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In Photosystem II, light-induced water splitting occurs via the S state cycle of the CaMn4O5-cluster. To understand the role of various possible conformations of the CaMn4O5-cluster in this process, the temperature dependence of the S1 → S2 and S2 → S3 state transitions, induced by saturating laser flashes, was studied in spinach photosystem II membrane preparations under different conditions. The S1 → S2 transition temperature dependence was shown to be much dependent on the type of the cryoprotectant and presence of 3.5% methanol, resulting in the variation of transition half-inhibition temperature by 50 K. No similar effect was observed for the S2 → S3 state transition, for which we also show that both the low spin g = 2.0 multiline and high spin g = 4.1 EPR configurations of the S2 state advance with similar efficiency to the S3 state, both showing a transition half-inhibition temperature of 240 K. This was further confirmed by following the appearance of the Split S3 EPR signal. The results are discussed in relevance to the functional and structural heterogeneity of the water oxidizing complex intermediates in photosystem II. [ABSTRACT FROM AUTHOR]

Calcium and chloride are activators of oxygen evolution in photosystem II (PSII), the light-absorbing water oxidase of higher plants, algae, and cyanobacteria. Calcium is an essential part of the catalytic Mn4CaO5 cluster that carries out water oxidation and chloride has two nearby binding sites, one of which is associated with a major water channel. The co-activation of oxygen evolution by the two ions is examined in higher plant PSII lacking the extrinsic PsbP and PsbQ subunits using a bisubstrate enzyme kinetics approach. Analysis of three different preparations at pH 6.3 indicates that the Michaelis constant, KM, for each ion is less than the dissociation constant, KS, and that the affinity of PSII for Ca2+ is about ten-fold greater than for Cl−, in agreement with previous studies. Results are consistent with a sequential binding model in which either ion can bind first and each promotes the activation by the second ion. At pH 5.5, similar results are found, except with a higher affinity for Cl− and lower affinity for Ca2+. Observation of the slow-decaying Tyr Z radical, YZ•, at 77 K and the coupled S2YZ• radical at 10 K, which are both associated with Ca2+ depletion, shows that Cl− is necessary for their observation. Given the order of electron and proton transfer events, this indicates that chloride is required to reach the S3 state preceding Ca2+ loss and possibly for stabilization of YZ• after it forms. Interdependence through hydrogen bonding is considered in the context of the water environment that intervenes between Cl− at the Cl−1 site and the Ca2+/Tyr Z region. [ABSTRACT FROM AUTHOR]

The green algal genus Picochlorum is of biotechnological interest because of its robust response to multiple environmental stresses. We compared the metabolic performance of P. SE3 and P. oklahomense to diverse microbial phototrophs and observed exceptional performance of photosystem II (PSII) in light energy conversion in both Picochlorum species. The quantum yield (QY) for O2 evolution is the highest of any phototroph yet observed, 32% (20%) by P. SE3 (P. okl) when normalized to total PSII subunit PsbA (D1) protein, and 80% (75%) normalized per active PSII, respectively. Three factors contribute: (1) an efficient water oxidizing complex (WOC) with the fewest photochemical misses of any organism; (2) faster reoxidation of reduced (PQH2)B in P. SE3 than in P. okl. (period-2 Fourier amplitude); and (3) rapid reoxidation of the plastoquinol pool by downstream electron carriers (Cyt b6f/PETC) that regenerates PQ faster in P. SE3. This performance gain is achieved without significant residue changes around the QB site and thus points to a pull mechanism involving faster PQH2 reoxidation by Cyt b6f/PETC that offsets charge recombination. This high flux in P. SE3 may be explained by genomically encoded plastoquinol terminal oxidases 1 and 2, whereas P. oklahomense has neither. Our results suggest two distinct types of PSII centers exist, one specializing in linear electron flow and the other in PSII-cyclic electron flow. Several amino acids within D1 differ from those in the low-light-descended D1 sequences conserved in Viridiplantae, and more closely match those in cyanobacterial high-light D1 isoforms, including changes near tyrosine Yz and a water/proton channel near the WOC. These residue changes may contribute to the exceptional performance of Picochlorum at high-light intensities by increasing the water oxidation efficiency and the electron/proton flux through the PSII acceptors (QAQB). [ABSTRACT FROM AUTHOR]

