The Basic Properties of the Electronic Structure of the Oxygen-evolving Complex of Photosystem II Are Not Perturbed by Ca2+ Removal*

Background: EPR/55Mn ENDOR spectroscopy of the oxygen-evolving complex (OEC) and Mn2+ in Ca2+-depleted photosystem II. Results: Electronic model of the Ca2+-depleted OEC; characterization of Mn2+ binding. Conclusion: Ca2+ is not critical for maintaining the electronic and spatial structure of the OEC. Its removal exposes a Mn2+ binding site supposedly in an extrinsic subunit. Significance: Mechanistic implications for water oxidation; Mn2+ in photoassembly/D1 protein repair. Ca2+ is an integral component of the Mn4O5Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S2′ state of the Ca2+-depleted cluster from spinach is examined by X- and Q-band EPR and 55Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca2+ removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S2′ state in spinach PS II is very similar to the native S2 state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca2+ a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between YZ and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel 55Mn2+ signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca2+-depleted PS II. Mn2+ titration, monitored by 55Mn ENDOR, revealed a specific Mn2+ binding site with a submicromolar KD. Ca2+ titration of Mn2+-loaded, Ca2+-depleted PS II demonstrated that the site is reversibly made accessible to Mn2+ by Ca2+ depletion and reconstitution. Mn2+ is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn4O5Ca cluster during photoassembly and/or D1 repair.

Mn ENDOR, revealed a specific Mn 2؉ binding site with a submicromolar K D . Ca 2؉ titration of Mn 2؉ -loaded, Ca 2؉ -depleted PS II demonstrated that the site is reversibly made accessible to Mn 2؉ by Ca 2؉ depletion and reconstitution. Mn 2؉ is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn 4 O 5 Ca cluster during photoassembly and/or D1 repair.
The oxygen-evolving complex (OEC) 5 of photosystem II (PS II) catalyzes the light-driven oxidation of water. The OEC contains an inorganic Mn 4 O 5 Ca metallocofactor that includes five -oxo bridge linkages and is coordinated by a framework of surrounding amino acids (1-6) in a highly defined manner that confers catalytic function. The redox-active tyrosine residue Y Z (D1-Tyr-161) enables electron transfer from the Mn 4 O 5 Ca cluster to P 680 . ϩ , the radical cation formed upon photon absorption and charge separation. The Mn 4 O 5 Ca cluster undergoes four successive oxidations, cycling through a series of different net valence states, referred to as the S i states (where i ϭ 0 -4 denotes the number of oxidizing equivalents stored in the cluster). The transient state S 4 spontaneously returns to S 0 upon regaining four electrons from the two substrate water molecules, which in the process form molecular oxygen. The release of O 2 is followed by the rebinding of at least one H 2 O molecule (for reviews, see Refs. [7][8][9][10][11][12][13][14]. X-ray crystallographic structures of the PS II protein complex provided an atomic picture of the structure of the OEC (1)(2)(3)(4)(5)(6), identifying all amino acids that ligate the Mn 4 O 5 Ca cluster. The metallocofactor resembles a distorted chair, consisting of the cuboidal moiety Mn 3 O 3 Ca (Mn B3 Mn C2 Mn D1 ), 6 with the fourth, outer manganese ion (Mn A4 ), connected to the cuboid via an additional -oxo bridge (O4) to one of the manganese vertices (Mn B3 ). The reported cluster is likely modified due to photoreduction of the Mn III and Mn IV ions, such that the Mn-Mn and Mn-Ca distances seen in the x-ray structure are all elongated as compared with those derived from extended x-ray absorption fine structure (EXAFS) measurements (15). Allowing for this, the basic topology of the x-ray structure is similar to earlier literature models, including the geometry-optimized density functional theory (DFT) models of Kusunoki (16), Siegbahn (17), and the recent model of Ames et al. (18), in which the cuboid exhibits an open conformation with Mn A4 connected to Mn B3 via a di--oxo bridge (Fig. 1).
The Ca 2ϩ ion of the Mn 4 O 5 Ca cluster, which can be removed from and reconstituted into the OEC (19 -21), is essential for catalytic function (19 -23). The non-catalytic Ca 2ϩ -depleted OEC cannot complete the S state cycle, advancing only to a modified S 2 state, termed S 2 Ј (24, 25). The reason for this remains unclear. However, four basic explanations exist in the current literature based on the proposed role(s) for the Ca 2ϩ ion during the S state cycle (for reviews, see Refs. 26 -28). These include the following: (i) As an integral component of the OEC (6), the Ca 2ϩ ion can be suspected to be of crucial structural importance. However, EXAFS experiments suggest that Ca 2ϩ depletion leads to only a small spatial reorganization of the remnant Mn 4 O 5 cluster (29). (ii) It facilitates fast one-electron transfer from the OEC to Y Z . ϩ (for reviews, see Refs. 11 and 30). The formation of the S 2 Ј state requires long visible light illumination at temperatures Ն0°C. This is in contrast to the native S 2 state, which can be generated via visible light illumination at Ϫ78°C. This apparent increase in the activation energy of OEC turnover upon Ca 2ϩ removal may represent a decoupling of the Y Z . ϩ from the OEC, such that Ca 2ϩ -mediated protein conformational changes and/or H ϩ translocations associated with physiological S state transitions are blocked. (iii) It is a binding/ staging site for substrate water and its deprotonation (26,31). The kinetics of substrate water binding to the OEC are affected by biochemical exchange of Ca 2ϩ with Sr 2ϩ , the only surrogate ion able to confer catalytic activity (19,23,32). It can presumably act in place of Ca 2ϩ as it has approximately the same size and a similar Lewis acidity (31). This result has been interpreted as evidence for Ca 2ϩ binding one of the substrate waters. Inhibition due to Ca 2ϩ depletion would then reflect the loss of a substrate binding site. (iv) Although the basic structural arrangement of manganese ions in the cluster is retained upon Ca 2ϩ removal, it is uncertain if their magnetic and/or electronic interactions are per-turbed, which could lead to a decoupling of the cluster or a rearrangement of the manganese valence states. Thus, Ca 2ϩ depletion could potentially change the redox properties as well as substrate and/or protein interactions of the complex, inhibiting catalytic function.
