the Ca ion, making it from 7-coordinated in the S 1 state to 8-coordinated in the S 3 state. In addition to the above structural changes, we also found a number of structural changes at the channels and H-bond networks in PSII. First, new water molecules are found in the O1-channel following 2F, suggesting that this channel may function to pump water into the reaction site. Second, different movements are found in the Cl-1 channel between 1F and 2F, suggesting that this channel may play different roles in the S 1 → S 2 and S 2 → S 3 transitions. Details can be found in the original publication [5]. References [1] Y. Umena et al. : Nature 473 (2011) 55. [2] M. Suga et al. : Nature 543 (2017) 131. [3] M. Suga et al. : Science 366 (2019) 334. [4] A. Bhowmick et al. : Nature 617 (2023) 629. [5] H. Li, Y. Nakajima, E. Nango, S. Owada, D. Yamada, K. Hashimoto, F. Luo, R. Tanaka, F. Akita, K. Kato, J. Kang, Y. Saitoh, S. Kishi, H. Yu, N. Matsubara, H. Fujii, M. Sugahara, M. Suzuki, T. Masuda, T. Kimura, T. N. Thao, S. Yonekura, L.-J. Yu, T. Tosha, K. Tono, Y. Joti, T. Hatsui, M. Yabashi, M. Kubo, S. Iwata, H. Isobe, K. Yamaguchi, M. Suga and J.-R. Shen: Nature 626 (2024) 670 . Jian-Ren Shen* and Michihiro Suga Research Institute for Interdisciplinary Science, Okayama University *Email: shen@cc.okayama-u.ac.jp Fig. 3. Structural changes of the Mn 4 CaO 5 cluster and its immediate environment following (a) 1F or (b) 2F. Residue names not encircled in a rectangle are of D1 subunit, whereas that encircled in a rectangle is of the CP43 subunit. Residues or water molecules encircled with red dashed lines indicate their disappearance, and those encircled with cyan dashed lines indicate their appearance. Fig. 2. Structural changes in the Y Z -P680 area after light excitation. (a) Structural changes at various time points after 1F. Green and red meshes represent positive and negative difference densities between 1F-0F, and arrows indicate movements of residues and water molecules. (b) Distance changes in the H-bond between D1-H190 and Y Z after 1F or 2F. (c) Schematic of the relationship between D1-H190 and Y Z after 1F or 2F. (a) (b) Datasets Shift of Y z (c) 20 ns 20 ns 200 ns 200 ns 1 1 μ μ s s 30 30 μ μ s s 200 200 μ μ s s 5 ms 5 ms 1F 2F 200 μ s 5ms 30 μ s 1 μ s 200ns 20ns 0 0.1 0.2 0.3 0.4 0.5 Distance (Å) S 1 →S 2 S 2 →S 3 30 – 200 μ s 1 – 30 μ s 20 – 200 ns 200 μ s 5 ms 200 ns – 1 μ s H + e – H + Y z ● Y z Y z ● Y z ●+ Y z OEC D1-H190 D1-H190 + D1-H190 D1-H190 + D1-H190 7 7 7 7 7 (a) (b) 20 nm 20 nm 200 nm 200 nm 1 1 μ μ m m 30 30 μ μ m m 200 200 μ μ m m 5 ms 5 ms 20 nm 20 nm 200 nm 200 nm 1 1 μ μ m m 30 30 μ μ m m 200 200 μ μ m m 5 ms 5 ms
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