Light-driven formation of manganese oxide by today’s photosystem II supports evolutionarily ancient manganese-oxidizing photosynthesis

Water oxidation and concomitant dioxygen formation by the manganese-calcium cluster of oxygenic photosynthesis has shaped the biosphere, atmosphere, and geosphere. It has been hypothesized that at an early stage of evolution, before photosynthetic water oxidation became prominent, light-driven formation of manganese oxides from dissolved Mn(2+) ions may have played a key role in bioenergetics and possibly facilitated early geological manganese deposits. Here we report the biochemical evidence for the ability of photosystems to form extended manganese oxide particles. The photochemical redox processes in spinach photosystem-II particles devoid of the manganese-calcium cluster are tracked by visible-light and X-ray spectroscopy. Oxidation of dissolved manganese ions results in high-valent Mn(III,IV)-oxide nanoparticles of the birnessite type bound to photosystem II, with 50-100 manganese ions per photosystem. Having shown that even today’s photosystem II can form birnessite-type oxide particles efficiently, we propose an evolutionary scenario, which involves manganese-oxide production by ancestral photosystems, later followed by down-sizing of protein-bound manganese-oxide nanoparticles to finally yield today’s catalyst of photosynthetic water oxidation.

N ature´s invention of photosynthetic water oxidation about three billion years ago (or even earlier 1 ) was a breakpoint in Еarth´s history because it changed the previously anoxic atmosphere to today´s composition with~21% O 2 , practically depleting the oceans of ferrous iron and divalent manganese due to metal-oxide precipitation 2,3 . Water oxidation is catalyzed by a unique bioinorganic cofactor, denoted Mn 4 CaO 5 according to its oxo-bridged metal core, which is bound to amino acids of the proteins of photosystem II (PSII) in the thylakoid membrane ( Fig. 1) [4][5][6][7][8] . This catalyst originally developed in (prokaryotic) cyanobacteria, which were later incorporated by endosymbiosis into the ancestor of the (eukaryotic) cells of algae and plants to yield the chloroplast organelles 9 . The central PSII proteins as well as Mn 4 CaO 5 (and its main catalytic performance features) are strictly conserved among photosynthetic organisms 10,11 .
Native PSII operates as a light-driven oxidoreductase (Fig. 1). Upon sequential excitation with four visible-light photons, four electrons from two bound water molecules are transferred from Mn 4 CaO 5 to a redox-active tyrosine (Y Z ) at the donor side and then via a cofactor chain to terminal quinone acceptors at the stromal side so that two reduced quinols as well as O 2 and four protons are released during each catalytic water oxidation cycle 5,7,[12][13][14] . Starting from a Mn(III) 3 Mn(IV)Y Z state, the catalytic cycle involves alternate electron and proton abstraction to reach a Mn(IV) 4 Y Z ox state followed by (concomitant) Mn rereduction, O-O bond formation and O 2 release ( Fig. 1) 15 . The exceptionally efficient Mn 4 CaO 5 catalyst has inspired development of synthetic water-oxidizing materials [16][17][18] . Among the wealth of findings on water oxidation by Mn-based catalysts, here the following two results are of particular importance: (i) Selfassembly of the Mn(III/IV) 4 CaO 5 core in PSII is a light-driven process, involving step-wise oxidation of four solvent Mn 2+ ions by Y Z ox coupled to electron transfer to the quinones 19,20 . (ii) Many amorphous Mn oxides of the birnessite type show significant water oxidation activity and share structural as well as functional features with the Mn 4 CaO 5 core of the biological catalyst [21][22][23][24][25] .
The evolutionary route towards the present water oxidation catalyst in PSII is much debated 2,[26][27][28][29][30][31] . It has been hypothesized that before the evolution of oxygenic photosynthesis an ancestral photosystem developed the capability for light-driven oxidation of dissolved Mn 2+ ions towards the Mn(III/IV) level, thereby providing the reducing equivalents (electrons) needed for primary biomass formation by CO 2 fixation 32,33 . Aside from the implications for biological evolution, photosynthetic Mn-oxide formation has significance in the context of recent hypotheses to account for geologic Mn deposits, for example from the early Paleoproterozoic in South Africa 2,33 . Notably, the process of continuous Mn 2+ oxidation is chemically not trivial, because suitable redox potentials alone are insufficient. Because solitary Mn(III/IV) ions are not stable in aqueous solution, the ability of the photosystem to stabilize high-valent Mn ions by the efficient formation of extended Mn (oxide) structures is pivotal.
