Structural Dynamics of the Oxygen Evolving Complex of Photosystem

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Structural Dynamics of the Oxygen Evolving Complex of Photosystem II in Water-Splitting Action Andrew J. Wilson, and Prashant K. Jain J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Journal of the American Chemical Society

Structural Dynamics of the Oxygen Evolving Complex of Photosystem II in Water-Splitting Action Andrew J. Wilson1 and Prashant K. Jain*1,2,3 1Department

of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Research Lab, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 3Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 2Materials

KEYWORDS. Photocatalysis, photosynthesis, homogeneous catalysis, Raman, SERS,

ABSTRACT: Oxygenic photosynthesis in nature occurs via water splitting catalyzed by the oxygen evolving complex (OEC) of photosystem II. To split water, the OEC cycles through a sequence of oxidation states (Si, i = 0-4), the structural mechanism of which is not fully understood under physiological conditions. We monitored the OEC in visible light-driven watersplitting action by using in-situ, aqueous-environment surface-enhanced Raman scattering (SERS). In the unexplored lowfrequency region of SERS, we found dynamic vibrational signatures of water binding and splitting. Specific snapshots in the dynamic SERS correspond to intermediate states in the catalytic cycle, as determined by density functional theory and isotopologue comparisons. We assign the previously ambiguous protonation configuration of the S0-S3 states and propose a structural mechanism of the OEC’s catalytic cycle. The findings address unresolved questions about photosynthetic water splitting and introduce spatially-resolved, low-frequency SERS as a chemically sensitive tool for interrogating homogeneous catalysis in operando.

Introduction Photosystem II (PSII) is a transmembrane protein complex responsible for the initiation of solar to chemical energy conversion in photosynthesis. PSII accomplishes this function by light driven splitting of water into protons, electrons, and molecular oxygen at a catalytic site known as the oxygen evolving complex (OEC), a metal-oxo cubane with the composition Mn4O5Ca.1 Not only is efficient watersplitting central in the photosynthesis of carbohydrates, but it is a key scientific and technological challenge for a sustainable hydrogen economy.2 Remarkably, PSII photochemically splits water near-thermodynamic potential with near-unity internal efficiency, producing few reactive intermediates, feats currently unattained by artificial water splitting catalysts.3 Understanding the mechanistic underpinning of water splitting at the OEC in PSII can guide major advancements in artificial photosynthesis and homogeneous catalysis. There is considerable understanding of PSII’s photosynthetic function, which begins with solar light absorption by chlorophyll and accessory carotenoid pigments followed by energy transfer to the reaction center, P680, a special pair of chlorophyll molecules. At the reaction center, a charge-separated state is generated when photoexcited electrons are transferred to pheophytin while holes

remain in P680. Photoexcited electrons in pheophytin are shuttled to PSI; concurrently, photoexcited holes are transferred to the OEC via a tyrosine residue. In sum, four oxidizing equivalents (i.e., holes) are stored within the OEC, which drive the extraction of four electrons from water, generating molecular oxygen and protons. Each accumulation of a photoexcited hole advances the OEC to a structurally unique intermediate state, Si, where i = 0 – 4.4,5 Apart from the S1 to S2 transition, all transitions between intermediate states involve the release of a proton. Several complementary tools including X-ray diffraction (XRD),1,6–8 X-ray spectroscopy,9–11 electron paramagnetic resonance (EPR),12 vibrational spectroscopy,13,14 and computational chemistry15,16 have been employed to determine the structures of the OEC intermediates and infer mechanisms of water splitting. Although such studies have provided valuable information, experimental limitations have left open questions about the OEC structure and consequently prevented a consensus on the water-splitting mechanism. For example, X-ray diffraction and spectroscopy studies have provided interatomic distances of the OEC in the S1 state as well as information on its ligand coordination,7 but the insensitivity of these methods to protons has led to a conflicting mechanistic picture of the Si structures formed by proton-coupled electron transfer steps. As a general limitation, conventional experimental techniques interrogate a

