Mechanism of O–O bond formation at the oxygen-evolving complex of photosystem II: substrate-oxygen identity and the electronic structure of the final (S4) oxidation state

The oxygen-evolving complex (OEC) of photosystem II, a Mn4CaO5 cluster, splits water into electrons, protons, and O2 through the light-driven Kok cycle of S-states, but the atomistic mechanism of the O–O bond-forming step remains unresolved. Building on Cox et al. (2014), who established by magnetic resonance and quantum chemistry that the S3 state — the last state before O–O bond formation — has all four manganese ions six-coordinate and formally Mn(IV), this problem asks three linked questions: (i) the identity and provenance of the two substrate oxygens, in particular whether the second substrate oxygen in S3 is a newly inserted water (the sixth ligand O6 seen in X-ray free-electron laser structures) or a repositioned existing ligand (W2 on Mn4 or W3 on Ca); (ii) the electronic nature of the final oxidation that generates the O–O bond-forming state — a ligand-centered Mn(IV)-oxyl radical versus a metal-centered Mn(V)=O species, or an S3TyrZ•-type configuration without a discrete S4 intermediate; and (iii) the coupled electron/proton transfer sequence and energetic barrier of the S3→S4 transition. Despite major advances — room-temperature XFEL structures of all S-states, microsecond time-resolved crystallography and infrared spectroscopy, and high-resolution X-ray spectroscopy — reviews through 2026 still describe the mechanism as unresolved, and the existence and substrate role of the sixth ligand are actively disputed.

Background

Photosystem II (PSII) is the membrane protein complex of oxygenic photosynthesis that converts light energy into chemistry by oxidizing water: $2\,\mathrm{H_2O} \rightarrow \mathrm{O_2} + 4e^- + 4\mathrm{H^+}$. This reaction, the source of Earth's atmospheric oxygen, is catalyzed by the oxygen-evolving complex (OEC), an inorganic $\mathrm{Mn_4CaO_5}$ cluster in which four manganese ions (conventionally numbered Mn1–Mn4) and one calcium ion are bridged by five oxo/hydroxo bridges and ligated by terminal waters (W1 and W2 on the outward 'dangler' manganese Mn4, W3 and W4 on calcium) and by protein residues. The cluster stores oxidizing equivalents one photon at a time through the Kok cycle, a ladder of oxidation states $S_0$–$S_3$ culminating in a fleeting $S_4$ state in which the O–O bond forms and O2 is released, resetting the catalyst to $S_0$. Each charge separation oxidizes the chlorophyll donor P680, which oxidizes the redox-active tyrosine TyrZ (D1-Tyr161), the immediate one-electron oxidant of the cluster. Successive flashes advance the cycle with a period-four oscillation in O2 yield.

Two structural motifs for the cluster core are debated: in the 'open-cubane' geometry seen in crystal structures the central $\mu_4$-oxo bridge O5 binds Mn1, Mn2, Mn3 and Ca but not Mn4, whereas in a 'closed-cubane' isomer O5 also binds Mn4, completing a cubic Mn4O4 unit. High-valent candidates for the O–O bond-forming species are distinguished by where the last oxidizing equivalent resides: a ligand-centered Mn(IV)-oxyl radical (an oxo ligand carrying unpaired spin density, formally Mn(IV)–O•) or a metal-centered Mn(V)=O. Cox et al. (2014) combined multifrequency electron paramagnetic resonance (EPR), including $^{55}\mathrm{Mn}$ hyperfine spectroscopy, with broken-symmetry density functional theory (DFT) to show that all four Mn ions of the $S_3$ state are six-coordinate and formally Mn(IV) — an assignment consistent with extended X-ray absorption fine structure (EXAFS) distances. If $S_3$ is $\mathrm{Mn(IV)_4}$, forming Mn(V)=O would require a coordination change, so a ligand-centered oxyl on the O5 bridge became the leading hypothesis, although a Mn(V)=O pathway or oxidation before full S4 assembly (an $S_3\mathrm{Y_Z^\bullet}$ configuration) could not be excluded.

Isotope-exchange mass spectrometry ($\mathrm{H_2{}^{18}O}$ labeling) shows that the two substrate waters exchange with very different rates (one slow, one fast), but does not identify their binding sites; candidates include W2, W3, the O5 bridge, and — after 2017 — a sixth oxygen ligand O6 that X-ray free-electron laser (XFEL) serial femtosecond crystallography (a 'diffraction-before-destruction' method that avoids radiation damage at room temperature) showed inserted between Mn1 and Ca during the $S_2 \rightarrow S_3$ transition. Mechanistic proposals include oxo–oxyl radical coupling between O5 and O6, nucleophilic water attack on a high-valent oxo (argued against by DFT), and coupling schemes involving other ligand pairs. Since 2016, room-temperature XFEL structures of the S-state intermediates, time-resolved pump–probe crystallography and X-ray emission spectroscopy (XES, sensitive to Mn oxidation state), high-energy-resolution fluorescence-detected X-ray absorption spectroscopy (HERFD-XANES), and microsecond time-resolved infrared spectroscopy have each constrained pieces of the mechanism without settling it.

Problem Statement

The open problem is the atomistic mechanism of O–O bond formation at the oxygen-evolving complex of photosystem II during the final step of the Kok cycle, as framed by Cox et al. (2014) and still unresolved. It comprises three linked questions:

1. Substrate-oxygen identity and provenance. Which oxygen atoms of the $\mathrm{Mn_4CaO_5(H_2O)_n}$ cluster and its ligand sphere are the two substrates that end up in the O2 product, and where does the second substrate oxygen come from? In the $S_3$ state, is it a newly bound water molecule from bulk solvent (the sixth ligand O6 observed inserted between Mn1 and Ca in XFEL structures, or a water bound later in the transition), or is it a repositioning of a ligand already present in the cluster — the terminal water W2 on Mn4 or the calcium ligand W3 — with the central bridge O5 as the other substrate?

2. Electronic nature of the final oxidation. When the last oxidizing equivalent is delivered (via oxidation of TyrZ), is it taken from a substrate oxygen, generating a Mn(IV)-oxyl radical, or from a manganese center, generating a Mn(V)=O species — or does the reaction proceed without a discrete $S_4$ intermediate through an $S_3\mathrm{Y_Z^\bullet}$-type configuration? This includes assigning the oxidation states, coordination numbers, and spin distribution of the cluster in the O–O bond-forming state.

3. The transition path and its energetics. What is the detailed atomic sequence of coupled electron- and proton-transfer events that converts $S_3$ into the O–O bond-forming species and then into a peroxide-level intermediate and O2 release — including which protons are removed from which ligands and when, and what the energetic barriers and kinetics of each elementary step are — such that the pathway is quantitatively consistent with the structural (XFEL crystallographic), spectroscopic (EPR/ENDOR, EXAFS, XES/HERFD-XANES, time-resolved infrared), and substrate-exchange ($\mathrm{H_2{}^{18}O}$) data?

A complete answer must identify the two substrate oxygens and their binding histories, assign the electronic structure of the O–O bond-forming state, and supply a mechanistic and energetic account of the $S_3 \rightarrow S_4 \rightarrow$ peroxo $\rightarrow \mathrm{O_2}$ sequence consistent with these observables.

References

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