Here, the experimental evidence is provided that today's PSII, depleted of its native Mn 4 CaO 5 complex and the membraneextrinsic polypeptides, can form a Mn(III/IV) oxide of the birnessite type. By optical (UV-visible) and X-ray absorption spectroscopy, we show that the light-driven oxidation of Mn 2+ ions results in Mn-oxide nanoparticles which are bound to the photosystem, thereby supporting that also ancient photosystems could have produced Mn oxides and suggesting a viable evolutionary route to today's catalyst of photosynthetic water oxidation.

Results
Spinach photosystems depleted of Mn 4 CaO 5 and extrinsic polypeptides. Figure 1 shows the arrangement of protein subunits and cofactors in PSII. A recent crystallographic study has revealed that the metal-binding amino acids are similarly arranged in PSII with or without Mn 4 CaO 5 , with the voids in the Mndepleted photosystem being filled by water molecules 34 . Only in the absence of the Mn-stabilizing extrinsic proteins, sufficient room for the incorporation of a Mn-oxide nanoparticle into the PSII structure may exist (Fig. 1). Therefore, we explored the ability of purified PSII, depleted of Mn 4 CaO 5 and the extrinsic proteins, to form Mn-oxide species in vitro. PSII-enriched membrane particles were prepared from spinach 35 and Mn depletion was achieved using an established protocol (see Supplementary Information) 36 . The resulting PSII preparation was inactive in light-driven O 2 -evolution and Mn was practically undetectable, i.e., <0.2 Mn ions per PSII were found (Table 1) concentrations, a clearly more rapid phase of DCPIP ox reduction grew in (Fig. 3, arrows; Fig. 4 and Supplementary Fig. 8). Its amplitude saturated at 240 µM MnCl 2 and indicates reduction of 17% (~10 µM) of the initial DCPIP ox (60 µM), which corresponds to up to 200 transferred electrons per PSII within about 1 min. Notably, at low MnCl 2 concentration DCPIP reduction continued at undiminished rate for a clearly longer illumination time (>5 min, Fig. 4 and Supplementary Fig. 7), suggesting that photoinhibitory damage does not rapidly terminate electron transfer in the Mn-depleted PSII. Based on the Mn concentration dependence ( Fig. 4 and Supplementary Fig. 8) and the results presented in the following, we can assign this rapid DCPIP  . Then DCPIP ox and either 5 µM MnCl 2 (in a) or 240 µM MnCl 2 (in b) were added, followed by continuous white-light illumination (1000 µE m −2 s −1 ) of the PSII suspensions and collection of spectra (one spectrum per minute; immediately prior to illumination, red lines, and after 3 min light, blue lines). The green and lightgreen lines correspond to the spectral contributions of DCPIP ox to the dark and 3-min light spectra; the amplitude decrease at 604 nm represents the loss of DCPIP ox due to its reduction by PSII (arrows). The dark-yellow dotted spectra are assignable to a Mn oxide, as verified by X-ray absorption spectroscopy (spectra obtained by weighted spectral deconvolution, see caption of Supplementary Fig. 6, and scaled by a factor of 5, for clarity). Note that significant spectral changes due to Mn-oxide formation were only observed with 240 µM MnCl 2 (in b), but not with 5 µM MnCl 2 (in a). Source data are provided as a Source Data file.
