Orientational Jahn–Teller Isomerism in the Dark‐Stable State of Nature's Water Oxidase

Abstract The tetramanganese–calcium cluster of the oxygen‐evolving complex of photosystem II adopts electronically and magnetically distinct but interconvertible valence isomeric forms in its first light‐driven oxidized catalytic state, S2. This bistability is implicated in gating the final catalytic states preceding O−O bond formation, but it is unknown how the biological system enables its emergence and controls its effect. Here we show that the Mn4CaO5 cluster in the resting (dark‐stable) S1 state adopts orientational Jahn–Teller isomeric forms arising from a directional change in electronic configuration of the “dangler” MnIII ion. The isomers are consistent with available structural data and explain previously unresolved electron paramagnetic resonance spectroscopic observations on the S1 state. This unique isomerism in the resting state is shown to be the electronic origin of valence isomerism in the S2 state, establishing a functional role of orientational Jahn–Teller isomerism unprecedented in biological or artificial catalysis.


Introduction
Theo xygen-evolving complex (OEC) is au nique system common to all oxygenic photosynthetic organisms that catalyzes the four-electron oxidation of water, powered by light-driven charge separation at the reaction center of photosystem II (PSII). TheM n 4 CaO 5 cluster at the heart of the OEC serves both as the charge accumulator that sequentially stores the four required oxidizing equivalents through asuccession of states S i (i = 0-4), and as the catalyst that activates substrate water molecules enabling OÀOb ond formation in the transient S 4 state ( Figure 1). Thee ssential structural details of the OEC have been painstakingly refined over the years. [1][2][3][4][5] Protein crystallography has focused primarily on the dark-stable (resting) S 1 state of the enzyme [6][7][8][9] yielding structural models of increasing reliability,a lthough uncertainty persists about important details. [5,[10][11][12][13][14][15] In the parallel efforts of spectroscopic and quantum chemical studies to provide experimentally consistent atomistic models for other catalytic states and intermediates,amost intriguing outcome has been the role of polymorphism in the OEC. Specifically,t he S 2 state of the Mn 4 CaO 5 cluster with oxidation states Mn III Mn IV 3 is known to exhibit two types of EPR signal, [16][17][18][19] at g = 2( multiline signal, spin S = 1/2) and g ! 4.1 (S ! 5/2). Several explanations for this phenomenon have been considered (see Supporting Information), but the most well-supported scenario suggests that the signals arise from two valence isomeric forms (referred to as S 2 A and S 2 B ) that differ in the position of the unique Mn III ion, Mn1 in S 2 A and Mn4 in S 2 B ,respectively ( Figure 1). [20][21][22] Thetwo valence isomers,which are close in energy and interconvertible, [20,[22][23] have distinct spin states and spectroscopic properties that correspond to the two known types of EPR signal [16][17][18][19] for the S 2 state:t he S 2 A with spin S = 1/2 and g = 2, and the S 2 B with S ! 5/2 and g ! 4.1. Orientation dependence of the g = 4.1 signal confirms the location of the Mn III ion at the outer Mn4 ion and the orientation of the zero-field splitting (ZFS) z-axis along the Mn4-W1 vector. [24] Theemergence of valence isomers in the S 2 state may be crucial for controlling access to the last experimentally observable S 3 state. [25][26][27] Thev alence isomers are part of agating mechanism in which the majority form (S 2 A )ismore easily deprotonated yet the minority form (S 2 B )c an be more easily oxidized to an all-Mn IV species before binding an additional water molecule. [26] This water-unbound form of the S 3 state (Figure 1) was recently identified by EPR spectroscopy on spinach PSII as ahigh-spin (S = 6) species with high effective anisotropy, [27] in contrast to water-bound S 3 intermediates that have intermediate-spin S = 3ground states. [27][28][29][30][31][32] Thedetails of the S 2 -S 3 transition and the precise composition of the heterogeneous S 3 state are subjects of intense current research efforts because of their direct implications for the mechanism of the final unresolved steps of O 2 formation and evolution. On the other hand, despite the functional relevance of the S 2 state valence isomerism for catalytic progression, the fundamental question of how the enzyme enables and controls its emergence remains entirely open. Although structural heterogeneity in the resting S 1 state of the OEC has been previously considered, [12,13,33] it remains unclear which intrinsic feature enables the cluster to generate distinct valence isomers upon oxidation. Thep resent work answers this question with the aid of quantum chemical and electron paramagnetic resonance (EPR) analysis of the S 1 state,b ye stablishing the existence and demonstrating the functional role of orientational Jahn-Teller isomerism in the OEC.