The Mn 4 O 5 Ca cluster in the S 2 state exhibits a characteristic multiline EPR signal centered at g Ϸ 2 (33) that arises from an S ϭ 1/2 ground spin state of the cluster. Under certain conditions (illumination, reactants), additional signals are observed at higher g values; in spinach, a second broad signal can be detected at g Ϸ 4.1 (34,35), attributed to an S ϭ 5/2 spin state (36). These signals are affected by the presence of small alcohols, foremost methanol (MeOH) (37)(38)(39)(40)(41), which enhance the intensity of the multiline signal at the expense of the g Ϸ 4.1 signal (37) (for a full discussion see Ref. 41). The Mn 4 O 5 cluster in the S 2 Ј state also exhibits a multiline signal; however, its hyperfine splitting pattern is perturbed. It contains a larger number of resolved lines as compared with the native S 2 multiline signal, with a smaller average line spacing (5.5-6 versus 8.8 mT). The magnetic interaction between Y Z ⅐ and the OEC is also perturbed in Ca 2ϩ -depleted PS II as evidenced by changes in the tyrosine split signal of the S 2 ЈY Z ⅐ state (24,25).
A detailed understanding of the electronic structure of the Mn 4 O 5 Ca cluster in the S 2 state has been developed from pulse EPR data (42)(43)(44)(45)(46), in particular 55 Mn electron nuclear double resonance (ENDOR). These experiments demonstrated that the four manganese ions contribute about equally to the ground electronic state of the S 2 state; i.e. all four manganese ions carry approximately the same spin density. This requirement allows an assessment of the electronic exchange interactions between the four manganese ions and the development of Mn 4 coupling schemes. These necessarily reflect the geometric structure of the OEC and allow the assignment of the individual manganese oxidation states. Our recently proposed model for the S 2 state (18) is described under "Discussion." This scheme places the only Mn III ion inside the cuboidal unit (Mn D1 ) (see also Ref. 47) and compares favorably with information from complementary spectroscopic measurements (48 -50).
Although it has not been directly observed by EPR spectroscopy, the possibility of another paramagnetic manganese species being able to bind to the Ca 2ϩ -depleted PS II has been suggested in an earlier study by Booth et al. (51). The additional species was suggested to be a Mn 2ϩ ion that can bind specifically to a site in the protein complex that is created or becomes accessible via structural changes in the course of Ca 2ϩ removal. This was based on the observation that, after equimolar amounts of Mn 2ϩ ions had been added to Ca 2ϩ -depleted PS II, no Mn 2ϩ was observed by X-band continuous wave (CW) EPR. Upon titrating Ca 2ϩ ions back into these samples, Mn 2ϩ was released as seen from the appearance of the six-line Mn 2ϩ EPR signal.
In this work, both the spin system of the Mn 4 O 5 cluster in the S 2 Ј state of Ca 2ϩ -depleted PS II and the binding of Mn 2ϩ ions to this protein were studied by EPR and ENDOR spectroscopy at X-and Q-band frequencies. The results provide new insight into the role of the Ca 2ϩ ion in the native OEC.

EXPERIMENTAL PROCEDURES
Sample Preparation-PS II-enriched thylakoid membranes were prepared from spinach based on the procedure of Berthold et al. (52) using detergent treatment by incubation with Triton X-100 for 15 min. All work was performed in the dark or very dim green light, and the PS II was kept at 4°C before storage in the dark at Ϫ80°C or in liquid N 2 . Chlorophyll concentrations were determined by assays using aqueous acetone (80%) extracts (53) with updated extinction coefficients (54) using an ATI Unicam UV-visible spectrometer UV2-300.
Ca 2ϩ depletion and reconstitution based on the low pH/citrate treatment method (21) was achieved as described previously (55). The final buffer used was 50 mM MES, 15 mM NaCl, 0.4 M sucrose, 1 mM EDTA, pH 6.5. Ca 2ϩ removal and, as a proof for the integrity of the OEC, Ca 2ϩ rebinding was confirmed both by enzymatic assays and by X-band CW EPR. The O 2 evolution rates of native PS II were ϳ400 mol O 2 /mg of chlorophyll/h (see the following section). O 2 evolution rates dropped to 5-10% in Ca 2ϩ -depleted and were reactivated to Ͼ80% in Ca 2ϩ -reconstituted samples. Similar percentages of the S 2 multiline signal were observed after white light illumination with a tungsten lamp through an aqueous 5% CuSO 4 IR filter of the respective samples at 200 K for 5 min (Table 1, Fig.  2A). These numbers are consistent with previous literature reports (25,29,56).
Advancement of dark-adapted S 1 Ј state EPR samples to the S 2 Ј and S 2 ЈY Z ⅐ states (25) was done by illumination at 0°C for 3 min, with 125 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea (10 mM in dimethyl sulfoxide) added to the samples advanced to the S 2 Ј state, which restricts the acceptor site and, thus, Y Z to one turnover.
For Ca 2ϩ and Mn 2ϩ titration experiments, dark-adapted Ca 2ϩ -depleted PS II membranes were rebuffered in EDTA-free buffer by three cycles of dilution, centrifugation at 39,000 ϫ g for 15 min, and resuspension using 50 mM MES, 15 mM NaCl, 5 mM MgCl 2 , 0.4 M sucrose, pH 6.5. The final concentration of PS II reaction centers (RCs) in the samples was 28 Ϯ 3 M based on a chlorophyll concentration of 6.3 Ϯ 0.8 mg ml Ϫ1 and assuming 250 chlorophylls/RC (57) after 15 min Triton X-100 treatment. The samples were incubated with known amounts of Mn 2ϩ ranging from 0 to 4 eq per RC for 2 h. For the Ca 2ϩ titration, samples containing 0.8 added eq of Mn 2ϩ were incubated with known amounts of Ca 2ϩ between 0 and 2400 eq for one additional hour. Mn 2ϩ and Ca 2ϩ ions were added from stock solutions of their chlorides.
Oxygen Evolution Measurements-Steady state PS II enzyme activity at 25°C was determined by polarographic measurement of the O 2 concentration in a PS II-containing assay mixture using a Clark-type Hansatech oxygen electrode with a high sensitivity Teflon membrane under continuous illumination with a tungsten lamp through an aqueous 5% CuSO 4 IR filter. The assay medium was the buffer of the samples lacking EDTA and with 5 mM MgCl 2 and 0.2 mM phenyl-p-benzoquinone (20 mM in dimethyl sulfoxide) added as an electron acceptor.
EPR/ENDOR Spectroscopy-X-band CW EPR spectra were recorded on a Bruker ELEXSYS E500 spectrometer equipped with an ESR900 liquid helium flow cryostat and an ITC503 helium flow temperature controller (Oxford Instruments Ltd.). X-band pulse experiments were performed with a Bruker ESP a Determined as an average of at least 8 single measurements at a minimum of 2 different chlorophyll concentrations from 5 to 25 g/ml. b Determined from the peak-to-trough distances of four prominent derivative peaks in the CW EPR spectrum (100).

FIGURE 2. EPR and ENDOR experimental spectra (black solid traces) and simulations (red dashed traces).
