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... Joliot et al. (1971) and Delosme (1971a, b) discovered period-4 oscillations in the initial fluorescence yield, F 0 , and in maximal fluorescence yield, F m ST , respectively. The oscillation patterns were found to closely relate to that of the yield of flash-induced oxygen evolution in a series of ST, which could be satisfactorily interpreted by the 4-step charge accumulation model of water-splitting proposed by Kok et al. (1970). The fluorescence data suggested that F 0 follows the sum of the S 0 + S 1 population and F m ST that of the S 2 + S 3 population briefly before an ST. ...
... In Fig. 5 an example of a typical measurement of an STK sequence (STKS) with dark-adapted Chlorella is presented: 10 consecutive ST were applied at 5% of maximal ST intensity, 40 µs width and 100 ms repetition time. Under these conditions pronounced period-4 oscillations of F 0 and F m ST are observed which reflect the 4-step cooperation of positve charges in the oxygen evolving complex (OEC) at the donor-side of PSII (Kok et al. 1970;Delosme 1971a, b) (see section below on 'Sequences of ST-kinetics and period-4 oscillations: Delosme (1971) revisited'). The Pam-Win-4 software provides a dedicated routine for plotting and analysing the complex information contained in an STKS recording. ...
... Based on the Kok model of water-splitting, i.e. the linear four-step accumulation of positive charges at the donor side of PSII (Kok et al. 1970), in principal the FR effect may be explained in two different ways: 1) If it is assumed that Chlorella after thorough dark-adaptation displays a significant population of S 0 , this would be shifted to S 1 by weak FR. 2) If Chlorella were in state S 1 after thorough dark-adaptation, this would be shifted to S 2 . ...
A new measuring system based on the already existing Multi-Color-PAM Fluorimeter (Schreiber et al. in Photosynth Res 113:127–144, 2012) was developed that in addition to standard PAM measurements enables pump-and-probe flash measurements and allows simultaneous measurements of the changes in chlorophyll fluorescence yield (F) during application of saturating flashes (ST). A high-power Chip-on-Board LED array provides ST flashes with close to rectangular profiles at wide ranges of widths (0.5 µs to 5 ms), intensities (1.3 mmol to 1.3 mol 440 nm quanta m⁻² s⁻¹) and highly flexible repetition times. Using a dedicated rising-edge profile correction, sub-µs time resolution is obtained for assessment of initial fluorescence and rise kinetics. At maximal to moderate flash intensities the flash-kinetics (changes of F during course of ST, STK) are strongly affected by ‘High Intensity Quenching’ (HIQ), consisting of Car-triplet quenching, TQ, and donor-side-dependent quenching, DQ. The contribution of TQ is estimated by application of a second ST after 20 µs dark-time. Upon application of flash trains (ST sequences with defined repetition times) typical period-4 oscillations in dark fluorescence yield (F0) and ST-induced fluorescence yield, FmST, are obtained which can be measured in vivo both with suspensions and from the surface of leaves. Examples of application with dilute suspensions of Chlorella and an intact dandelion leaf are presented. It is shown that weak far-red light (730–740 nm) advances the S-state distribution of the water-splitting system by one step, resulting in substantial lowering of FmST and also of the I1-level in the polyphasic rise of fluorescence yield induced by a multiple-turnover flash (MT). Based on comparative measurements of STK and the polyphasic rise kinetics with the same Chlorella sample, it is concluded that the generally observed lower values of maximal fluorescence yields using ST-protocols compared to MT-protocols are due to a higher extent of HIQ (mainly DQ) and the contribution of variable PSI fluorescence to FmST.
... A breakthrough in the elucidation of the function of photosystem II was made, independently, by Pierre Joliot et al. [5] and Bessel Kok et al. [6]. When they exposed Chlorella algae or spinach chloroplasts that had been in darkness for some time to repeated very short and intense light flashes, they observed no oxygen after a single flash, a maximum of oxygen (O 2 ) production after the third flash, and thereafter on every fourth flash ( Figure 2). ...
... The molecular structure in which the oxidation of water to molecular oxygen takes place is called the Mn 4 CaO 5 cluster ( Figure 3A). The structure was first reported by Shen's group, Umena et al. [6] and Suga et al. [7]. Figure 4 shows a schematically of how the Mn 4 CaO 5 cluster forms part of the large photosystem II complex, which is one of the proteins in the thylakoid membranes in cyanobacteria and in the chloroplasts of algae and other plants. The molecular structure in which the oxidation of water to molecular place is called the Mn4CaO5 cluster ( Figure 3A). ...