UV-vis spectra point towards Mn-oxide formation. To search for evidence of Mn-oxide formation, informative absorption difference spectra of Mn-depleted PSII before and after illumination were calculated ( Fig. 3 and Supplementary Fig. 6). For 60 µM MnCl 2 , after completion of rapid DCPIP ox reduction (3 min), there was a broad absorption increase (ranging from 350 to 700 nm), which is similar to the wide-range absorption of Mn oxides 25 . For higher concentrations of MnCl 2 , the absorption assigned to Mn oxides gained strength and became maximal at 240 µM MnCl 2 (Fig. 3). Using alternative electron acceptors ( Supplementary Fig. 11), similar or even higher Mn-oxide amounts were detected with DCBQ (2,5-dichloro-1,4-benzoquinone) or PPBQ (phenyl-p-benzoquinone), resembling the native quinone acceptor (Q B ), but the slow (hydrophilic) acceptor ferricyanide (K 3 Fe III (CN) 6 ) did not yield significant Mn-oxide formation.
PSII with 50-100 bound Mn ions prepared for analysis by Xray spectroscopy. To investigate the Mn 2+ oxidation products and identify their atomic structure, we employed X-ray absorption spectroscopy (XAS) at the Mn K-edge ( Fig. 5 and Supplementary Figs. 12-16). Mn-depleted PSII was illuminated for 3 min with 240 µM MnCl 2 and 60 µM PPBQ ox at pH 8.5 or pH 7 (Fig. 5, Supplementary Figs. 14 and 15), the reaction was terminated by rapid sample cooling in the dark, and the PSII membranes were pelleted by centrifugation and then transferred to XAS sample holders, followed by freezing in liquid nitrogen and later collection of X-ray spectra at 20 K (see SI). The metal content was determined by X-ray fluorescence analytics (Table 1,  Supplementary Table 1), revealing 65 ± 19 Mn ions per initially Mn-depleted PSII after illumination in the presence of 240 µM MnCl 2 . The calcium content in the PSII-formed Mn oxide could not be reliably determined because CaCl 2 was present in the buffer and Ca is known to bind nonspecifically to the used PSII membrane particle preparation (Supplementary Table 1) 43 .
X-ray spectroscopy reveals extended Mn(III/IV) oxides. The shape of the XANES (X-ray absorption near-edge structure) pronouncedly differed from hexaquo-Mn 2+ , micro-crystalline Mn oxides (Mn III  2 O 3 , Mn II,III  3 O 4 , β-Mn IV O 2 ), and native PSII, but was similar to layered Mn(III,IV) oxides denoted as birnessite [44][45][46] (Fig. 5a). The K-edge energy indicated a mean redox level of about +3.5, suggesting equal amounts of Mn(III) and Mn (IV) ions (Fig. 5a, Supplementary Fig. 12). EXAFS (extended Xray absorption fine structure) analysis revealed the atomic structure of the PSII-bound Mn oxide (Fig. 5b 21,22,25,47,48 . We note that the long-range order in the oxide particles produced by PSII even exceeds that of the herein used reference oxides of the birnessite-type, as indicated by the magnitudes of the Fourier peaks assignable to the 2.87 and 5.54 Å distances, verifying formation of a comparably extended and well-ordered Mn oxide. Notably, according to the similar XAS spectra, a similar birnessite-type Mn(III,IV)-oxide was formed (i) in the presence as well as absence of CaCl 2 in the illumination buffer and (ii) at pH-values of 8.5 as well as 7.0 ( Fig. 5 and Supplementary Fig. 15). The number of 50-100 Mn ions per PSII suggests that the Mn oxides could be bound to PSII in form of small nanoparticles (<2 nm).
Mn-oxide nanoparticles are bound to the PSII core complex. In the experiments reported above, PSII membrane particles were investigated. These are comparably large membrane fragments containing numerous PSII units per fragment and therefore can be collected by centrifugation at comparably low speed (20,000 × g for 10 min). These centrifugation conditions do not allow for pelleting of unbound oxide nanoparticles of only about 100 Mn ions (diameter <2 nm), suggesting that the Mn-oxide nanoparticles are bound to the PSII membrane particles. To exclude that the Mn-oxide nanoparticles were bound to the lipid bilayer membrane or trapped between stacked membrane sheets, we also investigated detergent solubilized, membrane-free PSII particles using the same protocol for light-induced Mn-oxide formation as used for the PSII membrane particles. Speciation by EXAFS spectroscopy (Supplementary Fig. 16) verified that the same Mn oxide is formed also for detergent-solubilized PSII, thereby providing support for association of Mn-oxide nanoparticles directly with the PSII proteins.