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Figure 1. Dynamics of PSII as monitored by SERS. (a) Waterfall plots (normalized SERS spectra vs. time) showing the response of PSII as the environment transitions from ambient air to an aqueous one. To highlight key modes, spectra are shown normalized separately across the low-frequency (400-800 cm-1) and fingerprint (800-2000 cm-1) regions. SERS spectra were acquired continuously from a PSII cluster on the SERS substrate enclosed in a flow cell under focused 514.5 nm laser excitation of 1 mW power and a 1 s acquisition time. The water was injected into the flow cell at t = 13 s, relative to the start of the experiment. (b) Stacked SERS spectra at select times from (a). (c) Representative waterfall plot of SERS spectra in the low-frequency region showing the structural dynamics of the OEC in water. SERS spectra in an aqueous environment were acquired continuously with a 200 ms acquisition time. (d) Stacked SERS spectra at select times and (e) baseline subtracted SERS intensity at 672 cm-1 from panel (c) highlight the structural dynamics. (f) Average % of spectral frames in which the 672 cm-1 (16OH2) and 664 cm-1 (18OH2) modes of PSII are observed in measurements over 15 distinct regions of the sample. Error bars represent the standard deviations of measurements across the 15 regions.

large ensemble of PSII crystals frozen in an intermediate state, the purity of which has recently been called into question.17 Mechanisms deduced from such ensembleaveraged measurements obtained in a non-physiological state have obvious drawbacks. These shortcomings call for new experimental approaches with high spatiotemporal resolution, proton sensitivity, and operando capabilities for probing the dynamic structure and catalytic mechanism of the OEC in the water-splitting reaction. For gaining new insights into the action of OEC, we employ surface-enhanced Raman scattering (SERS), which is already recognized as a powerful operando tool for the study of heterogeneous catalysis.18,19 In our particular case, SERS is advantageous due to its suitability for aqueous environments (unlike the case for infrared spectroscopy), its sensitivity to the protonation states of analytes, and its ability to be employed in a high-spatial resolution mode, offering major improvement over bulk-level probing tools that yield averages over a large ensemble. SERS has been employed for the determination of the structural organization of pigment-protein complexes in PSII.20 In the current advance, we use SERS as a dynamic reporter of the OEC structure in the course of catalytic action. We employ a Ag nanoparticle (NP) film as the SERS substrate. The SERS enhancement of such films is known to decay to 1/e of the maximum at-surface enhancement over a 5–10 nm distance range.21,22 A PSII complex has a size1 of ca. 10 nm on

the same order as this distance range of SERS on a Ag NP film. The OEC is located ~ 5 nm from the periphery of the PSII complex, placing the OEC within the distance range of SERS enhancement. As compared to spherical Ag NPs, anisotropic nanostructures offer considerably larger enhancement factors; however, the SERS enhancement at sharp features of such nanostructures drops off steeply with distance away from the NP surface, resulting in a distance range on the order of 1 nm,23 which is not practical for the current study. We found low-frequency SERS of PSII to be highly dynamic under water-splitting conditions. Using isotopologue studies in water and density functional theory (DFT) calculations, we found vibrational modes associated with water binding and turnover within the OEC. Specific snapshots of the dynamic SERS spectra are found to correspond to intermediate states S0 – S3 of the water-splitting cycle, from which we determine the protonation configuration of the S1 state and likely structures for S2 and S3 intermediates. A structural mechanism of the OEC catalytic cycle is proposed as an outcome. Our study demonstrates the power of in-situ, high-spatial resolution SERS for structural elucidation of the biologically and technologically important water-splitting reaction and homogeneous catalysts, in general.