Photosystem II (PSII) is a multi‐subunit protein‐pigment complex with diverse redox‐active cofactors, which enabled the biological availability of O2 on Earth. The substrate specificity and the underlying redox chemistry of the Mn4CaO5 catalytic center are investigated using alternate substrates such as small molecules (ammonia and methanol) and halides (Cl‐, Br‐, I‐) instead of the natural substrate water. Changes in the kinetic profiles of steady‐state O2 evolution and of dichlorophenolindophenol (DCIP) photochemical reduction by PSII as well as the detection of modified sites by proteomic analysis implied the possibility of alternate substrate photooxidation. Of particular interest is the role of two chlorides bound close to the putative water channels in the native system. The mutation of D2‐K317 to alanine is believed to impair the binding of a catalytically relevant chloride, eliminating the chloride requirement for water oxidation catalysis. The efficiency of small molecule photooxidation by the OEC is enhanced by the mutated D2‐K317A PSII complex without the competition from chloride. These results provide insight into the role of bound chloride in native PSII as a filter for enhancing the selectivity of water oxidation. The design principles for PSII may be extended to new strategies for developing highly selective catalysts. [ABSTRACT FROM AUTHOR]

Photosystem II (PSII) uses light energy to oxidize water and to reduce plastoquinone in the photosynthetic electron transport chain. O2 is produced as a byproduct. While most members of the PSII research community agree that O2 originates from water molecules, alternative hypotheses involving bicarbonate persist in the literature. In this perspective, we provide an overview of the important roles of bicarbonate in regulating PSII activity and assembly. Further, we emphasize that biochemistry, spectroscopy, and structural biology experiments have all failed to detect bicarbonate near the active site of O2 evolution. While thermodynamic arguments for oxygen-centered bicarbonate oxidation are valid, the claim that bicarbonate is a substrate for photosynthetic O2 evolution is challenged. [ABSTRACT FROM AUTHOR]

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. This process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn4CaO5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the 'water wheel' to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn4CaO5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S0 → S1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421-425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831-15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

X-ray crystallography and X-ray spectroscopy using X-ray free electron lasers plays an important role in understanding the interplay of structural changes in the protein and the chemical changes at the metal active site of metalloenzymes through their catalytic cycles. As a part of such an effort, we report here our recent development of methods for X-ray absorption spectroscopy (XAS) at XFELs to study dilute biological samples, available in limited volumes. Our prime target is Photosystem II (PS II), a multi subunit membrane protein complex, that catalyzes the light-driven water oxidation reaction at the Mn4CaO5 cluster. This is an ideal system to investigate how to control multi-electron/proton chemistry, using the flexibility of metal redox states, in coordination with the protein and the water network. We describe the method that we have developed to collect XAS data using PS II samples with a Mn concentration of

Photosystem II (PSII) enables global-scale, light-driven water oxidation. Genetic manipulation of PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 has provided insights into the mechanism of water oxidation; however, the lack of a high-resolution structure of oxygen-evolving PSII from this organism has limited the interpretation of biophysical data to models based on structures of thermophilic cyanobacterial PSII. Here, we report the cryo-electron microscopy structure of PSII from Synechocystis sp. PCC 6803 at 1.93-Å resolution. A number of differences are observed relative to thermophilic PSII structures, including the following: the extrinsic subunit PsbQ is maintained, the C terminus of the D1 subunit is flexible, some waters near the active site are partially occupied, and differences in the PsbV subunit block the Large (O1) water channel. These features strongly influence the structural picture of PSII, especially as it pertains to the mechanism of water oxidation.