Discussion
Mn-oxide formation by PSII. We have obtained the first direct experimental evidence that PSII devoid of Mn 4 CaO 5 is capable of forming Mn(III,IV)-oxide particles of the birnessite type by lightdriven oxidation of Mn 2+ ions. Presumably these are nanoparticles of 50-100 Mn ions that are bound to the PSII protein complex, as suggested by their presence in PSII membrane particle pellets, due to co-sedimentation using a comparably mild centrifugation protocol, as well as in solubilized, membrane-free PSII core particles after their precipitation. Is it possible that larger Mn-oxide particles are formed (e.g., several thousand Mn ions), which would sediment also at moderate centrifugation speed? The cooperation of many PSII centers in formation of a single large Mn-oxide nanoparticle is unlikely, inter alia because efficient electron transfer from Mn 2+ ions to the redox-active tyrosine cannot occur over distances that are as long as the distance between neighboring PSII dimers in PSII membrane particles and even more so for solubilized PSII. Similarly, also the fast spontaneous fusion of Mn-oxide nanoparticles to more extended oxide particles is highly unlikely. Aggregation mediated by nonbonding interactions cannot be rigorously excluded, but is disfavored by the expected concentrations of oxide particles in the sub-micromolar range. On these grounds and supported by the inhibitory effect of Mn-oxide formation on electron donation ( Fig. 4 and Supplementary Fig. 8), we assume that Mn-oxide nanoparticles are bound to the PSII core complex, likely in the vicinity of the redox-active tyrosine (Y Z in Fig. 1).
Light-driven Mn 2+ oxidation also can promote self-assembly of the functional Mn 4 CaO 5 complex, which is a comparably inefficient (low quantum yield) low-light process denoted as photoactivation 19,20 Supplementary Fig. 14). The presence of 5 mM CaCl 2 allows for photoactivation, although a higher concentration is required for optimal photoactivation yield 49,50 . The about 20 times higher light intensities we used likely promoted the oxidation and binding of numerous Mn ions at the expense of formation of a single native Mn 4 CaO 5 cluster, because the latter requires low-light intensities presumably due to the presence of a slow 'dark rearrangement' step for assembly 19 .
Since Cheniae's work 49 , it had remained an open question in what form a larger number of Mn ions can bind to PSII membrane particles. Coordination of individual high-valent Mn ions to protein groups is one possibility (as often observed for divalent cations and trivalent Fe ions); the formation of extended protein-bound Mn-oxide nanoparticles is another possibility. Under our high-light conditions, Mn(III,IV)-oxide formation clearly is dominant. The Mn-oxide cluster size seems to be limited to around 100 Mn ions, which may correspond to a nanoparticle of about 20 Å in diameter (Fig. 6a). Such a particle may well be formed within the PSII cavity that becomes solvent-exposed upon removal of the extrinsic protein subunits (Fig. 1). These subunits are evolutionarily younger than the PSII core proteins 51 and are absent in related anoxygenic photosystems 52 . Thus, it is well conceivable that an early PSII ancestor would have lacked these extrinsic proteins and therefore could accommodate a Mn-oxide nanoparticle. Furthermore, an ancient autotroph, capable of exploiting Mn 2+ as a metabolic reductant 32,33 , would be expected to be configured so that the donor side of the early PSII ancestor would be exposed to the environment as opposed to being sequestered within the lumen of modern thylakoids. In this context, the cyanobacterium Gloeobacter violaceous provides an interesting example. Gloeobacter occupies a basal phylogenetic position and evolved before the appearance of thylakoids. It possesses photosynthetic reaction centers that are located in the cytoplasmic membrane with the oxidizing domain of PSII facing the periplasmic space and thus the exterior of the cell 53 . Thus Gloeobacter provides an example of how a primordial reaction center might have been arranged to facilitate the photochemical utilization of Mn 2+ as a reductant source, as originally proposed by Zubay 32 .