A, X-band CW EPR of PS II isolated from spinach. Shown are the Ca 2ϩ -depleted OEC poised in the S 2 Ј state (a) and the native (b), Ca 2ϩ -depleted (c), and Ca 2ϩ -reconstituted OECs (d) illuminated at 200 K. In the experimental spectrum, the region of the overlapping Y D ⅐ signal (g Ϸ 2) was omitted for clarity. In a, a fourth order polynomial, and in b-d, a background signal of the resonator cavity were subtracted from the raw data. . a and b were smoothed using a 9-and 5-point moving average, respectively. b is the difference of an S 2 Ј state spectrum after illumination at 0°C minus an S 1 Ј state spectrum after dark adaptation (supplemental Fig. S2) to remove an overlapping Mn 2ϩ signal. The Mn 2ϩ signal is attributed to residual Mn 2ϩ ions stemming from a small fraction of damaged manganese clusters. In X-band pulse EPR (not shown) and ENDOR spectra (a), Mn 2ϩ contributions were avoided by optimizing the MW pulse lengths for the S 2 Ј state signal of the Mn 4 O 5 spin system with an S ϭ 1/2 ground spin state. Experimental parameters: a and b, MW frequencies, 9.717 GHz (X-band), 34.033 GHz (Q-band); shot repetition rate, 5 ms; MW pulse lengths , 12 ns (X-band), 72 ns (Q-band); , 200 ns (X-band), 440 ns (Q-band); magnetic fields, 380 mT (X-band), 1208 mT (Q-band); RF pulse length RF , 4 s; temperature, 5 K; c-e, see Refs. 45 and 49. 380E spectrometer equipped with a dielectric ring resonator, an Oxford ITC liquid helium flow system, and a temperature controller. Q-band pulse experiments were performed using a Bruker ELEXSYS E580 spectrometer equipped with a laboratory-built cylindrical ENDOR resonator (58), a CF935 cryostat, and an ITC5025 temperature controller (Oxford Instruments Ltd.). Field-swept electron spin echo (ESE)-detected experiments were performed at Q-band frequencies using the pulse sequence /2--/2--echo with ϭ 72 ns and ϭ 440 ns. For 55 Mn Davies ENDOR, the pulse sequence was -RF -T-/2--/2--echo, with ϭ 12 ns (X-band), 72 ns (Q-band), or 16 ns (Q-band Mn 2ϩ titration/quantification), RF ϭ 4 s (X-, Q-band) or 4.5 s (Q-band Mn 2ϩ titration/quantification), T ϭ 3.4 s (X-, Q-band) or 1 s (Q-band Mn 2ϩ titration/quantification), and ϭ 200 ns (X-band), 440 ns (Q-Band), or 320 ns (Q-band Mn 2ϩ titration/quantification). The radio frequency (RF) was varied randomly in the desired range, and the RF pulses were amplified by an ENI 5100L amplifier. Except for Mn 2ϩ titration/quantification, 55 Mn Davies ENDOR spectra were collected using a home-built computer console with Spec-Man control software (59) coupled to an SMT02 external RF pulse generator.
EPR/ENDOR Spectral Simulations-Simulations of EPR and 55 Mn ENDOR spectra were performed numerically using the EasySpin software package (60). The fitting procedures employed a least squares minimization routine. All tensors were set to be collinear. The Ca 2ϩ -depleted Mn 4 O 5 cluster in the S 2 Ј state was treated as an effective electronic spin S ϭ 1/2 ground state coupled to the four 55 Mn nuclei, described by the following spin Hamiltonians for the EPR (Equation 1) and 55 Mn ENDOR (Equation 2) spectra.
The EPR spectrum was calculated using second order perturbation theory, neglecting nuclear Zeeman terms and forbidden transitions. The 55 Mn ENDOR spectra were calculated exactly, including nuclear Zeeman terms and considering all transitions. For the monomeric Mn 2ϩ ion (S ϭ 5/2, I ϭ 5/2) bound to the Ca 2ϩ -depleted PS II, the following spin Hamiltonian was solved exactly both for the ESE and ENDOR spectra: For details on the simulation procedure and the theoretical background, see Refs. 46, 49, and 61.
Temperature-dependent CW EPR Signal Intensity-The temperature was calibrated using a thermometer in place of the sample in the EPR tube. To guarantee that the actual unsaturated intensity I 1 of the S 2 Ј state modified multiline, as the ground state signal, was measured at all temperatures, the saturation behavior was studied at the lowest temperature employed. As a result, the non-saturating microwave (MW) power of 0.1 milliwatt was used throughout. The intensities I 1 of the derivative signals were measured by means of the heights of 19 peaks throughout the spectral range, thereby minimizing statistical errors and contributions of underlying broader signals, such as from cytochrome b 559 and the semiquinone-iron complex. How the ground-to-first excited state energy difference ⌬ is determined from the temperature dependence of I 1 is outlined in the supplemental data.
Quantification of the Relative Concentrations of PS II-bound Mn 2ϩ and Hexaquo-Mn 2ϩ -The Mn 2ϩ species in Ca 2ϩ and Mn 2ϩ titration samples were quantified by means of their Q-band 55 Mn Davies ENDOR spectra in two ways, and the results were averaged. (i) The relative contributions of the spectra from the pure Mn 2ϩ species needed to reproduce the spectra from the various titration points were determined. The spectra from Mn 2ϩ already present in the Ca 2ϩ -depleted PS II samples without the addition of Mn 2ϩ ions and from 40 M MnCl 2 dissolved in the titration buffer represented PS II-bound and hexaquo-Mn 2ϩ , respectively. (ii) The relative amplitudes of the 55 Mn ENDOR m S ϭ Ϫ3/2 transitions, which appear in different RF ranges characteristic for the two Mn 2ϩ species, were quantified in the regions of 353-376 MHz for PS II-bound Mn 2ϩ and 390 -395 MHz for hexaquo-Mn 2ϩ .

EPR and 55 Mn ENDOR of the Ca 2ϩ -depleted Mn 4 O 5 Cluster
in the S 2 Ј State-The characteristic modified multiline CW EPR signal (24,25) was observed for Ca 2ϩ -depleted PS II samples poised in the S 2 Ј state. It is centered at g Ϸ 2 and spans the magnetic field range from ϳ260 to ϳ430 mT, resolving at least 27 hyperfine interaction (HFI) lines with an average peak-topeak spacing of ϳ6 mT ( Fig. 2A). The central HFI lines are superimposed by the signal of the stable tyrosyl radical Y D ⅐ centered at g Ϸ 2, which is not depicted for clarity of presentation. The broad underlying signal of the reduced Q A Ϫ · Fe 2ϩ complex (62) contributes in the 350 -375-mT region (24,25,29).