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Results and Discussion PSII complexes were extracted from spinach thylakoids using a known procedure.24 It was ensured that PSII complexes extracted in this fashion were active toward photosynthetic water splitting. Under excitation of 1.6 mW/cm2 of 514.5 nm laser light, an oxygen evolution activity of 1.8 ± 0.9 µmol O2/mg Chl/h was measured for PSII in an aqueous buffer in the presence of 2 mM [Fe(CN)6]3- serving as the electron scavenger (Table S1). For SERS studies, extracted PSII complexes were dropcast from solution onto a Ag NP film supported by a glass substrate mounted in an inverted microscope. A 514.5 nm laser was focused using a 100X objective to a neardiffraction-limited spot centered on a cluster of PSII complexes found on the substrate. In addition to serving as the source of SERS, this blue-green light is absorbed by accessory carotenoid pigments of PSII (see Fig. S2). Resulting energy transfer to the P680 reaction center triggers photocatalytic water splitting25 in the presence of an electron acceptor and the aqueous environment maintained in our study. In addition, the excitation wavelength is chosen to be off-resonance from chlorophyll absorption (Fig. S2), thereby minimizing fluorescence, which would otherwise outcompete the weaker Raman scattering. The lower catalytic turnover under blue-green excitation also increases the likelihood of capturing discrete states of PSII with our 200-ms experimental time resolution. In a control study, PSII, drop cast from solution onto a glass substrate and exposed to ambient air, exhibited stable Raman spectra (Fig. S3a and S3c). The key vibrational modes observed at 1524, 1157, and 1009 cm-1 are signatures of β-carotene.26 In an aqueous environment, the βcarotene modes persist, but they decrease in intensity due to partial dissolution of β-carotene (Fig. S3b and S3d). Vibrational modes from the metal-oxo complex are expected to lie in the low-frequency (200-800 cm-1) region,13,27,28 but no such modes are seen in either air or an aqueous environment. SERS of PSII in water-splitting conditions. Next, a film of Ag NPs underlying the PSII clusters was employed (see Supporting Information) for enhancing the sensitivity of Raman scattering and further quenching the chlorophyll fluorescence. The Ag NP film was irradiated with UV light to photo-oxidize surface ligands prior to PSII deposition. This procedure ensured that all SERS signals in measurements from the sample-bearing substrates originated from PSII (Fig. S4). In addition to providing SERS enhancement, Ag NPs also serve as exogenous electron acceptors required to maintain charge separation and enable watersplitting by PSII under 514.5 nm excitation. In the presence of Ag NPs and 514.5 nm excitation, PSII in solution showed an oxygen evolution activity of 5.7 ± 1.0 µmol O2/mg Chl/h in aqueous buffer (Table S1). Ag NPs alone in solution do not show oxygen evolution activity under 514.5 nm excitation, ruling out direct plasmon-excitation-catalyzed water oxidation. These bulk oxygen evolution measurements, summarized in the Supporting Information and Table S1, ensure that native PSII water oxidation activity is being probed in our SERS measurements.

In ambient air, SERS spectra of a representative PSII cluster (supported on a Ag NP film) display the signature β-carotene modes (Fig. S5), but no SERS modes are observed in the low-frequency region, where OEC vibrations would be expected. When an aqueous environment is introduced, the PSII SERS spectra become dynamic in the fingerprint region (800–2000 cm-1), which is likely a manifestation of PSII activity, as the protein complex shuttles photoexcited holes and electrons to the OEC and Ag NPs, respectively. Most strikingly, distinct low-frequency modes emerge upon the introduction of water (Fig. 1a and 1b). PSII is quite sensitive and it can undergo damage in the course of SERS sample preparation and/or SERS study. Given this scenario, it was important to ensure that the PSII is active (even if partially) and that the observed spectral dynamics are indeed a result of PSII catalytic activity and not simply from photolytic side reactions of PSII components. Fluorescence emission of PSII is diagnostic of its structural integrity on Ag surfaces.20,29 We found that PSII supported on the Ag NP film showed a fluorescence emission maximum of 686 nm, identical to that for native PSII complexes in solution (Fig. S2). This comparison ensures that SERS-substrate supported PSII probed in our measurements is structurally intact. Further, we performed a control SERS study on deliberately heat-denatured (and therefore inactivated) PSII in an aqueous environment. This heat-inactivated PSII rarely showed any modes in the fingerprint or low-frequency regions (Fig. S6). The fact that low-frequency modes are observed only in the case of active (non-denatured) PSII in the presence of an aqueous environment and an electron acceptor (Ag NPs here) strongly indicates that the observed modes are vibrational signatures of water binding to the OEC. This claim is further supported by an isotopologue (16OH2 vs. 18OH2) comparison. The control study also proves that active PSII did not undergo laser-induced photothermal denaturation in the SERS studies; otherwise the PSII would cease to exhibit the low-frequency SERS modes associated with water binding. Signature SERS modes of water coordination. We compared low-frequency SERS spectra from PSII in 16OH2 and 18OH2 environments. In each case, we measured SERS spectra from 15 unique spatial locations on the PSII sample. Most SERS spectra exhibit a single mode in the lowfrequency region: 672 cm-1 in the case of 16OH2 and 664 cm1 in the case of 18OH . The shift to lower frequency for the 2 heavier 18OH2 isotopologue affirms that the detected mode involves water molecules in some form within PSII. The relatively large isotope shift of 8 cm-1 is consistent with OEC geometries with the O atoms of water bound to the Mn4 and Ca atoms of the OEC.1 Thus, the most prominent low-frequency SERS mode is a signature of water molecules coordinated with the OEC, specifically its S1 state, an assignment further verified by DFT simulations. Dynamic nature of water coordination modes. Fig. 1c–e shows that the emergent low-frequency SERS modes are dynamic under the extant water splitting conditions.