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In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

To date, cyclic electron flow around PSI (PSI-CEF) has been considered the primary (if not the only) mechanism accepted to adjust the ratio of linear vs cyclic electron flow that is essential to adjust the ratio of ATP/NADPH production needed for CO2 carboxylation. Here we provide a kinetic model showing that cyclic electron flow within PSII (PSII-CEF) is essential to account for the accelerating rate of decay in flash-induced oscillations of O2 yield as the PQ pool progressively reduces to PQH2. Previously, PSII-CEF was modeled by backward transitions using empirical Markov models like Joliot-Kok (J-K) type. Here, we adapted an ordinary differential equation methodology denoted RODE1 to identify which microstates within PSII are responsible for branching between PSII-CEF and Linear Electron Flow (LEF). We applied it to simulate the oscillations of O2 yield from both Chlorella ohadii, an alga that shows strong PSII-CEF attributed to high backward transitions, and Synechococcus elongatus sp. 7002, a widely studied model cyanobacterium. RODE2 simulations reveal that backward transitions occur in microstates that possess a QB− semiquinone prior to the flash. Following a flash that forms microstates populating (QAQB)2−, PSII-CEF redirects these two electrons to the donor side of PSII only when in the oxidized S2 and S3 states. We show that this backward transition pathway is the origin of the observed period-2 oscillations of flash O2 yield and contributes to the accelerated decay of period-4 oscillations. This newly added pathway improved RODE1 fits for cells of both S. elongatus and C. ohadii. RODE2 simulations show that cellular adaptation to high light intensity growth is due to a decrease in QB availability (empty or blocked by Q2−B), or equivalently due to a decrease in the difference in reduction potential relative to QA/QA−. PSII-CEF provides an alternative mechanism for rebalancing the NADPH:ATP ratio that occurs rapidly by adjusting the redox level of the PQ:PQH2 pool and is a necessary process for energy metabolism in aquatic phototrophs. [ABSTRACT FROM AUTHOR]

The understanding of light‐induced biological water oxidation in oxygenic photosynthesis is of great importance both for biology and (bio)technological applications. The chemically difficult multistep reaction takes place at a unique protein‐bound tetra‐manganese/calcium cluster in photosystem II whose structure has been elucidated by X‐ray crystallography (Umena et al. Nature 2011, 473, 55). The cluster moves through several intermediate states in the catalytic cycle. A detailed understanding of these intermediates requires information about the spatial and electronic structure of the Mn4Ca complex; the latter is only available from spectroscopic techniques. Here, the important role of Electron Paramagnetic Resonance (EPR) and related double resonance techniques (ENDOR, EDNMR), complemented by quantum chemical calculations, is described. This has led to the elucidation of the cluster's redox and protonation states, the valence and spin states of the manganese ions and the interactions between them, and contributed substantially to the understanding of the role of the protein surrounding, as well as the binding and processing of the substrate water molecules, the O‐O bond formation and dioxygen release. Based on these data, models for the water oxidation cycle are developed. [ABSTRACT FROM AUTHOR]

James Barber, known to colleagues and friends as Jim, passed away in January 2020 after a long battle against cancer. During his long and distinguished career in photosynthesis research, Jim made many outstanding contributions with the pinnacle achieving his dream of determining the first detailed structure of the Mn cluster involved in photosynthetic water oxidation. Here, colleagues and friends remember Jim and reflect upon his scientific career and the impact he had on their lives and the scientific community. [ABSTRACT FROM AUTHOR]

Photosystem II (PSII) has a number of hydrogen-bonding networks connecting the manganese cluster with the lumenal bulk solution. The structure of PSII from Thermosynechococcus vulcanus (T. vulcanus) showed that D1-R323, D1-N322, D1-D319 and D1-H304 are involved in one of these hydrogen-bonding networks located in the interfaces between the D1, CP43 and PsbV subunits. In order to investigate the functions of these residues in PSII, we generated seven site-directed mutants D1-R323A, D1-R323E, D1-N322R, D1-D319L, D1-D319R, D1-D319Y and D1-H304D of T. vulcanus and examined the effects of these mutations on the growth and functions of the oxygen-evolving complex. The photoautotrophic growth rates of these mutants were similar to that of the wild type, whereas the oxygen-evolving activities of the mutant cells were decreased differently to 63–91% of that of the wild type at pH 6.5. The mutant cells showed a higher relative activity at higher pH region than the wild type cells, suggesting that higher pH facilitated proton egress in the mutants. In addition, oxygen evolution of thylakoid membranes isolated from these mutants showed an apparent decrease compared to that of the cells. This is due to the loss of PsbU during purification of the thylakoid membranes. Moreover, PsbV was also lost in the PSII core complexes purified from the mutants. Taken together, D1-R323, D1-N322, D1-D319 and D1-H304 are vital for the optimal function of oxygen evolution and functional binding of extrinsic proteins to PSII core, and may be involved in the proton egress pathway mediated by YZ. [ABSTRACT FROM AUTHOR]

