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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.

Herein, we report the synthesis, characterization, crystal structure, density functional theory calculations, and water-oxidizing activity of a pivalate Mn-Ca cluster. All of the manganese atoms in the cluster are Mn(iv) ions and have a distorted MnO6 octahedral geometry. Three Mn(iv) ions together with a Ca(ii) ion and four-oxido groups form a cubic Mn3CaO4 unit which is similar to the Mn3CaO4 cluster in the water-oxidizing complex of Photosystem II. Using scanning electron microscopy, transmission electron microscopy, energy dispersive spectrometry, extended X-ray absorption spectroscopy, chronoamperometry, and electrochemical methods, a conversion into nano-sized Mn-oxide is observed for the cluster in the water-oxidation reaction.

Information on binding and rearrangement of pivotal water molecules could support understanding of light-driven water oxidation at the catalytic Mn 4 CaO 5 cluster of photosystem II (PSII). To address this point, the binding of ammonia (NH 3 )-a possible substrate-water analogue-has been investigated and discussed in the context of putative reaction mechanisms. By time-resolved detection of O 2 formation after light-flash excitation, we discriminate three NH 3 /NH 4 + binding sites jointly characterized by a K m value around 25 mM (of NH 4 + ), but differing in their influence on the O 2 -formation step. At 100 mM NH 4 Cl (pH 7.5), we observe (1) a PSII fraction with complete inhibition of O 2 -formation, (2) fast O 2 -formation with a time constant of 1.7 ms at 20 °C (Fast-PSII), and (3) slow O 2 -formation with a time constant of 36 ms at 20 °C (Slow-PSII). For the Fast-PSII, we determine an activation enthalpy of 223 ± 11 meV. Activation enthalpy and entropy of the Fast-PSII are essentially identical to the corresponding figures in the absence NH 3 /NH 4 + binding. For the Slow-PSII, the activation enthalpy is 323 ± 11 meV and thus significantly increased, whereas the activation entropy remains essentially unchanged. We conclude: (1) The fully-inhibitory binding site could relate to bound NH 3 replacing one of the two substrate-water molecules. (2) The Fast-PSII may relate to NH 3 /NH 4 + binding in the S 2 -state of PSII followed by unbinding before onset of the OO bond formation step, but also more intricate mechanisms are not excluded. (3) In the Slow-PSII, NH 3 /NH 4 + binding increases the energetic barrier of the OO bond formation step significantly., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH 4 + /NH 3 binding. Time-resolved detection of O 2 formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NH 4 Cl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O 2 formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn(IV) 4 (S 3 ) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) Partial inhibition can be explained by anticooperative (mutually exclusive) NH 3 binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the "dangling" Mn ion. (4) Unexpectedly strong modifications of the FTIR difference spectra suggest that in the non-inhibited PSII, ammonia binding obliterates the need for some of the nuclear rearrangements occurring in the S 2 -S 3 transition as well as their reversal in the O 2 formation transition, in line with the carousel mechanism [Askerka, M., et al. (2015) Biochemistry 54, 5783]. (5) We observe the same partial inhibition of PSII by NH 4 Cl also for thylakoid membranes prepared from mesophilic and thermophilic cyanobacteria, suggesting that the results described above are valid for plant and cyanobacterial PSII.