Relation to water-oxidizing synthetic Mn oxides. Birnessite and buserite are layered, typically non-crystalline metal-oxides with sheets of edge-sharing MnO 6 octahedra (which corresponds to diµ-oxo bridging between neighboring Mn ions) separated by water and cations, e.g., Na + or Ca 2+ , in the interlayer space 44 24 . The presence of Ca ions is especially favorable for water oxidation activity by synthetic manganese oxides, pointing towards similar wateroxidation mechanisms in the synthetic oxides and the biological Mn 4 CaO 5 cluster of PSII 21,22,25,62 . Regarding their high degree of structural order, the Mn-oxide particles formed by PSII resemble electrodeposited Mn oxides that are able to undergo Mn(III)-Mn(IV) redox transitions, but exhibit low electrochemical water oxidation activity 24 . The structural characteristics that have been identified for transforming a largely inactive Mn oxide into an oxide with sizeable water-oxidation activity 24 are apparently lacking in the Mn-oxide particles formed by PSII, which may explain the absence of detectable water-oxidation activity by the herein investigated PSII-bound Mn-oxide particle.
Mechanism of Mn-oxide formation. The basic biochemical mechanism of the here described light-induced Mn-oxide formation likely involves the initial binding of Mn 2+ ions followed by Mn oxidation and stabilization of the oxidized Mn(III/IV) ions by di-µ-oxo bridging, in analogy to both the photoassembly process of today's Mn 4 CaO 5 cluster 19,20 and the oxidative selfassembly process in the electrodeposition of non-biological birnessite-type Mn oxides 25,47 . The formation of extended oxide particles likely involves a nucleation-and-growth mechanism. In the photosystem, the initial site of Mn 2+ binding and formation of an oxide nucleus likely is provided by carboxylate and possibly imidazole sidechains of protein residues followed by an oxide growth that does not require further ligating residues. Alternative hypothesis on the evolution of the Mn 4 CaO 5 cluster. We see a close relation between inorganic Mn oxides and today's Mn 4 CaO 5 cluster of PSII that differs distinctively from the Mn-oxide incorporation hypotheses outlined above. In our study, the facile formation of birnessite-type Mn-oxide particles by PSII is reported. They (i) share structural motifs with the biological cluster in PSII 10,65 and biogenic Mn oxides in general 59,66 and (ii) resemble synthetic Mn oxides closely that have been investigated as synthetic catalyst materials 24 . On these grounds we propose a scenario illustrated in Fig. 6: Rather than Mn-oxide incorporation, Mn-oxide nanoparticles were formed by an evolutionary precursor of PSII, inter alia enabling the formation of early geological Mn-oxide deposits. Initially, dissolved Mn 2+ ions served as a source of reducing equivalents eventually needed for CO 2 reduction, as has been suggested first by Zubay 32 and later by others 31,33 . At a later stage, down-sized oxide particles developed into today's water-oxidizing Mn 4 CaO 5 cluster. Whether the early photosynthetic reaction centers initially exhibited a sufficiently high potential for the oxidation of aqueous Mn 2+ ions 67,68 or such a potential was acquired during evolution starting from a low-potential ancestor 31 has to remain an open question at the present stage (Fig. 6). Olson has already favored the high-potential first hypothesis 50 years ago 68 , and recently such ideas have gained support based on structural and genomic comparisons from Cardona and coworkers 69 . We note that our hypothesis that Mn-oxide incorporation precedes the formation of the present water oxidation catalyst in PSII is independent on the earlier way of evolution of a high-potential reaction center, because such a species is needed for both processes.
Are there evolutionary relicts that may support our above hypothesis? Extensive studies on the diversity of the PSII reaction center protein D1 have revealed several atypical variants that can be distinguished phylogenetically 70,71 . These early evolved forms lack many residues needed for the binding of today's Mn 4 CaO 5 cluster and could relate to ancient Mn-oxide-forming photosystems, even though today they might play other physiological roles (e.g., in the synthesis of chlorophyll f 72 ).