Historically, two modeling approaches have been developed independently to describe photosynthetic electron transport (PET) from water to plastoquinone within Photosystem II (PSII): Markov models account for losses from finite redox transition probabilities but predict no reaction kinetics, and ordinary differential equation (ODE) models account for kinetics but not for redox inefficiencies. We have developed an ODE mathematical framework to calculate Markov inefficiencies of transition probabilities as defined in Joliot–Kok-type catalytic cycles. We adapted a previously published ODE model for PET within PSII that accounts for 238 individual steps to enable calculation of the four photochemical inefficiency parameters (miss, double hit, inactivation, backward transition) and the four redox accumulation states (S-states) that are predicted by the most advanced of the Joliot–Kok-type models (VZAD). Using only reaction kinetic parameters without other assumptions, the RODE-calculated time-averaged (e.g., equilibrium) inefficiency parameters and equilibrium S-state populations agree with those calculated by time-independent Joliot–Kok models. RODE also predicts their time-dependent values during transient photochemical steps for all 96 microstates involving PSII redox cofactors. We illustrate applications to two cyanobacteria, Arthrospira maxima and Synechococcus sp. 7002, where experimental data exists for the inefficiency parameters and the S-state populations, and historical data for plant chloroplasts as benchmarks. Significant findings: RODE predicts the microstates responsible for period-4 and period-2 oscillations of O2 and fluorescence yields and the four inefficiency parameters; the latter parameters are not constant for each S state nor in time, in contrast to predictions from Joliot–Kok models; some of the recombination pathways that contribute to the backward transition parameter are identified and found to contribute when their rates exceed the oxidation rate of the terminal acceptor pool (PQH2); prior reports based on the assumptions of Joliot–Kok parameters may require reinterpretation. [ABSTRACT FROM AUTHOR]

The immobilization of natural photosystem II (PSII) enzyme onto an artificial electrode offers an ingenious and promising avenue for semiartificial solar energy conversion. However, this process is significantly limited by the poor stability and the short life of PSII. Here, a new prototype of a semiartificial system is reported by anchoring PSII on polyethylenimine‐coated macroporous carbon electrode with a high load. Good electronic communication is established at the biointerface of this PSII electrode, enabling excellent photoelectrochemical (PEC) water oxidation and lasting electricity generation. The maximum turnover number of 10 200 ± 1380 mol O2 per mol PSII dimer is obtained in this system at around 10 h before complete deactivation, reaching high current‐to‐O2 conversion efficiencies. The functions of PSII to release O2 both in light and dark conditions as well as for H2O2 formation are revealed. Under periodic irradiation (AM 1.5G 1 sun), this PSII electrode allows for stable mediated photocurrent output of ≈4.31 µA cm−2 after five days, which represents the most stable photoelectric performance achieved so far for PSII‐related electrodes. [ABSTRACT FROM AUTHOR]

We derive a model that provides an exact solution to the substrate-water exchange kinetics in a double-conformation system and use this model to interpret recently published data for Ca2+- and Sr2+-containing PSII in the S2 state, in which the g = 2.0 and g = 4.1 conformations coexist. The component concentrations derived from the kinetic model provide an analytic description of the substrate-water exchange kinetics, allowing us to more accurately interpret the results. Based on this model and the previously reported data on the S2 state g = 2.0 conformation, we obtain the substrate-water exchange rates of the g = 4.1 conformation and the conformational change rates. Two conclusions are made from the analyses. First, contrary to previous reports, there is no significant effect of substituting Sr2+ for Ca2+ on any of the exchange rate constants. Second, the exchange rate of the slowly-exchanging water (Ws) in the S2 state g = 4.1 conformation is much faster than that in the S2 state g = 2.0 conformation. The second conclusion is consistent with the assignment of Ws to W1 or W2 bound as terminal ligands to Mn4; Mn4 has been proposed to undergo an oxidation state change from Mn(IV) in the g = 2.0 conformation to Mn(III) in the g = 4.1 conformation.