The choreography of electron transfer (ET) and proton transfer (PT) in the S-state cycle at the manganese-calcium (Mn 4 Ca) complex of photosystem II (PSII) is pivotal for the mechanism of photosynthetic water oxidation. Time-resolved room-temperature X-ray absorption spectroscopy (XAS) at the Mn K-edge was employed to determine the kinetic isotope effect (KIE = τ D 2 O /τ H 2 O ) of the four S transitions in a PSII membrane particle preparation in H 2 O and D 2 O buffers. We found a small KIE (1.2-1.4) for manganese oxidation by ET from Mn 4 Ca to the tyrosine radical (Y Z •+ ) in the S 0 n → S 1 + and S 1 n → S 2 + transitions and for manganese reduction by ET from substrate water to manganese ions in the O 2 -evolving S 3 n → S 0 n step, but a larger KIE (∼1.8) for manganese oxidation in the S 2 n → S 3 + step (subscript, number of accumulated oxidizing equivalents; superscript, charge of Mn 4 Ca). Kinetic lag phases detected in the XAS transients prior to the respective ET steps were assigned to S 3 + → S 3 n (∼150 μs, H 2 O; ∼380 μs, D 2 O) and S 2 + → S 2 n (∼25 μs, H 2 O; ∼120 μs, D 2 O) steps and attributed to PT events according to their comparatively large KIE (∼2.4, ∼4.5). Our results suggest that proton movements and molecular rearrangements within the hydrogen-bonded network involving Mn 4 Ca and its bound (substrate) water ligands and the surrounding amino acid/water matrix govern to different extents the rates of all ET steps but affect particularly strongly the S 2 n → S 3 + transition, assigned as proton-coupled electron transfer. Observation of a lag phase in the classical S 2 → S 3 transition verifies that the associated PT is a prerequisite for subsequent ET, which completes Mn 4 Ca oxidation to the all-Mn(IV) level.

In oxygenic photosynthesis, water is oxidized and dioxygen is produced at a Mn4Ca complex bound to the proteins of photosystem II (PSII). Valence and coordination changes in its catalytic S-state cycle are of great interest. In room-temperature (in situ) experiments, time-resolved energy-sampling X-ray emission spectroscopy of the Mn Kβ1,3 line after laser-flash excitation of PSII membrane particles was applied to characterize the redox transitions in the S-state cycle. The Kβ1,3 line energies suggest a high-valence configuration of the Mn4Ca complex with Mn(III)3Mn(IV) in S0, Mn(III)2Mn(IV)2 in S1, Mn(III)Mn(IV)3 in S2, and Mn(IV)4 in S3 and, thus, manganese oxidation in each of the three accessible oxidizing transitions of the water-oxidizing complex. There are no indications of formation of a ligand radical, thus rendering partial water oxidation before reaching the S4 state unlikely. The difference spectra of both manganese Kβ1,3 emission and K-edge X-ray absorption display different shapes for Mn(III) oxidation in the S2 → S3 transition when compared to Mn(III) oxidation in the S1 → S2 transition. Comparison to spectra of manganese compounds with known structures and oxidation states and varying metal coordination environments suggests a change in the manganese ligand environment in the S2 → S3 transition, which could be oxidation of five-coordinated Mn(III) to six-coordinated Mn(IV). Conceivable options for the rearrangement of (substrate) water species and metal-ligand bonding patterns at the Mn4Ca complex in the S2 → S3 transition are discussed.

Photosynthetic water oxidation chemistry at the unique manganese-calcium complex of photosystem II (PSII) is of fundamental importance and serves as a paragon in the development of efficient synthetic catalysts. A recent crystal structure of PSII shows the atoms of the water-oxidizing complex; its Mn4CaO5 core resembles inorganic manganese-calcium oxides. Merging of crystallographic and spectroscopic information reverses radiation-induced modifications at the Mn-complex in silico and facilitates discussion of the O-O bond chemistry. Coordinated proton movements are promoted by a water network connecting the Mn4CaO5 core with the oxidant, a tyrosine radical and one possibly mobile chloride ion. A basic reaction-cycle model predicts an alternating proton and electron removal from the catalytic site, which facilitates energetically efficient water oxidation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The active site of photosynthetic water oxidation by Photosystem II (PSII) is a manganese-calcium cluster (Mn(4)CaO(5)). A postulated catalytic base is assumed to be crucial. CP43-Arg357, which is a candidate for the identity of this base, is a second-sphere ligand of the Mn(4)-Ca cluster and is located near a putative proton exit pathway, which begins with residue D1-D61. Transient absorption spectroscopy and time-resolved O(2) polarography reveal that in the D1-D61N mutant, the transfer of an electron from the Mn(4)CaO(5) cluster to Y(Z)(OX) and O(2) release during the final step of the catalytic cycle, the S(3)-S(0) transition, proceed simultaneously but are more dramatically decelerated than previously thought (t(1/2) of up to ~50 ms vs a t(1/2) of 1.5 ms in the wild type). Using a bare platinum electrode to record the flash-dependent yields of O(2) from mutant and wild-type PSII has allowed the observation of the kinetics of release of O(2) from extracted thylakoid membranes at various pH values and in the presence of deuterated water. In the mutant, it was possible to resolve a clear lag phase prior to the appearance of O(2), indicating formation of an intermediate before the onset of O(2) formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant, indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regard to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations led to the conclusion that proton expulsion during the initial phase of the S(3)-S(0) transition starts with the deprotonation of the primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O(2) formation chemistry.

