The electron–proton bottleneck of photosynthetic oxygen evolution

Photosynthesis fuels life on Earth by storing solar energy in chemical form. Today’s oxygen-rich atmosphere has resulted from the splitting of water at the protein-bound manganese cluster of photosystem II during photosynthesis. Formation of molecular oxygen starts from a state with four accumulated electron holes, the S4 state—which was postulated half a century ago1 and remains largely uncharacterized. Here we resolve this key stage of photosynthetic O2 formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combining these results with computational chemistry reveals that a crucial proton vacancy is initally created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in a single-electron, multi-proton transfer event. This is the slowest step in photosynthetic O2 formation, with a moderate energetic barrier and marked entropic slowdown. We identify the S4 state as the oxygen-radical state; its formation is followed by fast O–O bonding and O2 release. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O2 formation emerges. Our results provide insights into a biological process that is likely to have occurred unchanged for the past three billion years, which we expect to support the knowledge-based design of artificial water-splitting systems. Main In all plants, algae and cyanobacteria, sunlight drives the splitting of water molecules into energized electrons and protons, both of which are needed for the reduction of CO2 and eventually carbohydrate formation2. Molecular oxygen (O2) is formed during this process, which transformed the Earth’s atmosphere during the ‘great oxygenation event’3, which began about 2.4 billion years ago. Light-driven water oxidation occurs at the oxygen-evolving complex, a Mn4CaO5 cluster bound to the proteins of photosystem II2,4 (PSII). The relationship between electron and proton transfer in the bottleneck steps of O2 formation has remained incompletely understood. We address this key step here using time-resolved Fourier transform infrared (FTIR) experiments 

Reaction cycle of photosynthetic oxygen evolution. figure 1 a, Model of the S-state cycle with sequential electron and proton removal from the oxygen-evolving site10,11,50. Starting in the dark-stable S1 state, each laser flash initiates oxidation of the primary chlorophyll donor (P680+ formation) followed by electron transfer from a tyrosine sidechain (TyrZ oxidation) and—in three of the four S-state transitions—manganese oxidation, until four electron holes (oxidizing equivalents) are accumulated by the Mn4Ca-oxo cluster in its S4 state. b, Example of tracing S-state transitions using IR absorption changes after excitation with visible-wavelength laser flashes (at zero on the time axis). The absorption changes (ΔA) are provided in optical density (OD) units. The IR transients at 1,384 cm−1 reflect symmetric stretching vibrations of carboxylate protein sidechains that sense changes in the oxidation state of manganese in the microsecond and millisecond time domain (coloured lines are simulations with time constants provided in Supplementary Table 2). Note that the scale on the x axis is linear below t = 0 and logarithmic above t = 0. c, The Mn4Ca cluster (Mn, violet; Ca, pink) in the S3 state with six bridging oxygens, the redox-active tyrosine (TyrZ), and further selected protein sidechains as well as water molecules (red spheres), based on crystal structures25. Assignment to polypeptide chains, numbering of the atoms of Mn4Ca-oxo and water molecules and hydrogen-bond distances are indicated in Supplementary Fig. 1. The two oxygens atoms that form the O–O bond in the oxygen-evolving S3 → S0 transition are indicated by red arrows.

Time-resolved tracking of O2 transition

To perform time-resolved infrared spectroscopy on PSII, we developed an FTIR step-scan experiment with automated exchange of dark-adapted PSII particles (Methods), thereby expanding previous experiments at individual wavenumbers towards detection of complete fingerprint spectra. The sample exchange system was refilled about every 60 h using PSII membrane particles with about 1.5 g of chlorophyll prepared from 40 kg of fresh spinach leaves for day and night data collection over a period of 7 months. We initiated the transitions between semi-stable S states by 10 visible light (532 nm) nanosecond laser flashes applied to the dark-adapted photosystems   Using a specific deconvolution approach based on Kok’s standard model1 (Fig. 1a), we obtained time-dependent S-state difference spectra for each of the individual transitions between the four semi-stable reaction-cycle intermediates S1, S2, S3 and S0 (for selected time courses see Extended Data Fig. 2).

We focus on the oxygen-evolution transition, S3 → S4 → S0 + O2, predominantly induced by the third laser flash, for which time courses at selected wavenumbers are shown in F (time-resolved spectra are shown in Extended Data Fig. 3). Multiexponential simulations of the time courses provided 5 time constants describing acceptor- and donor-side PSII processes, including the expected time constants of 340 µs and 2.5 ms. The 2.5-ms time constant (tO2${�}_{{\mathrm{O}}_2}$) corresponds to the reciprocal rate constant of the rate-determining step in O–O bond formation and O2 release8,9. The 340 µs time constant (tH+${�}_{{\mathrm{H}}^{+}}$) corresponds to an obligatory step of proton removal from the oxygen-evolving complex of PSII, as shown recently by time-resolved detection of X-ray absorption, UV-visible spectroscopy, recombination fluorescence and photothermal signals  resulting in a specific Mn(IV)4 Tyrox∙Z${\mathrm{T}\mathrm{y}\mathrm{r}}_{\mathrm{Z}}^{\mathrm{o}\mathrm{x}\bullet }$ metalloradical intermediate that was also trapped in low-temperature magnetic resonance experiments  ‘Obligatory’ here signifies that the O–O bond formation chemistry can proceed only after proton removal is complete, as verified by the delayed onset of signals that trace manganese oxidation states or, generally, the O2 formation chemistry , which is also visible in the top time course of Fig.  For systematic analysis of the 2D time–wavenumber data array obtained by the FTIR step-scan experiment, we exploited that the requirement for wavenumber independence of the time constants of proton removal (tH+${�}_{{\mathrm{H}}^{+}}$ = 340 µs) and the electron transfer associated with O2 formation (tO2${�}_{{\mathrm{O}}_2}$ = 2.5 ms), because they always reflect the same reaction (the same rate constant). The time constants can thus serve as a kinetic tag of the reaction in the time-resolved spectroscopic data. By simultaneous simulation of the time courses at 2,582 wavenumbers (1,800 cm−1 to 1,200 cm1) using the same set of time constants at each wavenumber, we obtained the amplitude spectra shown in Fig.  , which are denoted as decay-associated spectra (DAS).

a, IR time traces at selected wavenumbers, demonstrating the delayed onset of O–O bond formation (1,381 cm−1) and reversible changes assignable to transient sidechain deprotonation (1,571 cm−1 and 1,707 cm−1). The corresponding wavenumbers in the spectra in b–d are marked with coloured asterisks. b–d, DAS corresponding to the proton release phase (tH+${�}_{{\mathrm{H}}^{+}}$ = 340 µs, blue line) and the oxygen-evolution phase (tO2${�}_{{\mathrm{O}}_2}$ = 2.5 ms, green) as well as the steady-state difference spectrum of the S3 → S0 + O2 transition (dashed black line). Red areas b,d mark inverted 340 µs DAS and 2.5 ms DAS, indicating reversible behaviour; purple shaded areas in c highlight the similarity of the 2.5 ms DAS and the steady-state spectrum, in line with the assignment to non-transient changes in Mn oxidation state. 

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