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Figure 2. S1 protonation state. Histogram of modes from 2250 SERS spectra (600-800 cm-1) of PSII in (a) 16OH2 and (b) 18OH2 solvents. Bin size is 5 cm-1 and two key modes are labeled. Raman spectra of the S1 intermediate calculated by DFT using various proposed configurations (see insets for geometry-optimized structures) of the W2 and O5 positions: c) W2 = H2O, O5 = O; d) W2 = O5 = OH; e) W2 = OH, O5 = O; f) W2 = H2O, O5 = OH. In c-e, red and blue curves correspond to Raman spectra calculated with 16OH2 and 18OH2 ligands, respectively. In c-e, red and blue vertical dashed lines represent the two key modes from the experimental histograms for 16OH2 and 18OH2, respectively.

The dynamics are not simply time-fluctuations commonly observed in SERS; rather they are a direct result of the OEC undergoing structural changes as it cycles through its intermediate redox states. This is evident from the isotopologue effect on the dynamics of the signature mode. Specifically, we determined how often the signature mode appeared in continuously acquired SERS spectra of PSII in 16OH2 as compared to SERS monitoring in 18OH2 (Fig. 1f). In each case, we combined SERS spectra from 15 different locations of the sample, so as to smear out the effect of any variations in local hot spot geometry and/or OEC activity. From 4500 total spectra (2250 for each isotopologue), we determined that the signature mode occurs less often in the 18OH2 environment (664 cm-1, 41% of the spectral frames) as compared to the case of 16OH2 (672 cm-1, 57% of the frames). Since the isotopologues are expected to have the same SERS activity,30,31 this difference in the frequency of appearance of the signature mode is a manifestation of a kinetic isotope effect (KIE) involving water turnover within the OEC. Using the data in Fig. 1f, the 16OH2/18OH2 KIE determined by SERS (N(672 cm-1)/N(664 cm-1)) is 1.39 ± 0.39. The relatively large standard deviation in this KIE determination is a result of spatial heterogeneity of PSII SERS. Although a precise KIE value is difficult to measure, our measurements show a KIE > 1, consistent with that known for the 16O/18O KIE determined experimentally and theoretically for a biomimetic oxomanganese complex.32 In the following sections, we demonstrate that the dynamics observed in the low-frequency SERS are due to the OEC sampling the intermediate Si states.

Figure 3. S0 and S1 states. For the (left) S0 and (right) S1 intermediate states of the OEC, (top) the geometry-optimized DFT model, (middle, red curves) representative SERS spectrum, and (bottom, black) DFT-calculated Raman activity spectrum are shown. Vertical dashed lines are guides to aid comparison of modes of experimental SERS and calculated Raman spectra. A representative spectrum assignable to each specific state was selected from continuously acquired SERS spectra with 200 ms acquisition time. More examples are shown in the Supporting Information.

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Figure 4. S2 and S3 intermediate state isomers. For the (left to right) S2 closed cubane isomer, S2 open cubane isomer, S3 RH Wx isomer, and S3 LH Wx isomer of the OEC, (top) the geometry-optimized DFT model, (bottom, red curve) representative SERS spectrum, and (bottom, black) DFT-calculated Raman activity spectrum are shown. A representative spectrum assignable to each specific state was selected from continuously acquired SERS spectra with 200 ms acquisition time. More examples are shown in the Supporting Information.