In oxygenic photosynthesis, solar energy drives the oxidation of water catalyzed by a Mn(4)Ca complex bound to the proteins of Photosystem II. Four protons are released during one turnover of the water oxidation cycle (S-state cycle), implying thermodynamic limitations at low pH. For proton concentrations ranging from 1 nm (pH 9) to 1 mm (pH 3), we have characterized the low-pH limitations using a new experimental approach: a specific pH-jump protocol combined with time-resolved measurement of the delayed chlorophyll fluorescence after nanosecond flash excitation. Effective pK values were determined for low-pH inhibition of the light-induced S-state transitions: pK(1)=3.3 ± 0.3, pK(2)=3.5 ± 0.2, and pK(3)≈pK(4)=4.6 ± 0.2. Alkaline inhibition was not observed. An extension of the classical Kok model facilitated assignment of these four pK values to specific deprotonation steps in the reaction cycle. Our results provide important support to the extended S-state cycle model and criteria needed for assessment of quantum chemical calculations of the mechanism of water oxidation. They also imply that, in intact organisms, the pH in the lumen compartment can hardly drop below 5, thereby limiting the ΔpH contribution to the driving force of ATP synthesis., (© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.)

Understanding the mechanisms of electron transfer (ET) in photosynthetic reaction centers (RCs) may inspire novel catalysts for sunlight-driven fuel production. The electron exit pathway of type II RCs comprises two quinone molecules working in series and in between a non-heme iron atom with a carboxyl ligand (bicarbonate in photosystem II (PSII), glutamate in bacterial RCs). For decades, the functional role of the iron has remained enigmatic. We tracked the iron site using microsecond-resolution x-ray absorption spectroscopy after laser-flash excitation of PSII. After formation of the reduced primary quinone, Q(A)(-), the x-ray spectral changes revealed a transition (t½ ≈ 150 μs) from a bidentate to a monodentate coordination of the bicarbonate at the Fe(II) (carboxylate shift), which reverted concomitantly with the slower ET to the secondary quinone Q(B). A redox change of the iron during the ET was excluded. Density-functional theory calculations corroborated the carboxylate shift both in PSII and bacterial RCs and disclosed underlying changes in electronic configuration. We propose that the iron-carboxyl complex facilitates the first interquinone ET by optimizing charge distribution and hydrogen bonding within the Q(A)FeQ(B) triad for high yield Q(B) reduction. Formation of a specific priming intermediate by nuclear rearrangements, setting the stage for subsequent ET, may be a common motif in reactions of biological redox cofactors.

The atmospheric dioxygen (O(2)) is produced at a tetramanganese complex bound to the proteins of photosystem II (PSII). To investigate product inhibition at elevated oxygen partial pressure (pO(2) ranging from 0.2 to 16 bar), we monitored specifically the redox reactions of the Mn complex in its catalytic S-state cycle by rapid-scan and time-resolved X-ray absorption near-edge spectroscopy (XANES) at the Mn K-edge. By using a pressure cell for X-ray measurements after laser-flash excitation of PSII particles, we found a clear pO(2) influence on the redox reactions of the Mn complex, with a similar half-effect pressure as determined (2-3 bar). However, XANES spectra and the time courses of the X-ray fluorescence collected with microsecond resolution suggested that the O(2) evolution transition itself (S(3)-->S(0)+O(2)) was not affected. Additional (nonstandard) oxidation of the Mn complex at high pO(2) explains our experimental findings more readily. Our results suggest that photosynthesis at ambient conditions is not limited by product inhibition of the O(2) formation step.