Traces a and b in Fig. 2B show the X-and Q-band Davies ENDOR spectra of the S 2 Ј state recorded at 5 K and magnetic fields of 380 and 1208 mT, respectively. The 55 Mn ENDOR spectrum of the Mn 4 O 5 cluster in the S 2 Ј state is essentially invariant across the corresponding EPR signal envelope (supplemental Fig. S1). It is ϳ130 MHz wide, extending over a range from ϳ60 to ϳ190 MHz. As compared with the 55 Mn ENDOR spectrum of the native S 2 state (Fig. 2Bc, supplemental Fig. S1), the Ca 2ϩ -depleted S 2 Ј state spectrum is broader. The edges of the spectrum change up to 10 MHz, resulting in a ϳ20 and ϳ10 MHz increase in the width of the X-and Q-band 55 Mn ENDOR spectra, respectively, as compared with the Ca 2ϩ -containing S 2 state of spinach PS II (42,45,46). The Q-band spectrum of the S 2 Ј state exhibits five clearly resolved peaks, as also seen for the native S 2 state spectrum from Thermosynechococcus elongatus (Fig. 2Bd); however, their positions differ slightly.
The X-band CW EPR and X-and Q-band 55 Mn Davies ENDOR spectra were simultaneously simulated using the spin Hamiltonian formalism (for details see "Experimental Proce-dures" and Refs. 46 and 49). In these simulations, the S 2 Ј state Mn 4 O 5 cluster is treated as an effective S ϭ 1/2 electronic spin state coupled to the four 55 Mn nuclei, the same as for the native S 2 state (41,42,46,48,49,(63)(64)(65)(66). This approach requires the ground electronic spin state to be well separated from higher states, as is experimentally observed (see the following section). The simulations reproduce all the major spectral features of the EPR and 55 Mn ENDOR spectra (Fig. 2, dashed red traces).
The isotropic and anisotropic values of the fitted effective 55 Mn HFI tensors A i (i ϭ 1-4) are given in Table 2. For means of comparison, the numbers for the native S 2 state from spinach (46) and the native and Sr 2ϩ -substituted S 2 states from T. elongatus (49) are also listed. A full set of G and HFI tensor components is listed in supplemental Table S1. As seen for the Mn 4 O 5 Ca/Sr clusters, four effective HFI tensors are required to simulate the Mn 4 O 5 cluster spectra. Their magnitudes are on the order seen for mono-and dimeric Mn 3ϩ and Mn 4ϩ complexes. Hence, their individual spin projection coefficients i must be on the order of 1 (see Ref. 49). In contrast to preliminary simulations of the S 2 Ј spectra (67) or others on the S 2 state from spinach PS II (46), the HFI tensors were not constrained to axial symmetry. However, as was found previously in simulations of the Mn 4 O 5 Ca/Sr clusters in T. elongatus (49), the tensors nevertheless show a considerable degree of axial symmetry. Moreover, these four OEC clusters show the same geometries of their HFI tensors, with larger axial than equatorial tensor components (A aniso Ͻ 0) for A 2 Ϫ A 4 and vice versa for the largest HFI A 1 (A aniso Ͼ 0).
Spin State Energies of the Ca 2ϩ -depleted Mn 4 O 5 Cluster in the S 2 Ј State-The energy difference ⌬ of the paramagnetic ground spin state and the first exited state was estimated from the temperature dependence of the unsaturated X-band CW modified multiline signal of the Ca 2ϩ -depleted S 2 Ј state. The measured intensities I 1 of the derivative signal at a series of temperatures are depicted in a Curie plot versus 1/T in Fig. 3. This relation is approximately linear over the measured range from 14.4 to 5.5 K and extrapolates to 0 for T 3 ∞. This Curie behavior of the temperature dependence indicates that the Ca 2ϩ -depleted S 2 Ј state features an S ϭ 1/2 ground spin state energetically well separated from states of higher spin multiplicity. The temperature dependence of the S 2 Ј modified multiline signal can be reproduced reasonably well with ⌬ Ն 35 cm Ϫ1 corresponding to J eff Ն 12 cm Ϫ1 (see "Experimental Procedures"). This relatively large separation from states of higher spin multiplicity allows the S 2 Ј state Mn 4 O 5 spin system to be treated in the strong exchange limit, i.e. as an effective S ϭ 1/2 spin state, as assumed in the previous section.
EPR and 55 Mn ENDOR of a Specifically Bound Mn 2ϩ Ion-The Ca 2ϩ -depleted PS II preparations exhibit an additional EPR and ENDOR signal in all accessible SЈ states that is not present in native PS II samples. At 5 K, Q-band ESE-detected field sweep EPR spectra of the dark-adapted Ca 2ϩ -depleted PS II preparations (S 1 Ј state), in which the Mn 4 O 5 cluster does not show a perpendicular mode EPR signal, displayed a broad EPR signal centered at g ϭ 1.99 with a full width at half-maximum of ϳ63 mT (Fig. 4, inset). A corresponding signal was not observed using CW X-band EPR spectroscopy; the signal is probably too broad to be discerned from the base-line drift in the CW EPR experiment (51). Q-band Davies ENDOR spectra were recorded at several magnetic fields in the RF frequency range of 30 to 400 MHz (Fig. 4). The 55 Mn ENDOR spectra are dominated by two broad peaks between 100 -195 MHz and another line centered at ϳ370 MHz. The two lines at 100 -195 MHz are  ‫؍‬ 1-4) used for the simulations of the X-and Q-band EPR and ENDOR spectra of the Ca 2؉ -depleted PS II from spinach in the S 2 state (Fig. 2) The anisotropy in the A i values is expressed as the difference A i,aniso ϭ A Ќ Ϫ A ʈ between the equatorial and axial components of the tensor. The equatorial and axial A i values are defined as  dependent on the magnetic field and shift to higher frequencies with increasing magnetic field. The spectra also contain sharp proton signals, one centered at the 1 H Larmor frequency (ϳ50 MHz) and a strongly coupled one at ϳ75 MHz with decreasing amplitude at increasing field positions. Its partner at low frequency (ϳ25 MHz) lies outside the spectral range. No further low frequency signals were detected for this species using either ENDOR or electron spin echo envelope modulation (ESEEM). These EPR and 55 Mn ENDOR signals can be readily assigned to high spin Mn 2ϩ with S ϭ 5/2, although their appearance is different from the spectra typically associated with Mn 2ϩ complexes (see Discussion and Fig. 5A). Simultaneous simulations of the EPR and of four ENDOR spectra at different magnetic fields (Fig. 4, dashed red traces) are consistent with this assignment. They reproduce both the spectral breadth and line shape of the EPR absorption signal and the peaks in the four 55 Mn ENDOR spectra. Besides a near-isotropic G tensor (principal values 1.983, 1.996, 2.002), the simulations yielded an almost isotropic HFI tensor with the principal components A x ϭ 256 MHz, A y ϭ 260 MHz and A z ϭ 257 MHz, resulting in an isotropic average A iso of 258 MHz. In addition, the simulations required a large fine structure parameter D ϭ Ϫ2355 MHz with a pronounced rhombicity ϭ E/D ϭ Ϫ0.38 of the zero-field splitting (ZFS). It is noted that the predominant contribution to the width of the EPR and ENDOR signals is the large and rhombic ZFS interaction (more information on the effect of the ZFS can be found in the supplemental data and Ref. 61). Hence, considering the good agreement of the measured and calcu-lated EPR and ENDOR signals (Fig. 4), the optimized fine structure parameter D can be considered robust; i.e. a single set of D and E values is sufficient to rationalize the data. The fact that the inclusion of a distribution of the ZFS parameters is not required indicates that there are only small site-to-site inhomogeneities of the Mn 2ϩ ligand sphere. Therefore, we propose that the Mn 2ϩ ion is bound to one specific site in Ca 2ϩ -depleted PS II.