... The molecular structure in which the oxidation of water to molecular place is called the Mn4CaO5 cluster ( Figure 3A). The structure was first repor group, Umena et al. [6] and Suga et al. [7]. The molecular structure in which the oxidation of water to molecular oxyge place is called the Mn4CaO5 cluster ( Figure 3A). ...
This review deals with the production of oxygen by photo-oxidation of water, which is a topic fitting a journal devoted to oxygen. Most of the present biosphere, including mankind, depends on oxygen. Elucidating the mechanism is of importance for solving the present energy crisis. Photosynthesis evolved in bacteria, first in a form that did not produce oxygen. The oxygen-producing version arose with the advent of cyanobacteria about three billion years ago. The production of oxygen by photo-oxidation of water requires the co-operative action of four photons. These are harvested from daylight by chlorophyll and other pigments (e.g., phycobiliproteins) and are channeled to photosystem II and photosystem I. The oxygen-evolving complex resides in photosystem II, surrounded by protein subunits, and contains one ion of calcium, four ions of manganese, and a number of oxygen atoms. For each quantum of energy it receives from absorbed light, it proceeds one step through a cycle of states known as the Kok–Joliot cycle. For each turn of the cycle, one molecule of oxygen (O2) is produced.
... These ash-induced oxygen oscillation patterns (FIOPs) were explained by Kok and coworkers in a model, which postulates that during water oxidation, the WOC cycles through ve intermediate redox states, collectively called the S states, labelled S 0 -S 4 (Fig. 1). 13,14 S 0 is the most reduced state while S 1, S 2 and S 3 represent sequentially higher oxidation states in the WOC. S 1 is the dominating state in the dark while the S 2 and S 3 states are metastable and decay back to the S 1 state in a few minutes at room temperature. ...
... 4,9 Thus, four consecutive charge separations and accompanying electron transfer events are necessary in PSII to oxidize two water molecules to molecular oxygen and four protons ( Fig. 1), which accounts for the period of four oscillations in FIOPs. 9,13,14 In Kok's S state cycle model, the dampening of the FIOP oscillations with the increasing ash number is explained by "double hits" and "misses". The double hit parameter accounts for centers that perform two consecutive charge separations and S state transitions as a result of a single ash excitation. ...
... The double hit parameter accounts for centers that perform two consecutive charge separations and S state transitions as a result of a single ash excitation. 13,14 Double hits are produced by the long ash tails from Xe ash-lamps and broad LEDs light pulses (tens to hundreds of ms long) and can be eliminated by using short nanosecond laser ashes which produce only a single charge separation in PSII. 21 The miss parameter is the most important factor in understanding the S state cycle advancement and dampening of the FIOPs. ...
Photosynthesis stores solar light as chemical energy and efficiency of this process is highly important. The electrons required for CO2 reduction are extracted from water in a reaction driven by light-induced charge separations in the Photosystem II reaction center and catalyzed by the CaMn4O5-cluster. This cyclic process involves five redox intermediates known as the S0-S4 states. In this study, we quantify the flash-induced turnover efficiency of each S state by electron paramagnetic resonance spectroscopy. Measurements were performed in photosystem II membrane preparations from spinach in the presence of an exogenous electron acceptor at selected temperatures between -10 °C and +20 °C and at flash frequencies of 1.25, 5 and 10 Hz. The results show that at optimal conditions the turnover efficiencies are limited by reactions occurring in the water oxidizing complex, allowing the extraction of their S state dependence and correlating low efficiencies to structural changes and chemical events during the reaction cycle. At temperatures 10 °C and below, the highest efficiency (i.e. lowest miss parameter) was found for the S1 → S2 transition, while the S2 → S3 transition was least efficient (highest miss parameter) over the whole temperature range. These electron paramagnetic resonance results were confirmed by measurements of flash-induced oxygen release patterns in thylakoid membranes and are explained on the basis of S state dependent structural changes at the CaMn4O5-cluster that were determined recently by femtosecond X-ray crystallography. Thereby, possible "molecular errors" connected to the e - transfer, H+ transfer, H2O binding and O2 release are identified.
... [50][51][52][53] The biological OEC consists of a Mn-Ca cluster that exhibits exceptional characteristics in water oxidation. [54][55][56][57] It displays an exceedingly low overpotential of only 20 mV and operates at a high turnover frequency, releasing between 25 and 90 molecules of O 2 per second. 54,55 The mechanism proposed by Kok, extensively accepted in the scientific community, outlines the progression of OEC through five distinct states: S 0 to S 4 . ...
... The absorption of photons by photosystem II triggers the transition of the system from state S 0 to S 4 . However, due to its instability, S 4 reacts with water, resulting in the formation of O 2 , as indicated in ref. 56 and 57. A recent study has focused 58 on the electrocatalytic activity of nickel (hydr)oxide in the OER at an extremely low overpotential. ...
