Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment-protein complex, couples the one-electron photochemistry at the reaction center with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC) (Fig. 1a, Extended Data Fig. 1). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, where S1 is the dark stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution2,3. A detailed understanding of the O-O bond formation mechanism remains a challenge, and elucidating the structures of the OEC in the different S-states, as well as the binding of the two substrate waters to the catalytic site4-6, is a prerequisite for this purpose. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage free, room temperature (RT) structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å structure of PS II7 at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, RT measurements are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analog, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10-13. Thus, this approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

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 ∼...

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This article explores the configural weighted average (CWA) hypothesis suggesting that extension biases, like conjunction and disjunction errors, occur because people estimate compound probabilities by taking a CWA of the constituent probabilities. The hypothesis suggests a process consistent with well-known cognitive constraints, which nonetheless achieves high robustness and bounded rationality in noisy real-life environments. Predictions by the CWA hypothesis are that in error-free data, conjunction and disjunction errors should be the rule rather than the exception when pairs of statements are randomly sampled from an environment, the rate of extension errors should increase when noise in data is decreased, and that adding a likely component should increase the probability of a conjunction. Four experiments generally verify the predictions by the hypothesis, demonstrating that extension errors are frequent also when tasks are selected according to representative design.

Nature’s water splitting cofactor passes through a series of catalytic intermediates (S0-S4) before O-O bond formation and O2 release. In the second last transition (S2 to S3) cofactor oxidation is coupled to water molecule binding to Mn1. It is this activated, water-enriched all MnIV form of the cofactor that goes on to form the O-O bond, after the next light-induced oxidation to S4. How cofactor activation proceeds remains an open question. Here, we report a so far not described intermediate (S3') in which cofactor oxidation has occurred without water insertion. This intermediate can be trapped in a significant fraction of centers (>50%) in (i) chemical-modified cofactors in which Ca2+ is exchanged with Sr2+; the Mn4O5Sr cofactor remains active, but the S2-S3 and S3-S0 transitions are slower than for the Mn4O5Ca cofactor; and (ii) upon addition of 3% vol/vol methanol; methanol is thought to act as a substrate water analog. The S3' electron paramagnetic resonance (EPR) signal is significantly broader than the untreated S3 signal (2.5 T vs. 1.5 T), indicating the cofactor still contains a 5-coordinate Mn ion, as seen in the preceding S2 state. Magnetic double resonance data extend these findings revealing the electronic connectivity of the S3' cofactor is similar to the high spin form of the preceding S2 state, which contains a cuboidal Mn3O4Ca unit tethered to an external, 5-coordinate Mn ion (Mn4). These results demonstrate that cofactor oxidation regulates water molecule insertion via binding to Mn4. The interaction of ammonia with the cofactor is also discussed.

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 H218O/H216O 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 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.

Probability theory has long been taken as the self-evident norm against which to evaluate inductive reasoning, and classical demonstrations of violations of this norm include the conjunction error and base-rate neglect. Many of these phenomena require multiplicative probability integration, whereas people seem more inclined to linear additive integration, in part, at least, because of well-known capacity constraints on controlled thought. In this article, the authors show with computer simulations that when based on approximate knowledge of probabilities, as is routinely the case in natural environments, linear additive integration can yield as accurate estimates, and as good average decision returns, as estimates based on probability theory. It is proposed that in natural environments people have little opportunity or incentive to induce the normative rules of probability theory and, given their cognitive constraints, linear additive integration may often offer superior bounded rationality.

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