Mn 2ϩ and Ca 2ϩ Titration Experiments-To further investigate the Mn 2ϩ species described in the previous section, Mn 2ϩ and Ca 2ϩ titration experiments of Ca 2ϩ -depleted PS II samples

. Q-band pulse ESE-detected field-swept EPR (inset) and Davies ENDOR experimental spectra (black solid traces) and simulations (Sim.; red dashed traces) of the Mn 2؉ ion bound to Ca 2؉ -depleted PS II isolated from spinach and poised in the S 1 state.
In the EPR spectrum, the region of the overlapping Y D ⅐ EPR signal (g Ϸ 2) is not displayed for clarity and was omitted in the simulations. The arrows indicate the four magnetic fields at which the ENDOR spectra were measured. Experimental parameters: MW frequency, 34.07 GHz; shot repetition rate, 5 ms; MW pulse length , 72 ns; , 440 ns; Davies ENDOR, magnetic fields, 1195, 1208, 1224, 1260 mT (top to bottom); RF pulse length RF , 4 s; temperature, 5 K. were performed, monitoring the CW EPR and ENDOR signal described above.
Mn 2ϩ /Ca 2ϩ Titration Monitored by CW EPR-Mn 2ϩ ions were added to Ca 2ϩ -depleted PS II samples and the characteristic S 2 ЈY Z ⅐ state split signal, S 2 Ј multiline signal, and hexaquo-Mn 2ϩ signal (not shown) were measured. The addition of Յ0.8 eq of Mn 2ϩ ions relative to the number of PS II RCs did not quantitatively alter the three signals. The Mn 2ϩ ions added are CW EPR-silent, as seen in the study of Booth et al. (51), which is consistent with a protein-bound Mn 2ϩ species. In addition, this species does not cause any line broadening or even splitting of the signals from the OEC or the tyrosyl radicals. The addition of Ն0.8 equivalents of Mn 2ϩ ions resulted in the appearance of the hexaquo-Mn 2ϩ signal. The subsequent addition of Ca 2ϩ to Ca 2ϩ -depleted, Mn 2ϩ -loaded PS II samples led to a loss of the S 2 ЈY Z ⅐ state split signal and of the multiline signal, as the Ca 2ϩreconstituted Mn 4 O 5 Ca cluster can proceed beyond the S 2 Ј state upon illumination. A concomitant increase of the Mn 2ϩ six-line signal was observed due to the release of the PS IIbound Mn 2ϩ into solution (51).
Mn 2ϩ /Ca 2ϩ Titration Monitored by 55 Mn ENDOR-Mn 2ϩ binding was also directly monitored by Q-band ENDOR. The concentrations of PS II-bound and solubilized Mn 2ϩ ions in each sample were quantified by means of the relative amplitudes of their characteristic 55 Mn ENDOR signals ( Fig. 5A; for the titration curve, see supplemental Fig. S3). Without the addition of MnCl 2 , dark-adapted Ca 2ϩ -depleted PS II (S 1 Ј state) always displayed the PS II-bound Mn 2ϩ signal shown in Fig. 4. The addition of ϳ0.8 eq of MnCl 2 led to a 4 -5-fold increase of this signal with only little free hexaquo-Mn 2ϩ (15 Ϯ 4%) present at the same time. This suggests that ϳ20% of RCs contain a bound Mn 2ϩ before exogenous addition of MnCl 2 so that in the end a total of ϳ1 eq Mn 2ϩ is in the sample. The basal Mn 2ϩ is likely derived from centers damaged during the Ca 2ϩ depletion procedure and nominally corresponds to the loss of ϳ5% Mn 4 O 5 (Ca) clusters. The high occupancy of the Mn 2ϩ site suggests that it is of high affinity, with a dissociation constant K D that is too small to be determined here. From the employed concentrations of the binding partner, K D is expected to be in the submicromolar/nanomolar range. It is also noted that the addition of the chelating agent EDTA did not remove or alter the appearance of the bound Mn 2ϩ signal, consistent with the protein site having a high affinity for Mn 2ϩ .
An additional Ca 2ϩ titration was performed on the fully Mn 2ϩ -loaded Ca 2ϩ -depleted PS II (ϩ0.8 eq of MnCl 2 , i.e. a final ratio of 1 Mn 2ϩ ion per PS II RC). The Ca 2ϩ concentrations ranged from 0 to 2400 eq Ca 2ϩ per RC (0 -60 mM). In Fig. 5B, the relative concentrations of the two Mn 2ϩ species (PS IIbound and solubilized) are plotted against the equivalents of Ca 2ϩ ions added. This behavior could be reproduced by a sigmoid curve with a half-saturation value of 700 Ca 2ϩ ions per RC. This value is similar to 1200 eq of Ca 2ϩ reported in Booth et al. (51). The difference may be due to the Ca 2ϩ depletion method used, the low pH/citrate treatment in this study versus a NaCl salt wash (24) in the study of Booth et al. (51). Their differing effects on the extrinsic PS II subunit composition could alter the Ca 2ϩ binding kinetics (see Refs. 24 and 51).