Results and Discussion
Orientational Jahn-Teller Isomers in the Dark-Stable State of the OEC Based on insights from previous modeling of the S 2 state, [34][35][36] large quantum mechanical cluster models were created starting from the coordinates of the 5B66 (monomer A) crystal structure [13] for exploring possible forms of the S 1 state.G eometry optimizations were carried out with density functional theory for various spin configurations,w ith the Mn4 ligand W2 in both the hydroxo and aquo forms,and the O5 bridge in both the oxo and hydroxo forms (Table S1). Tw o different motifs of the inorganic core within the same coordination environment and valence distribution III-IV-IV-III for the Mn1-Mn2-Mn3-Mn4 cluster were identified ( Figure 2). Thestructures of the distinct S 1 forms indicate that they both contain aM n1(III) ion with its (pseudo-)Jahn-Te ller axis of tetragonal elongation oriented along Asp342-O5, but differ in the Jahn-Teller distortion of the Mn4(III) ion. In the form labeled S 1 A the Jahn-Teller elongation axis of Mn4 lies along the Asp170-Glu333 vector, perpendicular to the Jahn-Teller axis of Mn1, whereas in the S 1 B isomer the Jahn-Teller elongation axis of Mn4 is collinear to that of Mn1 and oriented along the W1-O5 vector. Thea xial Mn-O elongations are so prominent that the orientations of Jahn-Te ller axes can be read directly from the geometric param- Figure 1. a) Structure of the oxygen-evolving complex suggested by crystallography (PDB ID:5 B66). [13] b) The S-state cycle of the OEC, illustrating the light-induced one-electron oxidation steps coupled to proton release, the uptake of substrate water molecules, and O 2 evolution.E lectron transfer from the OEC to the P680 radical cation of the PSII reaction center is mediated by aredox-active tyrosine (Tyr161 or Y Z )g iving rise to metalloradical S i Y Z C intermediates. c) Spectroscopically consistent S 2 state valence isomers S 2 A and S 2 B ,w hich gate progression to ah igh-spin water-unbound form of the S 3 state. [27] The orientation of Jahn-Teller elongation for the Mn III ion in each S 2 isomer is indicated with green arrows.