Summary of potential evolutionary implications. The ability for light-driven Mn-oxide formation by an ancient photosystem represents an important touchstone for evaluation of three interrelated hypotheses that each addresses a remarkable facet of the evolution of the Earth's biosphere and geosphere: (i) The ability for the direct and facile photosynthetic formation of stable Mn(III/IV)-oxide particles supports that early Mn deposits 33 resulted directly from photosynthetic activity. (ii) Structural and functional similarities between wateroxidizing synthetic Mn oxides and the here described Mn-oxide formation by PSII suggests that in the evolution of PSII, there may have been a transition from extended Mn-oxide nanoparticles towards the Mn 4 CaO 5 cluster of today's PSII, as illustrated by Fig. 6. (iii) An early quasi-respiratory cycle has been proposed that involves the formation of Mn(III/IV) oxide particles followed by utilization of the oxidizing equivalents stored in the Mn oxide for an efficient quasi-respiratory activity in the Archean or early Paleoproterozoic, when the Earth's atmosphere had been essentially O 2 -free, as detailed in ref. 65 .
By showing that today's PSII can form birnessite-type Mnoxide particles efficiently, even without any specific protein subunits that would support Mn-oxide formation, the general biochemical feasibility is verified. This finding renders it highly likely that similarly also an ancient photosystem, the PSII ancestor, had the ability for the light-driven formation of Mn oxides from hexaquo Mn 2+ ions. In conclusion, we believe that our successful demonstration of the photosynthetic formation of Mn(III/IV)-oxide particles provides relevant support for the above three hypotheses.

Methods
Preparation of PSII membrane particles. Native PSII-enriched thylakoid membrane particles were prepared from fresh market spinach following our established procedures 35 . Their typical O 2 -evolution activity (as determined by polarography with a Clark-type electrode at 27°C) was~1200 µmol O 2 mg −1 chlorophyll h −1 , which proved the full integrity of the PSII proteins and the water-oxidizing Mn 4 CaO 5 complex. We have shown earlier that this type of PSII preparation contains~200 chlorophyll molecules per PSII reaction center 73,74 . When kept for prolonged time periods in the dark, the Mn 4 CaO 5 complex is synchronized in the S 1 state of its catalytic cycle, which is established to represent a Mn(III) 2 Mn(IV) 2 oxidation state 75,76 .
Mn-depletion of PSII. Removal of Mn 4 CaO 5 and of the three extrinsic proteins of PSII (PsbQ, PsbP, and PsbO) was carried out using a literature procedure and evaluated using metal quantification and gel electrophoresis (see below) 36 . PSII membranes were dissolved at 200 µg chlorophyll mL −1 in a high-salt buffer (30 mL) containing 20 mM TEMED (N,N,N',N'-tetramethylethylenediamine) as a reductant for the PSII-bound Mn(III,IV) ions, 20 mM MES (2-(N-morpholino)ethane-sulfonic-acid) buffer (pH 6.5), and a high-salt concentration (500 mM MgCl 2 ) and incubated in the dark on ice for 10 min. PSII membranes were pelleted by centrifugation (Sorvall RC26, 12 min, 50,000 × g, 4°C), the pellet was three times washed by dissolution in a buffer (30 mL) containing 35 mM NaCl and 20 mM TRIS (tris (hydroxymethyl)aminomethane) buffer (pH 9.0) and pelleting by centrifugation as above, and the final pellet of Mn-depleted PSII membranes was dissolved at~1 mg chlorophyll mL −1 in a buffer containing 1 M glycine-betaine, 15 mM NaCl, 5 mM CaCl 2 , 5 mM MgCl 2 , and 25 mM MES buffer (pH 6.3). The PSII preparations (~2 mg chlorophyll mL −1 ) were thoroughly homogenized by gentle brushing and frozen in liquid nitrogen for the spectroscopic experiments. The Mn-depleted PSII showed zero O 2 -evolution activity as revealed by polarography.