Location of the Mn 2ϩ Binding
Site-Based on the observations described above (see "Results"), a preliminary assignment can be made as to where the binding site of the Mn 2ϩ ion is located. No strong magnetic interaction was observed between the Mn 2ϩ ion and the Ca 2ϩ -depleted Mn 4 O 5 cluster or the tyrosyl radical Y Z ⅐ in the form of a broadening or splitting of the corresponding EPR signals. Thus, Mn 2ϩ binding directly to the Ca 2ϩ site of the OEC can be excluded. This Mn 2ϩ ion must be at least 10 Å away not to be detectable via dipolar magnetic interaction. A similar argument holds for Y D ⅐ (D2-Tyr-160), as it also displays an unperturbed EPR lineshape when Mn 2ϩ is bound. These "exclusion zones" are indicated by green and violet spheres in Fig. 6A. There is, however, a long range dipolar interaction between the Mn 2ϩ ion and Y D ⅐ as evidenced by the relaxation enhancement of its EPR signal (51). Being smaller than the enhancement resulting from the Mn 4 O 5 Ca cluster in the S 2 state suggests a weaker Mn 2ϩ -Y D ⅐ interaction and thus a longer distance than the 31 Å measured between the cluster and Y D (6).
The binding and titration behavior can either be rationalized by a significant allosteric effect of Ca 2ϩ on the Mn 2ϩ site, or Mn 2ϩ binding could take place directly at a depleted Ca 2ϩ site. The recent crystal structure (6) of PS II from Thermosynechococcus vulcanus exhibits three additional Ca 2ϩ sites at distances greater than 30 Å from the paramagnetic species monitored, i.e. the Mn 4 O 5 (Ca) cluster, Y Z ⅐ , and Y D ⅐ (Fig. 6A). In the structure of PS II from T. elongatus, a different Ca 2ϩ site in PsbO has been identified (4,5,68), not found in the T. vulcanus crystals. All these Ca 2ϩ sites are located on the lumenal/donor side of PS II in the subunit CP47, the cytochrome b 559 subunit ␤ (PsbF), and the extrinsic protein PsbO, and are solvent-accessible. It is not clear, however, whether Ca 2ϩ binding at these sites is solely a crystallization artifact under the conditions used or of physiological relevance. With the exception of the two sites in PsbO, the Ca 2ϩ sites appear to be of low affinity, as the Ca 2ϩ ions are ligated to a large part by H 2 O and glycerol. In contrast, the two Ca 2ϩ sites seen in the PsbO possess at least three ligands from amino acid side chains (Fig. 6, B and C) and thus are potentially of high affinity. In the homologous PsbO from spinach, which has also been reported to bind Ca 2ϩ (69 -71), Asn-197 and Val-198 of the binding motif in Fig. 6B correspond to the conserved residues Ser-286 and Val-287, whereas there is no equivalent for Thr-135. Glu-81, Glu-140, and His-257 in the other binding motif (Fig. 6C) correspond to Glu-146, Glu-205, and Glu-317 (for a sequence alignment, see supplemental Fig. S4). Mn 2ϩ binding to PsbO has indeed been demonstrated previously in isolated PsbO from higher plants (72)(73)(74). As in the present study and Ref. 51, protein-bound Mn 2ϩ did not show a CW EPR signal, but a six-line signal was observed after denaturation of the protein (73). PsbO was reported to show carbonic anhydrase activity, which was maximal in the presence of Mn 2ϩ (74).
The magnetic properties of the Mn 2ϩ ion provide information about the immediate ligand environment in this binding pocket. The D and E values of Mn 2ϩ complexes of higher symmetry, such as Mn 2ϩ -EDTA and hexaquo-Mn 2ϩ , are significantly smaller than those for the PS II-bound Mn 2ϩ described

Electronic Structure of the Ca 2؉ -depleted OEC of Photosystem II
here (see supplemental data and Ref. 61). The large and highly rhombic ZFS reflects an asymmetric coordination sphere. Both the 7-and the 5-fold coordination geometries of the Ca 2ϩ ions in PsbO from the two cyanobacterial species exhibit considerable asymmetry (Fig. 6, B and C). In addition, the large proton coupling seen also suggests the Mn 2ϩ ion to have at least one water ligand. The absence of any smaller coupling, such as from 14 N (not shown), indicates that the Mn 2ϩ ion does not bind to a N-containing ligand residue like histidine. Thus, the absence of a (visible) water and the presence of His-257 as ligands of the Ca 2ϩ ion in T. elongatus PsbO (Fig. 6C) favor Mn 2ϩ binding to the Ca 2ϩ site in PsbO identified in the T. vulcanus crystal structure (Fig. 6B).
PS II from higher plants exhibits an extrinsic subunit composition different from that of the cyanobacterial system. Higher plant lumenal PsbP has been reported to be capable of binding Mn 2ϩ stoichiometrically (75,76). Similar to Ca 2ϩ -depleted PS II in this study and in Ref. 51, isolated PsbP loaded with Mn 2ϩ did not show a Mn 2ϩ X-band CW EPR signal unless it was denatured. A bound Mn 2ϩ could be detected by high field EPR spectroscopy and distinguished from non-specifically attached Mn 2ϩ , similar to the present study. It is noted though that the binding constant reported in Bondarava et al. (76) is probably incorrect; for a discussion, see Ref. 77. Moreover, the Mn 2ϩ ion in PsbP could be (partially) replaced by Ca 2ϩ , and Zn 2ϩ has been found to bind at one of the two proposed Mn 2ϩ sites in PsbP crystals from spinach (PDB accession number 2VU4) and its cyanobacterial homologue CyanoP (78). Mn 2ϩ bound to the PsbP would be at least 30 Å from either the OEC or Y D , again consistent with the distance constraints identified above. Thus, PsbP could also contain the putative site of specific Mn 2ϩ binding in Ca 2ϩ -depleted higher plant PS II.
The physiological role of the putative Mn 2ϩ binding site is the delivery of Mn 2ϩ to the OEC during photoassembly and/or the storage of Mn 2ϩ during the damage/repair cycle of the D1 protein (see Refs. 79 -82). Ca 2ϩ is essential for photoactivation of the OEC. It was suggested to bind at a site within the PS II complex, which leads to a conformational change of the protein pocket where the OEC is assembled (i.e. the C terminus of D1).
Thus, it appears reasonable that in the absence of Ca 2ϩ , it is favorable for the PS II supercomplex to sequester in a site Mn 2ϩ that can be rapidly delivered upon an increase in Ca 2ϩ concentration. In this scenario the lumenal Ca 2ϩ concentration would be a signaling mechanism for OEC assembly and repair.
Spectral Properties of the Mn 4 O 5 Cluster in the S 2 Ј State Compared with Other S 2 State Systems-The appearance of the 55 Mn ENDOR spectra, the fitted 55 Mn HFI tensors, and the ground-to-first excited state energy separation of the Ca 2ϩdepleted S 2 Ј state all fall within the natural spectral variations observed for the native S 2 states in different species (41). This demonstrates that the basic electronic and thus also spatial structure of the Mn 4 O 5 cluster remains intact upon Ca 2ϩ removal. This confirms and further refines observations on the interatomic distances of the manganese ions from earlier EXAFS experiments (29).