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Forschungsartikel eters,b ut the electronic structure also perfectly mirrors the above description. TheK ohn-Sham molecular orbitals (Figure 2b)show that both terminal Mn III ions of S 1 A and S 1 B have occupied d z 2 orbitals that are s-antibonding with respect to the ligands aligned with the structurally identified Jahn-Teller axes.
Thea diabatic potential energy surface for an octahedral homoleptic Mn III high-spin d 4 system (Ee coupling problem) in the space defined by the tetragonal and orthorhombic distortion coordinates Q q and Q e has three minima at the level of quadratic interaction corresponding to the three directions of tetragonal distortions ( Figure S1 and Figure 3a). In heteroleptic, asymmetrically coordinated Mn III complexes,e nvironment anisotropy lifts the degeneracyo fthe three minima and typically results in localization and alignment of the (pseudo) Jahn-Teller axis along as ingle "privileged" direction. In the present case the S 1 A and S 1 B are assigned as local Jahn-Teller minima with respect to the Mn4 ion, whereas at hird isomer, S 1 C ,w as also located and assigned as the transition state that connects them ( Figure S2). In S 1 C the Mn4 ion has as ingly occupied d x 2 Ày 2 orbital and aJ ahn-Teller compression axis along the Mn4-W2 bond, similar to amodel previously discussed by Paul et al. [37] Thec oordination sphere of the Mn4 ion of the OEC is extremely unusual in enabling,d espite the asymmetric ligation, the presence of two distinct minima with the Mn4 elongation axis either along Glu333- ). Af actor enabling this type of isomerism may be the unusual ligation mode of D1-Asp170, ar esidue that bridges Mn4 and Ca but interacts with the latter in alopsided fashion that allows bifurcated H-bonding interaction with CP43-Arg357 ( Figure 1). Thethird possible orientation for Mn4 and alternative Jahn-Teller orientations for Mn1 could not be located even when starting the self-consistent Kohn-Sham procedure with appropriately manipulated orbital guesses and geometries.
In terms of energetics,i somer S 1 A is computed to be 3.2 kcal mol À1 lower than S 1 B .Conversion of S 1 A to S 1 B occurs via S 1 C ,which is 5.3 kcal mol À1 higher than S 1 A and where the octahedron becomes compressed along the W2-Mn4-O4 axis. In view of the nature of the problem and the limitations of DFT it is inadvisable to treat these energy values as quantitative predictions.T he present description is not an exact theoretical treatment of the vibronic coupling problem and only highlights obvious analogies between the formal problem and the actual situation in the OEC as approximated by DFT.N evertheless,t he computed energies suggest that interchange of the different minima can be inhibited at cryogenic temperatures used for EPR spectroscopic studies of catalytic intermediates,w hich is important for our structural interpretation of the corpus of S 1 state EPR spectroscopy developed in the following.
Thei mportance of the Jahn-Teller effect for shaping the geometric structure of Mn III -containing clusters is recognized, [34,[38][39][40][41][42][43] but orientational Jahn-Teller isomerism is unexpected for fully heteroleptic Mn III centers.C ontrary to spin-state interconversions and redox isomerism, Jahn-Teller isomerism is rare in metalloenzymatic catalysis, [44] while specifically the orientation switching described here is without precedence in biology.Apossibly unique analogue in synthetic inorganic chemistry is the report by Christou and coworkers of two Jahn-Teller axis orientations for the same Mn III ion in aM n12 single molecule magnet, with each orientational Jahn-Teller isomer displaying distinct relaxation behavior. [45][46] Correlation with Experimental Structural Information All S 1 models discussed above are consistent with available crystallographic data on the OEC,giving root mean square deviation (RMSD) of less than 0.17 with respect to the highest-resolution crystallographic model of Photosystem II (Table S1). S 1 A Mn-Mn distances are closer to the corresponding crystallographic and X-ray absorption spectroscopy (EXAFS) derived values than S 1 B ,w hich is attributed to the elongation of Mn3-Mn4 distance and decrease of the Mn1-Mn3 distance that take place as the Mn4-O5 distance increases ( Figure S3). Thei somers reported here resemble forms of the cluster reported by Narzi et al. [12] and we similarly find that a combination of the S 1 A and S 1 B isomeric forms provides the best agreement with EXAFS and X-ray crystallography structural data (Table S1 and Figure S3). it would also appear in better agreement with structural data than each of the minima individually,b ut the energetics and Figure 3. a) Potential energy surface resulting from the quadratic Ee coupling for an idealized octahedralM n III ion with illustrationo ft he two types of distortion (see also Figure S1). b) Schematic depiction of the partial potential energy trough cross-section suggested to survive for the orientational Jahn-Teller S 1 isomers identified here with respect to configurations of the terminal Mn4(III) ion. Jahn-Teller elongation axes depicted with green arrows.
nature of the stationary points support S 1 A /S 1 B heterogeneity rather than predominance of the S 1 C form. S 1 state heterogeneity combined with apercentage of the S 0 state, [12] in which O5 is protonated and Mn3 is reduced to Mn III compared to the S 1 state, [15,34,[47][48][49] best rationalize the X-ray free electron laser crystallographic model of PSII and the unusual apparent position of the O5 bridge. [9] However,t he heterogeneity described here is unlikely to be discernible by any experimental structural method at presently achievable levels of resolution.
Theexistence of the orientational Jahn-Teller isomers S 1 A and S 1 B in the S 1 state provides au nique yet straightforward interpretation of Fourier transform infrared (FTIR) spectroscopic studies that documented spontaneous changes in carboxylate vibrational modes during dark adaptation of the S 1 state of the OEC. [50] Possible causes were suggested to include changes in carboxylate ligation to Mn, changes in secondary structure,a nd/or changes in polarity attributed to an unspecified isomer of the S 1 state.The isomerism proposed here accounts for the FTIR observations because rotation of the Jahn-Teller axis of Mn4 directly affects the carboxylate Asp170 and Glu333 ligands by toggling the antibonding character of the respective Mn4 À Ob onds.