TXRF analysis. X-ray emission spectra were recorded on a Picofox instrument (Bruker) and metal contents of PSII samples were determined from the data using the (fit) routines available with the spectrometer 39 . PSII membranes were adjusted to a chlorophyll concentration of 1-2 mM and to a 20 µL aliquot, a gallium concentration standard (1 mg mL −1 , 20 µL; Sigma-Aldrich) was added, and samples were homogenized by brief sonication (see Supplementary Fig. 1). A 5 µL aliquot of the samples was pipetted on clean quartz discs for TXRF, dried on a heating plate, loaded into the spectrometer, and TXRF spectra were recorded within 10-30 min. At least three repetitions of each sample and three independently prepared samples of each PSII preparation were analyzed. The TXRF data on Mn-oxide formation by Mn-depleted PSII shown in Table 1 were obtained using the same illumination and centrifugation protocol also used for preparation of the X-ray spectroscopy samples of Fig. 5.
Optical absorption spectroscopy and illumination procedures. For the optical absorption spectroscopy experiments, stock suspensions of the PSII preparations were diluted at 20 µg chlorophyll mL −1 (~0.1 µM PSII centers) in a buffer (3 mL) containing 1 M glycine-betaine, 15 mM NaCl, 5 mM CaCl 2 , 5 mM MgCl 2 , and 25 mM MES buffer (pH 6.3-8.5) and reactants (oxidized 2,6-dichlorophenol-indophenol = DCPIP ox from Fluka, other electron acceptors as in Supplementary  Fig. 11, MnCl 2 ) were added at indicated concentrations. The pH was routinely controlled prior to and after the illumination assays in the actual cuvette and found to be stable within ±0.2 pH units within a time period of at least 20 min. Optical absorption spectra of the samples in a 300-900 nm range were recorded within about 10 s at given time intervals (about 0.3-1.0 min) in a 3 mL quartz cuvette (Helma QS1000, 1 cm pathlength) using a Cary 60 spectrometer (Agilent). Alternatively, time traces of absorption were recorded at selected wavelengths (i.e., 604 nm to monitor DCPIP ox reduction) for up to 30 min. Temperature logging revealed that the sample temperature varied by <2°C within the extended illumination periods. PSII-sample filled cuvettes in the spectrometer were continuously illuminated from the top side using a white-light lamp (Schott KL1500, halogen light bulb with cold-light reflector) with attenuation option, which was directed through a heat-protection filter (Schott KG5) to the cuvette by a~20 cm light-guide (the full cuvette volume was homogenously illuminated). The combination of light source, KG5 filter, light guide, and cuvette material resulted in an effective spectral range of about 400-750 nm (limits correspond to the 10% level) effectively excluding UV irradiation, thereby minimizing potential interferences due to peroxide formation resulting from direct Mn-oxide excitation, and sample heating due to infrared light (thermal radiation). Several spectra (or time points) were recorded in the dark (prior to and after addition of, e.g., DCPIP), the light was switched on (or off) at indicated time points, and data were recorded on a PC linked to the spectrometer. Evaluation and fit analysis of absorption data was carried out using the Origin software (OriginLab). Light intensities at the sample center position were determined using a calibrated sensor device inserted in the spectrometer.
X-ray absorption spectroscopy. XAS at the Mn K-edge was performed at beamline KMC-3 at the BESSY-II synchrotron (Helmholtz Zentrum Berlin) with the storage ring operated in top-up mode (250 or 300 mA), using a standard set-up as described in refs. 25,77 . A double-crystal Si[111] monochromator was used for energy scanning, the sample X-ray fluorescence was monitored with an energyresolving 13-element germanium detector (Canberra) or a 13-element silicon-drift detector (RaySpec), and samples were held in a liquid-helium cryostat (Oxford) at 20 K (in a 0.2 bar He heat-exchange gas atmosphere at an angle of 55°to the incident X-ray beam). The X-ray spot size on the sample was shaped by slits to about 1 (vertical) × 5 (horizontal) mm 2 , the X-ray flux was~10 10 photons s -1 , the EXAFS scan duration was~10-20 min. The energy axis was calibrated (±0.1 eV accuracy) using a Gaussian fit to the pre-edge peak (6543.3 eV) in the transmission spectrum of a permanganate (KMnO 4 ) powder sample, which was measured in parallel to the PSII samples. For XAS data evaluation, up to 30 deadtime-corrected, energy-calibrated (I/I 0 ) XAS monochromator scans (each on a fresh sample spot) were averaged and normalized XANES and EXAFS spectra were extracted after background subtraction using in-house software 78 . EXAFS simulations in k-space were carried out using in-house software (SimX) and scattering phase functions calculated with FEFF9.0 79 (S 0 2 = 0.8). Calculation of the filtered R-factor (R F , the difference in % between fit curve and Fourier-backtransform of the experimental data in a 1-5 Å region of reduced distance) 80 facilitated evaluation of the EXAFS fit quality. Fourier-transforms of EXAFS spectra were calculated with cos windows extending over 10% of both k-range ends.