Table 1. Experimental SERS and calculated Raman frequencies (in cm-1 units) of the OEC. S0

S1

S2 open

S2 closed

S3 RH

S3 LH

SERS

DFT

SERS

DFT

SERS

DFT

SERS

DFT

SERS

DFT

SERS

DFT

-

788

-

736

-

777

771

765

-

774

-

769

-

756

706

700

-

747

710

713

-

733

-

721

687

683

666

668

735

726

-

694

-

704

646

644

-

641

604

600

686

691

-

680

684

680

593

605

613

619

-

572

-

682

-

664

644

642

-

573

588

584

529

527

-

654

621

611

-

613

-

558

-

533

-

478

630

633

-

574

-

590

537

538

-

505

-

460

-

614

564

562

572

578

-

518

485

480

-

448

-

587

-

530

530

529

-

497

448

441

-

425

-

561

-

504

499

510

-

470

539

536

-

465

458

461

495

498

-

442

-

444

443

440

-

404

The S1 state. A histogram of low-frequency SERS modes across 2250 spectra obtained from 15 PSII locations in an 16OH2 (18OH2) environment reveal two key maxima at 672 (664) cm-1 and 778 (767) cm-1 (Figs. 2a and 2b). To assign these low-frequency SERS modes, we turned to established models of the OEC.1,33–35 Due to dark- adaptation of our PSII samples and excitation off-resonance of chlorophyll, we expect the S1 state to be the most prevalent intermediate detected in our spectra. The interatomic distances of the OEC in the S1 state are available from XRD,1,8,36 but XRD is insensitive to the protonation of the OEC. Since many experimental and theoretical interpretations of the OEC are based on XRD structures of the S1 state, the uncertainty in the protonation configuration of S1 has precluded consensus on intermediate structures and the overall water-splitting mechanism. Batista and coworkers have summarized protonation combinations at

the W2 and O5 positions (see Supporting Information for atomic labels) of the OEC in the S1 state, which are subject to debate.37 To provide clarity on this issue, we combine the ligand and proton sensitivity and specificity of SERS with DFT calculations of Raman vibrational frequencies using existing OEC models. For our Raman calculations, we employ only the essential OEC cluster and water ligands, interatomic distances of which have been measured/optimized in complex protein matrices. Using this strategy, we determine the protonation configuration of the S1 state in an aqueous medium and also refine models for upstream and downstream intermediate states. To identify the protonation state of the S1 intermediate, we constructed a structural model of S1 using interatomic distances measured by XRD.1 Keeping the interatomic distances fixed, the geometry of the model was optimized using DFT. We examined models with water (16OH2

