Arrested Substrate Binding Resolves Catalytic Intermediates in Higher‐Plant Water Oxidation

Abstract Among the intermediate catalytic steps of the water‐oxidizing Mn4CaO5 cluster of photosystem II (PSII), the final metastable S3 state is critically important because it binds one substrate and precedes O2 evolution. Herein, we combine X‐ and Q‐band EPR experiments on native and methanol‐treated PSII of Spinacia oleracea and show that methanol‐treated PSII preparations of the S3 state correspond to a previously uncharacterized high‐spin (S=6) species. This is confirmed as a major component also in intact photosynthetic membranes, coexisting with the previously known intermediate‐spin conformation (S=3). The high‐spin intermediate is assigned to a water‐unbound form, with a MnIV 3 subunit interacting ferromagnetically via anisotropic exchange with a coordinatively unsaturated MnIV ion. These results resolve and define the structural heterogeneity of the S3 state, providing constraints on the S3 to S4 transition, on substrate identity and delivery pathways, and on the mechanism of O−O bond formation.


Simulation of the EPR spectra
The theoretical simulation of the EPR spectrum was performed with EasySpin. [4] For the approximation of an isolated spin state, the following spin Hamiltonian was used: (S1) In equation S1 the first term is the Zeeman interaction. The second and third terms represent the zero field interaction, were D and E are the axial and rhombic zero field splitting parameters, respectively.
For the detailed investigation on the S3 possible spin states of the MeOH containing spinach PSII preparations, apart from the second order zero field splitting parameters, additional fourthorder zero field splitting parameters were taken into account and the following more detailed spin Hamiltonian was used.

(S2)
For the approach of the two exchange coupled spins, the spin Hamiltonian shown in equation S3, which contains the exchange interaction terms was used: Theoretical EPR spectra obtained by assuming isolated spin states of S=1-6 In order to determine the spin configuration of the S3 state that gives rise to the low-field EPR feature at Q-band, we performed a detailed theoretical investigation of the S3 EPR spectrum in methanol-containing PSII membranes. First of all, in Figure S1 we show the superposition of the S3 experimental EPR spectrum at Q-band with the simulated one obtained by using a unique spin configuration of S=3, g=2, |D|=0.179 cm -1 , E/D=0.28, σD=0.018 cm -1 . It is obvious that the theoretical spectrum that results from the aforementioned parameters that were fitted to the Xband spectrum cannot match several features of the Q-band EPR spectrum. This indicates that an additional spin configuration is necessary to fit the experimental spectrum. Figure S1. Superposition of the S3 experimental EPR spectrum at Q-band (black trace) with the simulated spectrum (blue) using a unique spin configuration of S=3, g=2, |D|=0.179 cm −1 , Considering that the electronic distribution of the Mn4CaO5 in the S3 state is Mn(IV)4 [5] we  Figure S2. By comparing the simulation spectra obtained by assuming S=1 and S=6 (traces magenta and blue of Figure S2, respectively) we observe that both theoretical curves can reproduce the derivative at ~300 mT. However, apart from this derivative the additional EPR features cannot be described by assuming a spin of S=1. Instead, these features can be sufficiently described by assuming the S=6 configuration. Overall, among the various theoretical spectra, the one obtained by assuming an isolated spin state with S=6 and the parameters of g=1.98, D=+1.523 cm −1 , E/D=0.14 describes best the experimental EPR features at geff~8. By using the aforementioned parameters, the theoretical spectrum at X-band presents no EPR signal, in agreement with the experimental data and with previous reports. [6] Figure S2. Comparison of the experimental EPR spectrum of the S3 state at Q-band in MeOHcontaining PSII with various theoretical spectra obtained by assuming integer spins of S=1-6.

S5
It should be noted that recent simulations on S3 cyanobacterial PSII showed that apart from the second order zero field splitting parameters, additional higher-order zero field splitting parameters were needed to reproduce the experimental EPR spectrum. [7] Therefore, fourth order zero field splitting parameters described in equation S2 were taken into account for the present detailed investigation on the simulation of the experimental EPR spectrum with spin states of S=1-5. However, the use of these additional terms for these spin values fails to reproduce the S3 experimental EPR spectrum.

Energy level diagrams at Q-band and respective resonance transitions for the S=6 configuration
In Figure S3 we present the energy levels diagrams with the resonance transitions that explain the S3 EPR signals of the S=6 and S=3 configurations.

Origin of the zero field splitting for the S=3 and S=6 ground state multiplets of the S3 oxidation state of the OEC
In exchange coupled clusters the zero field splitting parameter, D, of the various spin multiplets originates from the local zero field splitting parameter Di, while there are cases where anisotropic exchange interactions have been also considered. [8][9] In the former case, D is given by the relationship [10] : Taking into account the previously reported nature of the spin-spin interactions within the OEC in the S3 state that give rise to the ground states of S=3 and S=6, as well as the local second order zero field splitting terms, [11][12] we applied equation S4 to examine whether the local contributions constitute the exclusive origin for the zero field splitting of the respective multiplets.
The most supported geometric structure of the S3 state that leads to the ground spin configuration of S=3 contains four octahedral Mn(IV) ions. [5,[13][14] A structure of the S3 state that was shown to have a ground-state spin of S=6 while still containing only Mn(IV) ions consists of three octahedral Mn(IV) ions of the Mn3CaO4 unit and an outer five-coordinated Mn4(IV), [11] i.e. a water-unbound form of the S3 state. Di values of the octahedral Mn(IV) ions are typical for this coordination geometry, [12] while that of the five-coordinated Mn4(IV) ion was computed to be 2.14 cm −1 from DFT. [11] We adopt this structure as the most suitable hydroxo S3 conformation. [5] Figure S5 depicts the same information [11] for the high-spin (S=6) water-unbound S3 conformation. The low-energy part of the Heisenberg ladder of the high-spin form can be simulated in the "3+1" representation of the OEC cluster with effective spins SA = 9/2 (for the trinuclear part) and  S9 Figure S5. Reduction of the 4-spin to an effective 2-spin representation. The scheme on the left depicts the computed isotropic exchange coupling constants for the water-unbound S3 state identified with the S = 6 signal (the exchange coupling values are taken from Retegan et al. [11] and converted to conform with our convention for the Heisenberg exchange Hamiltonian, H = +ΣJijSiSj , where negative J values denote ferromagnetic interaction). The Heisenberg spin ladder of the 4-spin system comprises 44 spin states. The four lowest states can be modelled with an effective 2-spin system shown on the rightthat represents the trinuclear and mononuclear Mn subunits of the cluster in this configuration. An effective exchange coupling of -5.6 cm −1 almost exactly reproduces the lowest spin levels of the 4-spin system. This spin ladder configuration is used in order to estimate the thermal occupation of the S = 6 ground state at the conditions of the EPR experiments (10K). S10