Sample preparation for XAS. Powder samples of manganese reference compounds (Mn oxides) were prepared from commercially available chemicals (MnCl 2 , Mn oxides) or from material (birnessite) that was kindly provided by the group of P. Kurz (Uni. Freiburg, Germany), diluted by grinding with boron-nitride (BN) to a level, which resulted in <15% absorption at the K-edge maximum to avoid flattening effects in fluorescence-detected XAS spectra, loaded into Kapton-covered acrylicglass holders, and frozen in liquid nitrogen. Aqueous MnCl 2 (20 mM) samples were prepared at pH 7.0. Unless otherwise specified, PSII samples were prepared as follows: Mn-depleted PSII samples (3 mL) were prepared similar to the samples for optical absorption spectroscopy (see above), the pH was adjusted to the desired value (pH 7 or 8.5), and samples were illuminated for 3 min at 1000 µE m −2 s −1 or kept in the dark as a control after addition of 240 µM MnCl 2 and 60 µM PPBQ ox . Thereafter, the cuvette volume was rapidly mixed with ice-cold MES buffer (7 mL, pH 7 or 8.5, see above for ingredients) on ice in the dark, the pH was measured using a pH electrode and, if necessary, readjusted to the desired value (+/−0.1 pH units), the PSII membranes were pelleted by centrifugation (10 min, 20,000 × g, 2°C), and kept on ice. Several of these sample types were rapidly merged on ice in the dark by loading (~30 µL) into XAS holders, which were immediately frozen in liquid nitrogen. Native PSII samples were prepared by pelleting of dark-adapted O 2evolving PSII membrane particles (~8 mg chlorophyll mL −1 , pH 6.3), loading of the pellet material into XAS holders, and freezing in liquid nitrogen 76 . The shown XAS data for the electrodeposited Mn oxides has been collected in the context of earlier studies 21,22,47 and replotted.
Solubilization of PSII membrane particles to yield membrane-free PSII complexes. Mn-depleted PSII membrane particles were solubilized as described by Haniewicz et al. 81 . In brief, 20 mM β-dodecylmaltoside (β-DM) was added to the membranes with a chlorophyll concentration of 2 mg mL −1 . After incubation for 30 min at 4°C in the dark, the solubilized material was separated from the insoluble fraction by centrifugation at 40,000 × g for 20 min at 4°C. From the supernatant containing the solubilized Mn-depleted PSII, XAS samples were prepared as follows. An aliquot of the solution with solubilized PSII was added to MES buffer (pH 7.0) containing 240 μM MnCl 2 , 5 mM CaCl 2 , 60 μM PPBQ, and 0.03 % (v/v) β-DM to a final chlorophyll concentration of 20 μg mL −1 and the sample was illuminated for 1 min at 1000 μE m −2 s −1 . To remove unbound Mn and electron acceptor, the sample was precipitated by adding polyethylene-glycol (PEG 6000) to a final concentration of 4.4% (w/v) and a subsequent centrifugation step (16,000 × g, 10 min, 4°C) 82 . The pellet was resuspended in MES buffer (pH 7.0) and the PEG precipitation and centrifugation procedure was repeated twice. After the final washing step, the pellet was resuspended in a small volume of buffer and transferred to the sample holder as described above.