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or 18OH2) coordinated at the Mn4 and Ca positions, but with two possible proton configurations of the W2 (H2O or OH) and O5 (O or OH) positions. For each of the four structural combinations, we calculated the Raman activity spectrum, shown in Fig. 2c–2f, for both water isotopologues. We compared the calculated Raman mode frequencies to our experimental distribution of low-frequency SERS modes (Fig. 2a and b). It must be noted that Raman spectra calculations are not expected to predict SERS intensities, since the latter depends on a host of physicochemical factors of surface enhancement. However, a comparison of vibrational mode frequencies can be reliably made. This comparison is summarized in Table 1. The protonation configuration with W2 = H2O & O5 = O (Fig. 2c) yields a primary Raman mode at 668 cm-1 (662 cm-1), offering the closest match to the experimental SERS mode at 672 cm-1 (664 cm-1) in 16OH2 (18OH2). Isotopic frequency shifts are reproduced for the two prominent modes seen in the experimental spectra (Fig. 2a and b). Another protonation configuration of W2 = O5 = OH (Fig. 2d) predicts a primary Raman mode at 676 cm-1 (675 cm-1), but it fails to reproduce the isotopic frequency shift observed in experiment. None of the other models of S1 or other intermediate Si states (Figs. S7-S11) reproduce the mode frequency and isotopic shift observed in the experimental SERS spectra (Fig. 2a and b). Therefore, we can confidently assign the 672 cm-1 (664 cm-1 in 18OH2) mode to a spectral signature of the OEC in the S1 state with a proton configuration of W2 = H2O and O5 = O. The primary component of this mode is a OH(W1)-Mn4 bending vibration, further confirming that the emergence of this low-frequency mode in an aqueous environment is directly associated with water binding to the OEC. Snapshots of OEC in S0 – S3 states. Building on the protonation configuration of S1 assigned above, we generate likely structural models for the S0 – S3 states by taking into account known facts about proton-coupled electron transfer and water insertion steps in the S0 – S3 transitions. To validate these models, we used DFT to calculate Raman activity spectra and correlate calculated frequencies with measured low-frequency SERS modes, e.g., Fig. 1c and d. Unlike dark-stable S1, the other intermediates of the OEC are less frequently sampled.38 Therefore, it is critical to minimize ensemble averaging to capture the OEC in these other intermediate states. Our adopted SERS probing strategy is aptly suited for this task, with its time resolution of 200 ms and diffraction-limited spatial resolution. Although the majority of our SERS spectra contain 0 or 1 low-frequency modes, we restrict our analysis to spectra that contain at least 3 low-frequency modes to improve our confidence in assignment of spectra to a specific intermediate state. Our spectral acquisition time of 200 ms is longer than the μs-timescale transitions between intermediate states;7 however, the acquisition time is shorter than the lifetimes of some of the intermediate states.39–41 Consequently, our continuously acquired spectra do not reflect the kinetics; rather snapshots of spectra stochastically capture the OEC as the metal-oxo complex cycles through its redox states.

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Starting with the S1 intermediate (Fig. 2c), we added an electron and a proton at the O5 position to generate an S0 model, which was then geometry-optimized by DFT. The OEC interatomic distances were kept fixed, according to Pal et al.34 Fig. 3 shows the optimized geometries of the S0 and S1 intermediates (top), low-frequency Raman spectra calculated for these geometries using 16OH2 ligands (black), and representative spectra from our dynamic SERS measurements (red). Out of 4500 experimental SERS spectra, we find 5 spectra of the S0 and 10 spectra of the S1 intermediate states, which exhibit multiple modes matching our calculated low-frequency Raman spectra. Additional examples are shown in Figs. S7 and S8. Our S0 and S1 models yield Raman modes within ≈ 5 cm-1 of the experimentally observed low-frequency SERS modes (Table 1). Thus, our models serve as good approximations for the structures of the OEC captured spectroscopically in S0 and S1 states of the water-splitting cycle. Moving downstream, we removed an electron from our S1 model to generate an S2 structure, which was then geometry-optimized by DFT. The S2 intermediate has two possible structures: an open cubane and a closed cubane isomer, both of which were separately interrogated by DFT. In each case, interatomic distances were maintained according to Pantazis et al.33 Using the aforementioned multiple-mode-match criterion, we found 6 experimental SERS spectra matching the calculated Raman spectrum of the S2 open cubane and 3 experimental spectra matching the S2 closed cubane (Figs. 4, S9, and S10). Thus, both S2 isomers are captured in the water-splitting cycle, but the open cubane form is two-fold more prevalent. Although there is a growing consensus on the structure of the S3 intermediate, there is an ongoing debate37 whether a new water molecule is inserted in the S2 to S3 transition. Starting with the assigned protonation configuration of S1, we generated a model of S3 incorporating a new water molecule and optimized the geometry using DFT. A right-handed (RH) and a left-handed (LH) form, corresponding to the final location of the newly inserted water molecule,35 were separately examined. We found 8 SERS spectra matching the calculated low-frequency Raman spectrum of the S3 RH isomer (Figs. 4 and S11). Only 1 SERS spectrum corresponded to the S3 LH isomer (Fig. 4). As with the S0 and S1 states, our models show Raman mode frequencies in close agreement with the experimental SERS modes (Table 1). S3 models without a new substrate water did not correspond with any of our experimental SERS spectra. Proposed structural mechanism of the watersplitting cycle. Informed by our OEC models and statistical sampling of SERS spectra, we propose a structural mechanism of the S0 – S3 transitions in the water-splitting catalytic cycle (Fig. 5). The fully reduced S0 state contains four substrate water molecules that are fully protonated, along with a protonated oxo bridge at the O5 position. In the transition to the S1 state, the OEC releases an electron and a proton from the O5 position, resulting in a deprotonated bridging oxo ligand at O5, but leaving W2 fully protonated. The S1 to S2 transition only involves the release an