The Ca 2ϩ -depleted S 2 Ј state from spinach resembles the native S 2 state from T. elongatus with regard to the spin state energies. Upon removal of the Ca 2ϩ ion, ⌬ increases to Ն35 cm Ϫ1 , which is much larger than for the native spinach S 2 state (⌬ ϭ 3-6 cm Ϫ1 ) but more similar to T. elongatus (⌬ ϭ 12-25 cm Ϫ1 ) (41,83,84). In intact spinach PS II, the energy ladder is sensitive to MeOH addition. The mechanism by which MeOH binding perturbs the electronic structure of the S 2 state was recently discussed in Su et al. (41). In the model proposed, MeOH binding to the OEC increases the electronic coupling of the pending manganese (Mn A4 ) to the cuboidal (Mn B3 ,Mn C2 , Mn D1 ) unit. It is this effective coupling that defines the groundto-first excited state energy difference ⌬ of the S 2 state. Ca 2ϩ depletion appears to have the same effect. However, the addition of MeOH did not modify the appearance of the S 2 Ј state ESE and ENDOR spectra (not shown). It is emphasized though that this effect is of the same size as that of the variation between species and thus is unlikely to be of physiological significance.
Electronic Structure/Exchange Coupling Scheme of the Ca 2ϩdepleted Mn 4 O 5 Cluster in the S 2 Ј State-To further rationalize the spectral results from the Ca 2ϩ -depleted Mn 4 O 5 cluster, a spin coupling scheme for the S 2 Ј state was developed. It was constructed to meet the following requirements: (i) a ground state of spin multiplicity S ϭ 1/2, (ii) a ground-to-first excited state energy difference ⌬ Ϸ 35 cm Ϫ1 , (iii) spin projection factors ͉ i ͉ Ϸ 1 for all four manganese electronic spins, and (iv) intrinsic ZFS constants d i of the manganese ions that lie within the range found for mono-and dimeric model complexes, i.e. 1 cm Ϫ1 Ͻ ͉d͉ Ͻ 5 cm Ϫ1 for Mn III and ͉d͉ Ͻ 0.1 cm Ϫ1 for Mn IV ions in an octahedral ligand environment (see Refs. 18,47,and 49). The inferred structural (29) and spectral similarity of the native and the Ca 2ϩ -depleted manganese cluster suggest that the spin coupling scheme for the native S 2 state (Fig. 7, c ϭ 1) (18), in which Mn D1 is the Mn III ion, can be used as a starting point. Calculated on the basis of the refined model of the OEC in the latest crystal structure (6), the basic arrangement of this scheme is in accordance with the spatial organization as described by Siegbahn and our group (17,18,47,85), in which Mn B3 , Mn C2 , and Mn D1 form a trimeric core unit connected to Mn A4 by a di-oxo bridge via Mn B3 (Fig. 1). Thus, this scheme represents an extension of the (3 ϩ 1)-or Y-coupling schemes, proposed earlier in EPR spectroscopic studies (42,46,47,49) The coupling topology fulfills criteria (i) and (iii) as ground spin state multiplicity and spin projection factors are the same for the two states, S 2 and S 2 Ј. In contrast, their ground-to-first excited state energy differences ⌬ and effective 55 Mn HFI tensors A i , relevant for (ii) and (iv), are different. Thus, the ⌬ ϭ 10.5 cm Ϫ1 calculated for the S 2 state coupling scheme also differs from the experimental ⌬ Ն 35 cm Ϫ1 determined for the S 2 Ј state. Correlations between the exchange coupling scheme and this energy difference have been investigated in previous studies (41,47). One mechanism by which ⌬ is influenced directly was shown to be the strength and the sign of the exchange coupling between Mn A4 and the trimeric unit comprising Mn B3 , Mn C2 , and Mn D1 . An increase or decrease in the magnitudes of the coupling constant J A4-B3 results in a larger or smaller energy gap, respectively. As the monomer-trimer joint is in the vicinity of a possible binding site of a MeOH molecule, this rationalizes the effect of MeOH on the electronic structure of the Mn 4 O 5 Ca cluster in the native S 2 state (41). For the Ca 2ϩdepleted S 2 Ј state, the coupling of Mn A4 to the trimeric unit was varied by multiplying the respective exchange coupling constants J A4-B3 , J A4-C2 , and J A4-C2 by a factor c (Fig. 7). It can be readily calculated that with c ϭ 1.65 (J A4-B3 ϭ Ϫ46 cm Ϫ1 , J A4-C2 ϭ 7 cm Ϫ1 , and J A4-D1 ϭ 10 cm Ϫ1 ), ⌬ is 35 cm Ϫ1 and thus in the desired range.
For testing whether the obtained model also reproduces reasonable estimates for the intrinsic ZFS values d i of the Mn III and Mn IV ions, a brief description on how those can be assessed based on the inferred coupling scheme and the fitted effective HFI tensors is given in the supplemental data. Because of their inherently small ZFSs, the d i values of the three Mn IV ions can be assumed to be 0 cm Ϫ1 for the calculations of the intrinsic HFI tensors a i from the fitted effective A i and the computed i tensors. Mn III ions generally exhibit an absolute isotropic HFI value ͉a iso ͉ in the range between 165 and 225 MHz and considerable anisotropy defined as the difference a i,aniso ϭ ͉a ʈ ͉ Ϫ ͉a Ќ ͉ between the absolute values. Mn IV ions tend to exhibit slightly larger isotropic HFI values (͉a iso ͉ ϭ 187-253 MHz) and only small intrinsic HFI anisotropies (͉a iso ͉ Ͻ ϳ30 MHz) (see Ref. 49  . Model for the electronic structure of the OEC in the native S 2 and Ca 2؉ -depleted S 2 states calculated based on a refined DFT structure of the OEC (18) in the latest crystal structure (6). A-D label the manganese ions in their respective oxidation state, and the numbers give the pair-wise exchange coupling J ij between the electronic spins of the Mn III and Mn IV ions in cm Ϫ1 . The constant c is 1 in the originally derived model but differs for the various clusters and conditions, such as the presence or absence of MeOH. The S 2 Ј state can be described by the scheme with c ϭ 1.65.

TABLE 3
Calculated spin projection tensor components Ќ and ʈ , intrinsic 55 Mn HFI tensor components a Ќ and a ʈ , and isotropic and anisotropic intrinsic HFI values a iso and a aniso for the Mn ions of the OEC in the Ca 2؉ -depleted S 2 state on the basis of the electronic exchange coupling scheme in Fig. 7  a The equatorial and axial a i values are defined as: a Ќ ϭ (a x ϩ a y )/2, a ʈ ϭ a z . b The isotropic a iso values are the averages of the individual components of the tensor a iso ϭ (a x ϩ a y ϩ a z )/3. c The anisotropy of the a tensor is expressed as the difference a aniso ϭ a ʈ Ϫ a Ќ between the parallel and perpendicular tensor components.
