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 MnIV3 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.

How Nature makes O2: an electronic level mechanism for water oxidation in photosynthesis.

“…This is a different assignment to that previously made for the S=6 form detected in spinach samples where the S=6 form was attributed to a closed cubane form of the WOC cluster with a pentacoordinated Mn 4 (IV) ionan intermediate formed prior to binding of the second substrate water. 25 This however, in striking contrast to the model proposed here, is not supported by the XFEL structural data. 23 In addition it has been shown that Mn(III) is required for NIR excitation 30 and the large D value of 1.523 cm -1 for the S=6 form strongly suggests the presence of Mn(III) ion in the complex.…”

“…Also shown are simulations for an S=6 form using the zero field splitting parameters reported for the spinach samples. 25 From the simulations it is clear that the spectral intensity of the S= 6 form is much less than that of the S=3 form. This suggests that the S=6 form would be difficult to detect in the Wband EPR experiment.…”

“…Intriguingly an S=6 species was proposed to be formed in the 2-flash S 3 state of spinach samples and indeed was proposed to be the major component (80%) of native samples. 25 The S=6 form was attributed to a so-called closed cubane form of the WOC cluster with a pentacoordinated Mn 4 (IV) ion formed before the second substrate binds. So far no experimental support for such a closed cubane structure of the WOC has been obtained for any S-state.…”

Probing the proton release by Photosystem II in the S1 to S2 high-spin transition

“…Nevertheless the non-detection of these two forms of S 3 by X-band EPR, it seems unlikely that they correspond to the EPR invisible S 3 mentioned above. Indeed, the new S 3 signals described in [46, 47] are detectable in the presence of glycerol and methanol, whereas the formation of the (S 2 Tyr Z ● )’ state upon a near-IR illumination in the centers in S 3 defined as EPR invisible is inhibited in the presence of glycerol (and in the presence of methanol in plant PSII) [48, 49]. Finally, computational works also suggested heterogeneities in S 3 [50–52] with also a S = 6 spin value [52, 53].…”

“…With X-and Q-band EPR experiments performed in the S 3 -state of plant PSII, both in the perpendicular and parallel modes, a high-spin, S = 6, was shown to coexist with the S = 3 configuration. This S = 6 form was attributed to a form of S 3 without O6/Ox bound and with the Mn IV part of the cluster in ferromagnetic interaction with the unsaturated dangler Mn IV [48]. Nevertheless these two forms of S 3 are not detected by X-band EPR, it seems unlikely that they correspond to the EPR invisible S 3 mentioned above.…”