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electron, and therefore is not expected to induce a large structural change in the OEC. Based on the structural similarity to the S1 state, we assign the open cubane isomer as the structure of the S2 intermediate state involved in the catalytic cycle. Once the OEC is in the S2 state, it may isomerize to the closed cubane form. In fact, both open and closed isomers are observed in our spectra with a 2:1 ratio of occurrence. However, we selected the open cubane isomer as the likely participant in the catalytic cycle because the open cubane is able to accommodate a new substrate water inserted in the S2 to S3 transition, making the open cubane form consistent with our SERS spectra. Secondly, the open cubane has a lower free energy than the closed cubane by 0.3 eV (Table S2), as determined by our DFT calculations of the optimized geometries.

isomer is consistent with the S3 structure that results from water insertion in the S2 to S3 transition.37,42,43 Our proposed intermediate structures support a recently proposed water insertion mechanism mediated by Ca starting with the S2 state in the open cubane isomeric form.42 Our SERS spectra do not capture the S4 intermediate, which is thought to transition rapidly to the S0 intermediate with the release of molecular oxygen. The highly transient nature of S4 has made the study of its structure elusive. Therefore, current models of the S4 intermediate state rely on chemical extrapolation from the S0 and S3 states. We foresee that improved SERS sensitivities will allow the necessary temporal resolution to uncover greater details of the structural dynamics of the OEC during watersplitting, such as the structure of S4 and the elementary steps involved in the Si to Si+1 transitions.

Conclusions The application of underexplored low-frequency SERS to PSII in the current study has enhanced the understanding of structural dynamics of the OEC during the water splitting reaction. A major key was the discovery of a lowfrequency vibrational mode that is a signature of water binding to the OEC. Snapshots of SERS spectra, combined with DFT calculations of possible models, allowed us to assign structures for the S0 – S3 intermediates states of the OEC’s catalytic cycle. The assigned structures lead to a mechanism of how water binding and turnover occurs within the OEC, information that was previously incomplete or unambiguous. In-situ, high-spatial resolution SERS successfully complements information from X-ray techniques and allows outstanding questions in natural photosynthesis to be addressed. As exemplified by its novel insight into the OEC, SERS imaging and spectroscopy can be a powerful tool for atomistic investigation of homogeneous catalysis, in operando conditions.

Figure 5. Proposed structural mechanism of the OEC watersplitting cycle. Intermediate structures are based on the mostlikely protonation state for S1 state, as determined in Fig. 2. The isomer of the S2 state (open cubane) likely involved in the catalytic cycle was chosen on the basis of its structural similarity with the S1 and S3 geometries and a lower free energy than the closed cubane isomer. The RH Wx configuration was chosen for S3 on the basis of its prevalence in experimental SERS spectra and consistency with a structure obtained by water insertion in the S2 → S3 transition, starting with the open cubane form of S2. The geometry-optimized structure from DFT is shown in each case.

Transitioning to S3 from the S2 open cubane, we insert a new substrate water molecule. The addition of the water molecule along with the release of a proton and an electron yield an S3 structure with a net gain of OH with respect to S2. We assign the RH Wx isomer as the most-likely structure of the S3 intermediate of the catalytic cycle for two reasons. First, the RH Wx isomer has a free energy 2.3 eV lower than the LH Wx isomer (Table S2), as also reflected by the prevalence in SERS spectra. Secondly, the RH Wx

ASSOCIATED CONTENT Supporting Information. Experimental methods and DFT calculations, PSII preparation and characterization, oxygen evolution activity measurements, control SERS study of heatdenatured PSII, additional SERS examples, and OEC interatomic distances. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT A.J.W. was supported by a Springborn Postdoctoral Fellowship. This research was supported by the National Science Foundation under Grant (NSF CHE-1455011).

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TABLE OF CONTENTS (TOC) GRAPHIC


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Structural Dynamics of the Oxygen Evolving Complex of Photosystem

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