This review gives a brief description of the theory and application of X-ray absorption spectroscopy, both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS), especially, pertaining to photosynthesis. The advantages and limitations of the methods are discussed. Recent advances in extended EXAFS and polarized EXAFS using oriented membranes and single crystals are explained. Developments in theory in understanding the XANES spectra are described. The application of X-ray absorption spectroscopy to the study of the Mn4Ca cluster in Photosystem II is presented.

Photosynthetic O2 evolution is catalyzed by the Mn4CaO5 cluster of the water oxidation complex of the photosystem II (PSII) complex. The photooxidative self-assembly of the Mn4CaO5 cluster, termed photoactivation, utilizes the same highly oxidizing species that drive the water oxidation in order to drive the incorporation of Mn2+ into the high-valence Mn4CaO5 cluster. This multistep process proceeds with low quantum efficiency, involves a molecular rearrangement between light-activated steps, and is prone to photoinactivation and misassembly. A sensitive polarographic technique was used to track the assembly process under flash illumination as a function of the constituent Mn2+ and Ca2+ ions in genetically engineered membranes of the cyanobacterium Synechocystis sp. PCC6803 to elucidate the action of Ca2+ and peripheral proteins. We show that the protein scaffolding organizing this process is allosterically modulated by the assembly protein Psb27, which together with Ca2+ stabilizes the intermediates of photoactivation, a feature especially evident at long intervals between photoactivating flashes. The results indicate three critical metalbinding sites: two Mn and one Ca, with occupation of the Ca site by Ca2+ critical for the suppression of photoinactivation. The longobserved competition between Mn2+ and Ca2+ occurs at the second Mn site, and its occupation by competing Ca2+ slows the rearrangement. The relatively low overall quantum efficiency of photoactivation is explained by the requirement of correct occupancy of these metal-binding sites coupled to a slow restructuring of the protein ligation environment, which are jointly necessary for the photooxidative trapping of the first stable assembly intermediate. [ABSTRACT FROM AUTHOR]

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ~350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated. [ABSTRACT FROM AUTHOR]

This review gives a brief description of the theory and application of X-ray absorption spectroscopy, both X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS), especially, pertaining to photosynthesis. The advantages and limitations of the methods are discussed. Recent advances in extended EXAFS and polarized EXAFS using oriented membranes and single crystals are explained. Developments in theory in understanding the XANES spectra are described. The application of X-ray absorption spectroscopy to the study of the Mn4Ca cluster in Photosystem II is presented.

The Mn 4 CaO 5 oxygen-evolving complex (OEC) in Photosystem II (PSII) is assembled in situ and catalyzes water oxidation. After OEC assembly, the PsbO extrinsic subunit docks to the lumenal face of PSII and both stabilizes the OEC and facilitates efficient proton transfer to the lumen. D1 residue R334 is part of a hydrogen bond network involved in proton release during catalysis and interacts directly with PsbO. D1-R334 has recently been observed in different conformations in apo- and holo-OEC PSII structures. We generated a D1-R334G point mutant in Synechocystis sp. PCC 6803 to better understand this residue's function. D1-R334G PSII is active under continuous light, but the OEC is unstable in darkness. Isolated D1-R334G core complexes have little bound PsbO and less manganese as the wild type control. The S 2 intermediate is stabilized in D1-R334G indicating that the local environment around the OEC has been altered. These results suggest that the hydrogen bond network that includes D1-R334 exists in a different functional conformation during PSII biogenesis in the absence of PsbO. • Hydrogen bond networks connect the PSII OEC to the lumen. • D1-R334 is observed in different conformations when PsbO is bound or absent. • A D1-R334G mutant has a dark unstable OEC. • D1-R334 helps direct the transition of a hydrogen bond network during PSII maturation. [ABSTRACT FROM AUTHOR]

Water oxidation to dioxygen in photosynthesis is catalyzed by a Mn4Ca cluster with O bridging in Photosystem II (PS II) of plants, algae and cyanobacteria. A variety of spectroscopic methods have been applied to analyzing the participation of the complex. X-ray spectroscopy is particularly useful because it is element-specific, and because it can reveal important structural features of the complex with high accuracy and identify the participation of Mn in the redox chemistry. Following a brief history of the application of X-ray spectroscopy to PS II, an overview of newer results will be presented and a description of the present state of our knowledge based on this approach.