Magnetism and Electron Paramagnetic Resonance Spectroscopy
Pairwise exchange coupling constants for structures S 1 A and S 1 B ( Figure S4) were computed with broken-symmetry density functional theory using TPSSh, an established functional for magnetic properties of manganese systems. [51,52] In both structures Mn1 is strongly antiferromagnetically coupled with Mn2 and weakly ferromagnetically coupled with Mn3. Mn2 and Mn3 interact ferromagnetically and Mn1 shows aw eak antiferromagnetic interaction with Mn4. S 1 A is diamagnetic with alow lying triplet first excited state,whereas S 1 B has an S = 3ground state with aquartet first excited state. Differences between the S 1 models arise from J 34 ,w hich is strongly antiferromagnetic when the Mn4-O5 distance is short, but weakens rapidly as the Mn4-O5 distance increases ( Figure S4b).
Most compelling is the connection of the present models with EPR spectroscopy,w hich provides rich and selective information on the electronic structure of the OEC.T he S 1 state is typically discussed as as ingle entity in structural studies,but observations from EPR spectroscopy have always pointed toward heterogeneity.T he first reported EPR signal of the S 1 state was af eatureless signal around g = 4.8, [53,54] observed in spinach. Adifferent signal at g % 12 with resolved Mn hyperfine interactions was later reported for cyanobacterial OEC. [55] This signal could also be induced in spinach by removal of two extrinsic PSII proteins, [56] showing that the spectroscopic response of the OEC in the S 1 state is sensitive to small structural perturbations.D exheimer and Klein suggested that the state giving rise to the S 1 g = 4.8 signal converts to the S 2 g = 2multiline state but does not correlate with the S 2 g = 4.1 signal, [53] while Gregor and Britt showed that the S 2 g = 4.1 signal preferentially decays to the S 1 multiline centered at g % 12. [57] Sioros et al. supported that the S 1 Y Z C component with the characteristic EPR signal at g = 2.035 advances to the S 2 multiline,w hile a2 6G wide signal advances to the g = 4.1 conformation. [58] EPR investigations of the g = 2.035 component of the S 1 Y Z C at X-band and Wband frequencies revealed that the major features of the spectra are due to the interaction of the S = 1state of the Mn 4 cluster with the S = 1/2 spin of Y Z C. [59] These observations are consistent with the idea that the S 1 state consists of two isomers that are the predecessors of the g = 2org = 4.1 forms of the S 2 state.T he two EPR signals described above remain without explicit atomistic explanation and even their common origin has been questioned. [55] Here we show that they are both attributable to the S 1 state and have ao ne-to-one correspondence with the Jahn-Teller isomers described above.
To examine possible connections between the S 1 A /S 1 B Jahn-Teller isomerism and available EPR data, we calculated local zero field splitting tensors (ZFS) of Mn1(III) and Mn4(III) ions using the multireference local complete active space configuration interaction (L-CASCI) approach. [60] The asymmetry of valence electronic configuration of Mn III is connected to ap ronounced Jahn-Teller distortion that produces sizable site anisotropy,c ontrary to the small site anisotropy (on the order of 0.3 cm À1 )o fM n IV ions.C onsequently,Mn1(III) and Mn4(III) largely determine the total anisotropy of the complex. Calculated on-site axial and rhombic anisotropy parameters are presented in Table S2 and orientations of the principal axes of the onsite D SOC for each Mn III ion are depicted in Figure 4a.The j D j values of all Mn III ions are ca. 3cm À1 ,w hich indicates that perturbations on  Figure S5. Right: Simulation of the S 1 EPR signal centered at g eff % 12, using S = 3, D = 6.67 cm À1 , E/D = 0.048.

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Forschungsartikel octahedral symmetry have limited impact on the axial part of the local anisotropy.D ifferences between the coordination environment of Mn1 and Mn4 are reflected in the rhombic ZFS values (E/D), as Mn1 has lower rhombicity than Mn4 due to higher asymmetry of the Mn4 coordination sphere. Additionally,the D i ðÞ z component of each Mn III ion is collinear with its Jahn-Teller axis.
According to the computed ladder of spin eigenstates, S 1 A is diamagnetic and the observed signal derives from al owlying magnetically excited state with S = 1, while S 1 B has an S = 3g round state and the first excited state is likely not populated at low temperatures.Based on the computed local ZFS tensors and the ground state spin configurations predicted by the pairwise exchange coupling constants,w e calculated total ZFS tensors for structures S 1 A and S 1 B (see Supporting Information) and further examined whether the aforementioned spin configurations of the S 1 isomeric forms and their ZFS parameters reproduce the experimental Xband EPR spectra in parallel mode.T he S 1 EPR spectrum at g eff % 4.8 is indeed reproduced for S = 1 (Figure 4b,left) with ar ange of D and E/D parameters shown in Figure S5 that agree with the computed ones.I ti sn oted that this signal cannot be described with E/D < 0.17. This is consistent with the rhombic symmetry expected for S 1 A owing to the presence of the Jahn-Teller axis of each Mn III at the xy-plane of the other. In addition, a S = 3configuration with E/D = 0.048 and D very close to the calculated value of 6.7 cm À1 reproduces the experimental derivative around g eff % 12 (Figure 4b, right). Theh yperfine structure of the multiline signal can additionally be reproduced using an effective hyperfine coupling constant for all Mn ions ( Figure S6).
Thepresent results establish that the Jahn-Teller isomers provide as imple and direct interpretation of both observed EPR signals and hence form ab asis for revisiting and reinterpreting al arge body of experimental spectroscopic work on anew atomistic basis.

