Structures of the intermediates of Kok’s photosynthetic water oxidation clock

Abstract

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3–7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 μs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

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

“…This implies that the right‐open structure is a suitable model for the S 2 (1F) state data under our experimental condition. Although a slight increase of B‐factors for Mn and bridging oxygens between the 0F and 1F data in both monomers can be seen, this can be explained mostly by the difference in resolution/data quality between the two data sets (2.05 vs 2.08 Å), and no clear evidence is observed under our experimental conditions for increased disorder in the S 2 (1F) state data (Kern et al ).…”

“…EPR (Cox et al , , Lubitz et al ) and theoretical studies (Bovi et al , Siegbahn , Narzi et al , Isobe et al , Shoji et al , Retegan et al , Ugur et al ) proposed that there is an insertion of a water molecule in the Mn 4 CaO 5 cluster during the S 2 to S 3 transition. As the quality of the room temperature crystallography data of the S 3 state has been improved compared to previous studies (Young et al , Suga et al ), the exact position of the inserted water molecule is becoming clear, although its protonation state is still uncertain (Kern et al ). It has been suggested from theory that this water insertion into the Mn 4 CaO 5 cluster is coupled with the interconversion of the two spin isomers, ‘open’ LS S 2 and ‘closed’ HS S 2 cubane, of the S 2 state (Fig.…”

“…Recently, we reported the structures of the catalytic intermediate states of PSII in the dark (0F; S 1 ‐rich), 1F (S 2 ‐rich), 2F (S 3 ‐rich) and 3F (S 0 ‐rich) states with resolutions of 2.04–2.08 Å (Kern et al ). Data were collected at an X‐ray Free Electron Laser (XFEL) with femtosecond X‐ray pulses at room temperature (RT), by in situ visible light excitation to advance the S‐states.…”

“…Improved resolution approaching 2 Å unambiguously reveals oxygen and metal atomic positions in the catalytic center in each state. Kβ 1,3 X‐ray emission spectra (XES) were simultaneously collected from crystals to provide confirmation of catalytic advancement by probing the changes in the oxidation state of the metal cluster (Kern et al , , , Fuller et al , Fransson et al ), together with the oxidation state changes of the mobile quinone Q B site, evidenced by its electron density changes. Availability of the structural data for each stable intermediate state now enables us to reexamine previous X‐ray spectroscopy data based on the new structural information.…”

“…Our improved crystallography datasets (Kern et al ) allow us to examine the chemical structures of the catalytic center in each S‐state, and in time‐intervals between the S‐state transitions. During the crystallography data acquisition, the S‐state advancement was also confirmed by Kβ 1,3 XES collected simultaneously with the diffraction data, which serves as a diagnostic tool for the oxidation state of the OEC.…”