On the simulation and interpretation of substrate-water exchange experiments in photosynthetic water oxidation

Abstract

Water oxidation by photosystem II (PSII) sustains most life on Earth, but the molecular mechanism of this unique process remains controversial. The ongoing identification of the binding sites and modes of the two water-derived substrate oxygens ('substrate waters') in the various intermediates (S_i states, i = 0, 1, 2, 3, 4) that the water-splitting tetra-manganese calcium penta-oxygen (Mn₄CaO₅) cluster attains during the reaction cycle provides central information towards resolving the unique chemistry of biological water oxidation. Mass spectrometric measurements of single- and double-labeled dioxygen species after various incubation times of PSII with H₂¹⁸O provide insight into the substrate binding modes and sites via determination of exchange rates. Such experiments have revealed that the two substrate waters exchange with different rates that vary independently with the S_i state and are hence referred to as the fast (W_f) and the slow (W_S) substrate waters. New insight for the molecular interpretation of these rates arises from our recent finding that in the S₂ state, under special experimental conditions, two different rates of W_S exchange are observed that appear to correlate with the high spin and low spin conformations of the Mn₄CaO₅ cluster. Here, we reexamine and unite various proposed methods for extracting and assigning rate constants from this recent data set. The analysis results in a molecular model for substrate-water binding and exchange that reconciles the expected non-exchangeability of the central oxo bridge O5 when located between two Mn(IV) ions with the experimental and theoretical assignment of O5 as W_S in all S states. The analysis also excludes other published proposals for explaining the water exchange kinetics.

Keywords: Mechanism of water oxidation; Membrane inlet mass spectrometry (MIMS); Oxygen-evolving complex; Photosystem II; Substrate-water exchange.

Fig. 1

The oxygen-evolving complex of PSII, its reaction cycle, and substrate-water exchange experiments. A Structure of the Mn₄CaO₅ cluster and its surrounding (PDB: 7RF1). Manganese is shown in magenta, oxygen in red, calcium in green, water molecules around the active site in blue. Water-filled channels are represented with red (O1 channel), blue (O4 channel), and green (Cl1 channel) areas. B S state cycle of the Mn cluster. The formal oxidation states of the four Mn atoms in the four quasi-stable states are given in the boxes next to the state. C, D TR-MIMS measurements of the water exchange in the S₂ state of Ca-PSII showing the single ¹⁸O-labeled (C) and double-labeled (D) O₂ yield at pH 6. E double-labeled O₂ yield of Sr-PSII. Points in C–E show individual experimental data points, and lines show exponential fits; redrawn from Ref. de Lichtenberg and Messinger (2020)

Fig. 2

Proposals for high spin (g ≥ 4.1) conformation of the OEC in S₂ state. A Closed cubane model in which the ‘dangling’ Mn4 is a pentacoordinate Mn(III) ion (Pantazis et al. ; Isobe et al. ; Bovi et al. 2013); B early water binding between Mn1 and Ca (Pushkar et al. ; de Lichtenberg and Messinger 2020) in which Mn2 or Mn3 maybe the only Mn(III) (Pushkar et al. 2019); C proton isomer of the open cubane conformation in which, compared to S₂^LS, a proton is moved from W1 to O4 (Corry and O’Malley 2019). Manganese atoms are shown in magenta, calcium in green, and oxygen in red with arabic numbers used as indices, whereas the roman numbers are showing the oxidation states of Mn ions

Scheme 1

Substrate-water exchange reactions in a double-conformation model of PSII. Modified after (Huang and Brudvig 2021). E_HS and E_LS signify the conformations of the HS and LS states of the Mn₄CaO₅ cluster in PSII, while the superscripts (E^Ws,Wf) indicate the oxygen isotope bound in the binding sites of the slowly (W_S) and fast (W_f) exchanging substrate waters. The exchange rates of W_f and W_S in E_LS, E_HS are denoted k_f1, k_f2 and k_s1, k_s2, respectively, while the rates for the E_HS/E_LS interconversion are noted as k_c1 and k_c2

Fig. 3

Substrate-water exchange data in two-conformation systems in the S₂ state taken from Ref. de Lichtenberg and Messinger (2020) and simulated using our extended form of the double-conformation model (Scheme 1) presented in Huang and Brudvig (2021). The single-labeled O₂ yield (³⁴Y) is shown on the left, and the double-labeled O₂ yield (³⁶Y) is shown on the right. Black dots show individual experimental data points. The black and the blue curves (mostly overlapping) show simulations using rate constants given in Table 1, for the cases when the slowest kinetic component corresponds either to the slow water exchange in E_LS ($k_{\text{s}1}^{\ast }$, black) or to the rate constant for the conversion of E_LS to E_HS ($k_{\text{c}2}$, blue). The intermediate kinetic component is explained in all simulations by the slow water exchange in E_HS. Corrections for initial enrichment (${\alpha }_{\text{in}}$ = 0.7%), non-instant injection (t_k = 3 ms), and exchange in S₃ (with additional 10 ms exchange using rate constants $k_{\text{f}}^{\ast }=19.5,k_{\text{s}}^{\ast }=0.25$ s⁻¹) are applied for all simulations. A Ca²⁺-PSII, pH 8.6; B Sr²⁺-PSII, pH 6.0; C Sr²⁺-PSII, pH 8.3

Fig. 4

A schematic presentation of the OEC conformations of the S states during the Kok cycle, based on previous spectroscopic, structural, and DFT results, see Text. The oxygens proposed to be the fast and slowly exchanging substrate waters are denoted as W_f and W_s, respectively, for states their exchange rates have been measured. N1 signifies a new water molecule that replaces W3 at Ca during the S₂ → S₃ transition. A second water binding event during the reconstruction of the cluster after O₂ release in the S₄ → S₀ transition, as well as proton and O₂ release are not indicated for simplicity of presentation. Manganese atoms are shown in magenta, calcium in green, and oxygen in red with Arabic numbers. Roman numbers indicate the oxidation states of manganese ions