Mechanistic Implications for Catalytic Progression
Themost important implication of Jahn-Teller isomerism in the S 1 state is the functional role in the emergence of valence isomerism in the S 2 state.T he S 1 isomers differ in ac rucial electronic structure feature that is relevant for catalytic progression to the next state.T he Mn1 d z 2 orbital is lower in energy in S 1 A because the Mn1-O5 antibonding interaction is minimized by movement of O5 away from Mn1 to form the Mn4-O5 bond. As the Mn1-O5 distance decreases, the Mn1 d z 2 orbital is destabilized and the Mn4-centered d z 2 orbital is similarly stabilized in S 1 B .T he relative stabilities between the Mn1 and Mn4 valence orbitals suggest that oxidation of S 1 A involves oxidation of Mn4, leading to the oxidation state distribution III-IV-IV-IV in the S 2 state (i.e. the S 2 A model), whereas oxidation of S 1 B favors oxidation of Mn1, leading to the S 2 B model with ap endant Mn4(III) ion that retains its Jahn-Teller axis oriented towards O5. .I n conclusion, the emergence of valence isomers in the S 2 state is ad irect consequence of orientational Jahn-Teller isomerism in the dark-stable S 1 state of the enzyme.T he diagram in Figure 5s ummarizes our suggestion for the correspondence of EPR observations with Jahn-Teller or valence isomeric forms in the S 1 and S 2 states of the OEC.
In addition to structural and spectroscopic implications, orientational Jahn-Teller isomerism in the S 1 state offers an ew perspective for the analysis of substrate exchange kinetics. [61] Thep osition of O5 is most affected by the Jahn-Te ller reorientation at Mn4, being at bonding distance to Mn4 in S 1 A but at non-bonding distance to either of the terminal Mn ions in S 1

B
.IfO5isasubstrate, [62,63] then it can be surmised that it would exchange faster in the S 1 B model, positioned along the Jahn-Teller axes of both Mn1 and Mn4, as opposed to the S 1 A model, where it is more strongly bound to Mn4.

Conclusion
Despite the asymmetric coordination sphere of the pendant manganese ion in the dark-stable S 1 state,t he OEC of PSII accommodates stabilization of two distinct minima that correspond to different orientations of the Mn4(III) Jahn-Teller axis of tetragonal elongation. Thet wo isomers provide as tructure-based interpretation of spectroscopic Figure 5. Left:C orrelation of Mn1 (blue line) and Mn4 (orange line) Mulliken spin populations with the Mn1-O5 distance for one-electron oxidized S 1 models derived from constrained geometry optimizations. Right:C orrespondence between S 1 and S 2 isomer structures, showing how orientational Jahn-Teller isomerism in the dark-stable S 1 state of the OEC leads upon oxidation to distinct oxidation state distributions in the S 2 state. The scheme indicates known EPR signals of the two states and metalloradical S 1 Y Z C intermediates, [58] showing how the isomeric forms accommodate spectroscopicobservations into one coherent structurali nterpretation.
observations relating to the known EPR heterogeneity of the S 1 state and of the S 1 -S 2 intermediates,b ecause they fully rationalize the combined effect of local anisotropies in producing distinct EPR signals.C rucially,t he locus of oxidation is different for each Jahn-Teller minimum and hence oxidation of each S 1 isomer leads to adistinct oxidation state distribution in the S 2 state.T his explains how valence isomerism arises in the S 2 state,aphenomenon implicated in control of substrate access in the later stages of the catalytic cycle.T he connection of orientational Jahn-Teller isomerism with redox isomerism suggests that the natural system may have leveraged aunique electronic feature of the Mn III ion to evolve an unprecedented mechanism for enabling and utilizing electronic structure fluxionality as an element of catalytic control.