Substrate-water exchange in photosystem II is arrested before dioxygen formation

Abstract

Light-driven oxidation of water into dioxygen, catalysed by the oxygen-evolving complex (OEC) in photosystem II, is essential for life on Earth and provides the blueprint for devices for producing fuel from sunlight. Although the structure of the OEC is known at atomic level for its dark-stable state, the mechanism by which water is oxidized remains unsettled. Important mechanistic information was gained in the past two decades by mass spectrometric studies of the H2(18)O/H2(16)O substrate-water exchange in the four (semi) stable redox states of the OEC. However, until now such data were not attainable in the transient states formed immediately before the O-O bond formation. Using modified photosystem II complexes displaying up to 40-fold slower O2 production rates, we show here that in the transient S3YZ state the substrate-water exchange is dramatically slowed as compared with the earlier S states. This further constrains the possible sites for substrate-water binding in photosystem II.

Figure 1. The water-oxidizing complex in PSII and its reaction sequence.

(a) Density functional theory-based model of the Mn₄CaO₅ cluster in the ‘open cube’ (S₂ EPR multiline) configuration together with its water derived ligands W1–W4. The model is inserted into the 1.9-Å crystal structure of photosystem II. Mn^III/IV, purple; Ca²⁺, yellow; oxo-bridges, red; water–oxygens, blue; Cl⁻, green; amino acid backbones, grey; carboxy-oxygen, red; and His-nitrogen, dark blue. (b) Kok cycle for water oxidation in PSII including proton release to the bulk (see also Supplementary Note 1) and water-binding events. The S_i states (i=0, …, 4) denote the oxidation state of the Mn₄CaO₅ cluster relative to the S₀ state, while the plus sign indicates a positive extra charge. (c) Detailed sequence of known and postulated states during molecular oxygen formation. Protons released in sequence c may in part be taken up by internal bases created in earlier S_i state transitions.

Figure 2. Conceivable O–O bond formation mechanisms in the ‘S₄’ state of PSII.

(a) Nucleophilic attack by a bulk water onto a Mn^V=O or Mn^IV-oxyl radical, (b) nucleophilic attack by a Ca bound water onto a Mn^V=O or Mn^IV-oxyl radical, (c) coupling of a Ca-hydroxyl radical with a Mn-bound radical substrate, (d) direct coupling between a terminal Mn-oxyl radical with an oxo bridge between Ca and Mn.

Figure 3. Substrate–water exchange in T. elongatus PSII core particles containing different ionic cofactors.

(a,b) The substrate water exchange in the S₃⁺Y_Z state at m/z 34 (^16,18O₂; a) and m/z 36 (^18,18O₂; b), while c displays the exchange in the state at m/z=34. Symbols mark the data points (green triangles, Ca/Cl-PSII; black squares, Sr/Br-PSII; blue circles, Sr/I-PSII), while full lines are fits representing the fast and slow substrate–water exchange (a,b; for rate constants see Table 1) or simulations of the expected experimental outcome assuming the exchange rates are identical in the S₃⁺Y_Z and states (c). The blue dashed and dotted lines in c represent simulations where either the slow (dashed) or fast (dotted) rate of exchange was set to be 1,000 times slower than that measured in the S₃⁺Y_Z state (Table 1). All data points (n=1) are normalized to values reached after complete isotopic equilibration. Each time course was measured once, but consists of many separately measured data points that were in part obtained on different days.