The membrane-protein complex photosystem II (PSII) catalyzes photosynthetic water oxidation. Proton transfer plays an integral role in the catalytic cycle of water oxidation by maintaining charge balance to regulate and ensure the efficiency of the process. The hydrogen-bonded amino-acid residues that surround the oxygen-evolving complex (OEC) provide an efficient pathway for proton removal. Hence, it is crucial to identify these pathways to provide deeper insights into the proton-transfer mechanisms. In this study, we have used bicarbonate as a mobile exogenous proton-transfer reagent to recover the activity lost by site-directed mutations in order to identify amino-acid residues participating in the proton-transfer pathway. We find that bicarbonate restores efficient S-state cycling in D2-K317A PSII core complexes, but not in D1-D61A and CP43-R357K PSII core complexes, indicating that bicarbonate chemical rescue can be used to differentiate single-point mutations affecting the pathways of proton transfer from mutations that affect other aspects of the water-oxidation mechanism. • A hydrogen-bonding network that surrounds the oxygen-evolving complex in photosystem II facilitates proton transfer. • Mutations of amino-acid residues around the oxygen-evolving complex cause loss of oxygen-evolution activity. • Bicarbonate restores efficient oxygen-evolution activity and S-state cycling in D2-K317A PSII core complexes. • Chemical rescue by bicarbonate after site-directed mutation enables identification of amino-acid residues that function in proton transfer. [ABSTRACT FROM AUTHOR]

The lumenal carbonic anhydrase (CA) CAH3 from green alga Chlamydomonas reinhardtii is the only one CA identified so far in close association with the photosystem II (PSII) multi-subunit protein complex. It was proposed earlier, that CAH3 could facilitate the H+ removal from the active center of the PSII water-oxidizing complex (WOC) under the light, thereby increasing its activity. In the present work, using PSII enriched membranes from the wild type of C. reinhardtii and from the CAH3-deficient mutant cia3, we demonstrate, that the suppression of the photosynthetic activity of PSII by increased pH is more pronounced in preparations from cia3 as compared to the wild type. Experiments with CA inhibitors show that the activity of CAH3 supports the function of PSII and prevents its irreversible inactivation under light upon increased pH. The photosynthetic activity of PSII from cia3 can be restored to the wild type level upon increased pH if an excess of HCO 3 – is added. These findings testify that the main role of CAH3 in the vicinity of PSII is the acceleration of the HCO 3 – dehydration reaction. Measurements of the photoinduced electron transfer rate in PSII from water or from an artificial electron donor indicate, that CAH3 has a direct influence on the WOC function. Based on the data obtained in this work we conclude, that in vivo CA-activity of CAH3 may support the photosynthetic activity of PSII at increased pH in the thylakoid lumen and can be observed under the dark to light transition. • CAH3 is associated with PSII multi-subunit protein complex. • CAH3 supports the photosynthetic activity of PSII under pH above 6.5. • CAH3 acts near WOC of PSII, where accelerates the HCO 3 – dehydration reaction. [ABSTRACT FROM AUTHOR]

The development of the X-ray free electron laser (XFEL) as a tool for crystallography started a new era in protein science with the intriguing possibility of determining time-resolved structural changes under physiological conditions. This chapter summarizes the current state of XFEL-based structural research on tetrapyrrole containing proteins including the photosystems of oxygenic photosynthesis, the purple bacterial reaction center, phycobilins, and phytochromes. Besides describing technical developments that were necessary to fully exploit the advantages of the method, emphasis is put on the task of producing suitable protein microcrystals for XFEL experiments. In particular, issues encountered in crystallizing membrane proteins are described with photosystem II as the prime example to underscore the importance of combining physical and biochemical knowledge, but also to point to unsolved problems. Finally, novel structural information obtained by XFEL experiments for the various systems is given with a focus on recent advances in understanding photosynthetic water oxidation. [ABSTRACT FROM AUTHOR]