The progress made in natural language processing (NLP) and large language models (LLMs) such as generative pre-trained transformer (GPT) has introduced exciting opportunities for enhancing research across various fields. Within the realm of catalysis studies, GPT-driven models present valuable support in expediting the exploration and comprehension of catalytic processes. The research underscores the significance of ChatGPT in catalysis research, emphasizing its prowess as a valuable tool for furthering scientific inquiries. It suggests that for an outstanding oxygen-evolution reaction (OER) catalyst as a case study, scientists can leverage ChatGPT to extract deeper insights and brainstorm innovative approaches to grasp the mechanism better and refine current systems.
... To account for these results, a schematic model, called the "S state mechanism", was proposed by Kok and colleagues in 1970s [9,10]. This model comprises of a series of five states, defined as S0 to S4 (Fig. 3a). ...
... (a) The S state model prepared by Kok and coworkers[9] (b) ...
Many inorganic elements play fundamental role in biochemical processes, such as photosynthesis, which is an incredibly important biological processes in this planet. This paper reviewed the role of Mg, Mn, Cu and Fe in photosynthesis. Magnesium plays a role in maintaining the structure of chlorophyll, due to its appropriate size, charge, and redox-inert feature. Manganese is required for photosynthetic O2 evolution. Copper and iron usually form metalloproteins with ligands such as halide and amino acid residual and serve as electron transporters in PSI and PSII. In the past a couple of decades, structures of major redox metalloproteins and principles of important photosynthetic reactions have been intensively studied, and significant progresses have been made. However, there are still a number of ambiguous points that need to be clarified.
... To yield one molecule of O 2 from two molecules of H 2 O, four successive flashes are required (illumination), therefore the reaction is believed to be a manifestation of five sequential events, named as state 0 through 4 (S 0 to S 4 ) ( Fig 4B) [28]. During the first four steps from S 0 to S 4 , oxidising ...
... The S 4 state upon catalysis releases one molecule of O 2 and thereby reverts back to the S 0 state [29]. This cycle is known as the 'Kok cycle' [28]. ...
Plants and other green organisms harvest sunlight by green chlorophyll pigments and covertit to chemical energy (sugars) and oxygen in a process called photosynthesis providing the foundation for life on Earth. Although it is unanimously believed that oceanic phytoplanktons are the main contributors to the global photosynthesis, the contribution of coniferous boreal forests distributed across vast regions of the northern hemisphere cannot be undermined. Hence boreal forests account signifificantly for social, economical and environmental sustainability. Not only do conifers thrive in the tundra regions with extreme climate, but they also maintain their needles green over the boreal winter. A question remains; what makes them so resilient? In this respect, we aimed to understand the remarkable winter adaptation strategies in two dominant boreal coniferous species,i.e., Pinus sylvestris and Picea abies. First, we mapped the transcriptional landscape in Norway spruce (Picea abies) needles over the annual cycle. Transcriptional changes in the nascent needles reflflected a sequence of developmental processes and active vegetative growth during early summer and summer. Later after maturation, transcriptome reflflected activated defense against biotic factors and acclimationin response to abiotic environmental cues such as freezing temperatures during winter. Secondly, by monitoring the photosynthetic performance of Scot pine needles, we found that the trees face extreme stress during the early spring (Feb-Mar) when sub-zero temperatures are accompanied by high solar radiation. At this time, drastic changes occur in the thylakoid membranes of the chloroplast that allows the mixing of photosystem I and photosystem II that typically remain laterally segregated. This triggers direct energy transfer from PSII to PSI and thus protects PSII from damage. Furthermore, we found that this loss of lateral segregation may be a consequence of triple phosphorylationof Lhcb1 (Light harvesting complex1 of photosystem II). The structural changes in thylakoid membranes also lead to changes inthe thylakoid macro domain organisationand pigment protein composition. Furthermore, we discovered that while PSII is protected by direct energy transfer, the protection of PSI is provided through photoreduction of oxygen by flavodiiron proteins, which in turn allows P700 to stay in an oxidised state necessary for direct energy transfer. These coordinated cascades of changes concomitantly protect both PSI and PSII to maintain the needles green over the winter.
... Hoganson (Scheme 4), [74] following the five-step Kok cycle. [75] The water oxidation cycle starts with the photoexcitation of the PSII primary electron donor P680, yielding the electronically excited P680* (17). Then an electron transfer occurs between P680* and the primary electron acceptor pheophytin (Pheo), ...
... In the next 30 sec in the dark, the solution was remeasured to obtain the blue absorption curve. The same experiments 75 with longer illumination time were carried out on the same solution. All the observed results were similar and therefore, are summarized in the same Figure 64 and 65. ...
