The assignment of the two substrate water sites of the tetramanganese penta-oxygen calcium (Mn 4 O 5 Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn 4 O 5 Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522-3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55-60]}; (ii) ammonia displaces a water ligand to the outer manganese (Mn A4 -W1); and (iii) as W1 is trans to O5, ammonia binding elongates the Mn A4 -O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.PSII | OEC | water oxidizing complex | water-oxidation | Mn cluster I n oxygenic photosynthesis, light-driven water splitting is catalyzed by the oxygen-evolving complex (OEC) of the membrane bound, pigment-protein complex photosystem II (PSII). The OEC consists of an inorganic tetra-manganese penta-oxygen calcium (Mn 4 O 5 Ca) cluster (1-3) and the nearby redox-active tyrosine residue Y Z (D1-Tyr161) that couples electron transfer from the Mn 4 O 5 Ca cluster to P680, the photo-oxidant of PSII. The cluster resembles a "distorted chair", where the base is formed by an oxygen-bridged (μ-oxo) cuboidal Mn 3 O 4 Ca unit (1) (Fig. 1A). The fourth Mn (Mn A4 ) is located outside of the cuboidal unit and is linked via a μ-oxo-bridged ligation (O4) to one of its corners (Mn B3 ). A second linkage between the outer Mn and the cube is provided by a fifth oxygen O5. The Mn 4 O 5 Ca cluster is also held together by six carboxylate ligands and has only one directly coordinating nitrogen ligand, D1-His332 (Fig. 1B).The OEC cycles through a series of five intermediate states that are known as S states (4) (Fig. 1A): S 0 , S 1 (dark stable), S 2 , S 3 , and S 4 (not yet isolated), where the subscript refers to the number of oxidizing equivalents stored in the OEC through successive electron withdrawals by Y Z • . In the 1.9-Å resolution structure, the S state of the cluster was assigned to be S 1 (1). However, this is unlikely as all Mn-Mn, Mn-Ca, and Mn-O/N distances of the crystal structure are ∼...

The S2 state of the oxygen-evolving complex of photosystem II, which consists of a Mn4O5Ca cofactor, is EPR-active, typically displaying a multiline signal, which arises from a ground spin state of total spin ST = 1/2. The precise appearance of the signal varies amongst different photosynthetic species, preparation and solvent conditions/compositions. Over the past five years, using the model species Thermosynechococcus elongatus, we have examined modifications that induce changes in the multiline signal, i.e. Ca(2+)/Sr(2+)-substitution and the binding of ammonia, to ascertain how structural perturbations of the cluster are reflected in its magnetic/electronic properties. This refined analysis, which now includes high-field (W-band) data, demonstrates that the electronic structure of the S2 state is essentially invariant to these modifications. This assessment is based on spectroscopies that examine the metal centres themselves (EPR, (55)Mn-ENDOR) and their first coordination sphere ligands ((14)N/(15)N- and (17)O-ESEEM, -HYSCORE and -EDNMR). In addition, extended quantum mechanical models from broken-symmetry DFT now reproduce all EPR, (55)Mn and (14)N experimental magnetic observables, with the inclusion of second coordination sphere ligands being crucial for accurately describing the interaction of NH3 with the Mn tetramer. These results support a mechanism of multiline heterogeneity reported for species differences and the effect of methanol [Biochim. Biophys. Acta, Bioenerg., 2011, 1807, 829], involving small changes in the magnetic connectivity of the solvent accessible outer MnA4 to the cuboidal unit Mn3O3Ca, resulting in predictable changes of the measured effective (55)Mn hyperfine tensors. Sr(2+) and NH3 replacement both affect the observed (17)O-EDNMR signal envelope supporting the assignment of O5 as the exchangeable μ-oxo bridge and it acting as the first site of substrate inclusion.