Ϫ1.2 cm Ϫ1 ) (49). It is, however, noted that the signs of the d D1 and of the HFI anisotropy of the Mn III ion do not change between the Ca 2ϩ -depleted S 2 Ј and the Ca 2ϩ -containing S 2 state. These parameters can be related to the ligand sphere of the Mn III ion (86 -88). Negative numbers for d D1 and a D1,aniso correspond to a 5 B 1g ground state, obtained in the cases of square pyramidal 5-coordinate or tetragonally elongated 6-coordinate ligand geometries. This suggests the coordination sphere of the Mn D1 III for the S 2 Ј and S 2 states to be similar. However, the increase in the magnitude of d D1 upon Ca 2ϩ removal does indicate modifications of the precise binding mode, e.g. altered ligand distances and angles. One possible mechanism for altering d D1 is protonation of one of the -oxo bridges ligating the Ca 2ϩ ion (Fig. 1) as a means of overall charge compensation of the cluster upon Ca 2ϩ removal. It is known from model complexes that Mn-Mn distances are elongated upon protonation of Mn-O-Mn bridges (89). However, within the trimeric cuboidal unit, this lengthening could be strongly impaired for the Mn C2 -Mn D1 distance. The fitted averaged distance of the Mn-Mn interactions at 2.7-2.8 Å from EXAFS on Ca 2ϩ -depleted PS II samples (29), however, does not allow for a conclusive assessment. Also, glutamate 189 of the D1 protein (D1-Glu-189), which directly coordinates the Mn D1 III (6,17,47) and potentially also the Ca 2ϩ ion (18), could be reoriented upon Ca 2ϩ depletion leading to a distortion of the coordination sphere and thus an altered d D1 .
In the latest crystal structure, all four manganese ions are 6-coordinate (6). This, however, requires the O5 -oxo bridge to be a ligand of Mn A4 , Mn B3 , and Mn D1 , engendering very long Mn-O5 bond distances well outside the range seen in model complexes (see Ref. 18) and by EXAFS spectroscopy of the Mn 4 O 5 Ca cluster in PS II (90,91). In most geometry-optimized DFT structures, such as those proposed by Siegbahn and our group (17,18,47), the position of O5 is significantly altered (Fig.  1). The O5 shifts toward the Mn A4 , forming a genuine -oxo bridge between Mn A4 and Mn B3 , and results in Mn D1 having an open coordination site. In this case, in the Ca 2ϩ -depleted S 2 Ј state, Glu-189 might function as a bidentate ligand in a then tetragonally elongated 6-coordinate Mn D1 III ligand sphere, leading to the observed change of d D1 .
Alternatively, the absence of Ca 2ϩ may allow this open site to be occupied by a water molecule in the S 2 Ј state (Fig. 8) forming a sixth ligand to Mn D1 . The Mn D1 -bound water molecule is the second substrate water in the mechanism proposed by Sieg-bahn (17), which potentially binds during the S 2 -to-S 3 transition. Thus, within this model, one of the roles of Ca 2ϩ in the active cluster would be to prevent the second substrate from binding too early in the reaction cycle (25,92). This activity would presumably avoid detrimental side product formation (reactive oxygen species) and lead to single product (O 2 ) formation. Consistent with this role for the Ca 2ϩ ion is the known S state dependence of the affinity of Ca 2ϩ to this site (93). It drops significantly in the S 3 state, suggesting that in this state Ca 2ϩ is less tightly bound, having a more flexible ligand sphere that potentially allows greater solvent access to the Mn D1 ion.
Besides -oxo bridge protonation, the loss of two positive charges is likely to be compensated by protonation of amino acid residues ligating the Ca 2ϩ ion in the intact cluster. Other possibilities are the replacement of Ca 2ϩ by monovalent Na ϩ in the samples or the absence of complete charge compensation, leaving the Mn 4 O 5 cluster with an additional negative charge. It is evident that any of these modifications could have a critical effect on the catalytic capabilities of the cluster, especially with regard to proton-coupled electron transfer to Y Z . In light of the proposed deprotonation sequence 1,0,1,2 for the individual oxidation steps starting from S 0 (94), this would explain the Mn 4 O 5 cluster being able to advance to S 2 Ј but not from S 2 ЈY Z ⅐ to S 3 Ј.
Conclusions-This study demonstrates that Ca 2ϩ is not required for conferring the critical electronic properties to the Mn 4 O 5 Ca cluster. This also confirms that Ca 2ϩ is not essential for structural maintenance of the OEC. Its presence or absence does not affect the position of the only Mn III ion of the cluster in the S 2 /S 2 Ј state (Mn D1 ), and the contribution of the four manganese ions to the electronic states S 2 and S 2 Ј does not differ considerably. Thus, the necessity for Ca 2ϩ in water splitting catalysis must be due to another functional role of the Ca 2ϩ ion.
Although the exact mechanism of inhibition upon Ca 2ϩ removal is still unclear, two models can be considered in terms of the two basic catalytic mechanisms proposed in the literature. (i) For mechanisms that involve O-O bond formation between a Ca 2ϩ -bound and a manganese-bound substrate water (be it a terminal ligand Mn V ϭ O or a -oxo bridge) (11,(95)(96)(97), inhibition due to Ca 2ϩ depletion is readily explained. The enzyme is inactive, as it has lost a substrate binding site. It should be noted though that this model provides no rationale for the fact that the catalytic cycle is blocked at the stage of S 2 ЈY Z ⅐ . (ii) Instead, O-O bond formation has been proposed to follow a mechanism that results in the coupling of substrates bound to two manganese sites (be it between two terminal bound Mn-O ligands or involving a -oxo bridge via oxyl radical coupling) (10,14,16,17,98). Then, inhibition due to Ca 2ϩ removal probably represents a secondary effect where the Ca 2ϩ ion is critical for maintaining the H-bond network between Y Z and the manganese cluster (6,11,30) as opposed (or in addition) to perturbation of substrate binding. Thus, Ca 2ϩ removal would disable proton-coupled electron transfer during the S 2 ЈY Z ⅐ -to-S 3 Ј transition, preventing substrate deprotonation and concomitant oxidation of Mn D1 III . Therefore, the elucidation of the mechanistic role of the Ca 2ϩ ion in the OEC is tightly linked to understanding the mechanism of photosynthetic water splitting. In the putative S 2 Јstate, the fast exchanging substrate water is already bound to Mn D1 III , filling the space of the Ca 2ϩ ion. W s and W f denote the slowly and fast exchanging substrate waters, respectively (96,99).