With this Tribute, we remember and honor Thomas John (Tom) Wydrzynski. Tom was a highly innovative, independent and committed researcher, who had, early in his career, defined his life-long research goal. He was committed to understand how Photosystem II produces molecular oxygen from water, using the energy of sunlight, and to apply this knowledge towards making artificial systems. In this tribute, we summarize his research journey, which involved working on 'soft money' in several laboratories around the world for many years, as well as his research achievements. We also reflect upon his approach to life, science and student supervision, as we perceive it. Tom was not only a thoughtful scientist that inspired many to enter this field of research, but also a wonderful supervisor and friend, who is deeply missed (see footnote*). [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Initial Mn2+ binding site in photoassembly of the Mn 4 CaO 5 cluster was studied. • QM/MM calculation showed that the Mn1 site is most stable for Mn2+ binding. • Mn2+ at the Mn2 site can be in equilibrium with that at the Mn1 site. • The Mn1 site is suggested to be the initial Mn2+ binding site in Mn 4 CaO 5 assembly. Abstract The initial Mn2+ binding site in photoassembly of the Mn 4 CaO 5 cluster, the catalytic center of water oxidation in photosystem II, was studied using quantum mechanics/molecular mechanics calculations. Among the five metal sites (Mn1–Mn4 and Ca) of the Mn 4 CaO 5 cluster, the Mn1 site involving D1-H332 was most stable for Mn2+ binding, while the Mn2 site showed a slightly higher energy comparable to the thermal fluctuation energy at room temperature. It is thus suggested that Mn2+ binding at the Mn1 site, likely in equilibrium with that at the Mn2 site, is the initial step of photoassembly of the Mn 4 CaO 5 cluster. [ABSTRACT FROM AUTHOR]

We have used the desiccation-tolerant lichen Flavoparmelia caperata , containing the green algal photobiont Trebouxia gelatinosa , to examine H/D isotope effects in Photosystem II in vivo. Artifact-free H/D isotope effects on both PSII primary charge separation and water oxidation yields were determined as a function of flash rate from chlorophyll- a variable fluorescence yields. Intact lichens could be reversibly dehydrated/re-hydrated with H 2 O/D 2 O repeatedly without loss of O 2 evolution, unlike all isolated PSII preparations. Above a threshold flash rate, PSII charge separation decreases sharply in both D 2 O and H 2 O, reflecting loss of excitation migration and capture by PSII. Changes in H/D coordinates further slow charge separation in D 2 O (−23% at 120 Hz), attributed to reoxidation of the primary acceptor Q A − . At intermediate flash rates (5–50 Hz) D 2 O decreases water oxidation efficiency (O 2 evolution) by −2–5%. No significant isotopic difference is observed at slow flash rates (<5 Hz) where charge recombination dominates. Slower D 2 O diffusion, changes in hydrogen bonding networks, and shifts in the pK a 's of ionizable residues may all contribute to these systematic variations of H/D isotope effects. Lichens' reversible desiccation tolerance allows highly reproducible H/D exchange kinetics in PSII reactions to be studied in vivo for the first time. [ABSTRACT FROM AUTHOR]

Photosynthetic oxygen evolution occurs through the oxidation of water at a catalytic Mn 4 CaO 5 cluster in photosystem II and is promoted by chloride, which binds at two sites near the Mn 4 CaO 5 cluster. Fluoride is a competitive inhibitor of chloride activation, but study of its effects is complicated by the possibility that it may form an insoluble CaF 2 complex. In this study, the effects of fluoride were studied using PSII lacking the PsbP and PsbQ subunits, which help to regulate the requirements for the inorganic cofactors Ca 2+ and Cl − . In this preparation, which allows easy exchange of ions, it was found that F − does not directly remove Ca 2+ even when catalytic turnovers take place, suggesting that fluoride is not able to access the inner coordination sphere of Ca 2+ . By monitoring the loss in O 2 evolution activity, the dissociation constant of F − was estimated to be about 1 mM in intact PSII, consistent with previous studies, and about 77 mM in PSII lacking the extrinsic subunits. The significantly higher value for PSII lacking PsbP and PsbQ is consistent with results for other ions. The effects of F − on electron transfer to Tyr Z was also studied and found to show similar trends in PSII with and without the two extrinsic subunits, but with a more pronounced effect in PSII lacking the extrinsic subunits. These results indicate that in PSII lacking PsbP and PsbQ, fluoride does not directly interact with or remove Ca 2+ and inhibits O 2 evolution in a manner comparable to PSII with the extrinsic subunits intact. [ABSTRACT FROM AUTHOR]