... After excitation of a PSII core Chl a dimer (P680), a pheophytin --P680 + intermediate is formed allowing a rereduction of P680 + by one single electron of the water-splitting complex via a tyrosine residue of D1 (Ferreira et al. 2004;Rappaport & Diner 2008;Telfer 2002). Water splitting happens at a Mn4Ca cluster which needs to abstract the four electrons from two water molecules in the lumen to form O2 and 4 H + in a single reaction (Ferreira et al. 2004;Kok et al. 1970). P680 + therefore is only able to oxidize the Mn4Ca-cluster partially, which is able to store the energy in four intermediate steps (S 1+ S 2+ S 3+ ) while only the S 4+ state (Mn4Ca 4+ ) is able to oxidize the two water molecules (Kok et al. 1970;Rappaport & Diner 2008;Suga et al. 2015). ...
... Water splitting happens at a Mn4Ca cluster which needs to abstract the four electrons from two water molecules in the lumen to form O2 and 4 H + in a single reaction (Ferreira et al. 2004;Kok et al. 1970). P680 + therefore is only able to oxidize the Mn4Ca-cluster partially, which is able to store the energy in four intermediate steps (S 1+ S 2+ S 3+ ) while only the S 4+ state (Mn4Ca 4+ ) is able to oxidize the two water molecules (Kok et al. 1970;Rappaport & Diner 2008;Suga et al. 2015). Pheophytinpasses its electron to the primary electron acceptor plastoquinone (QA) (Ferreira et al. 2004;Rappaport & Diner 2008;Telfer 2002). ...
... Wu's mechanism (see Fig. 4) indeed explains part of the bicarbonate effect in photosystem II, and some puzzles in the Kok-Joliot cycle [27] for oxygen evolution in photosynthesis. Whether the stimulation of oxygen evolution by bicarbonate (bicarbonate effect) occurs on the electron acceptor side, the electron donor side, or both of photosystem II has been studied by many in the past (see review [28,29]). ...
Whether rapid oxygen isotopic exchange between bicarbonate and water occurs in photosynthesis is the key to determine the source of oxygen by classic 18O-labeled photosynthetic oxygen evolution experiments. Here we show that both Microcystis aeruginosa and Chlamydomonas reinhardtii utilize a significant proportion (>16%) of added bicarbonate as a carbon source for photosynthesis. However, oxygen isotopic signal in added bicarbonate cannot be traced in the oxygen in organic matter synthesized by these photosynthetic organisms. This contradicts the current photosynthesis theory, which states that photosynthetic oxygen evolution comes only from water, and oxygen in photosynthetic organic matter comes only from carbon dioxide. We conclude that the photosynthetic organisms undergo rapid exchange of oxygen isotope between bicarbonate and water during photosynthesis. At the same time, this study also provides isotopic evidence for a new mechanism that half of the oxygen in photosynthetic oxygen evolution comes from bicarbonate photolysis and half comes from water photolysis, which provides a new explanation for the bicarbonate effect, and suggests that the Kok-Joliot cycle of photosynthetic oxygen evolution, must be modified to include a molecule of bicarbonate in addition to one molecule of water which in turn must be incorporated into the cycle instead of two water molecules. Furthermore, this study provides a theoretical basis for constructing a newer artificial photosynthetic reactor coupling light reactions with the dark reactions.
... A few picoseconds after the excitation reaches Chl D1 , a charge separation occurs resulting in the formation of the Chl D1 + Phe D1 and then of the [P D1 P D2 ] + Phe D1 radical pair states, with the positive charge mainly located on P D1 , e.g. (Holzwarth et al. 2006, Romero et al. 2017, Sirohiwal and Pantazis 2023 (Joliot et al. 1969, Kok et al. 1970. ...
Flash-induced absorption changes in the Soret region, which originate from the [PD1PD2]+ state, the chlorophyll cation radical formed upon Photosystem II (PSII) excitation, were investigated in Mn-depleted Photosystem II. In wild-type PSII from Thermosynechococcus elongatus, the [PD1PD2]+-minus-[PD1PD2] difference spectrum shows a main negative feature at 434 nm and a smaller negative feature at 446 nm [Boussac et al. Photosynth Res (2023), https://doi.org/10.1007/s11120-023-01049-3]. While the main feature at 434 nm is associated with PD1+ formation, the origin of the dip at 446 nm remains to be identified. For that, we have compared the [PD1PD2]+-minus-[PD1PD2] difference spectra from the PsbA3/H198Q PSII mutant in T. elongatus and D2/H197A PSII mutant in Synechocystis sp. PCC 6803 with their respective wild type strains. By modifying the PD1 axial ligand with the H198Q mutation in the D1 protein in T. elongatus, the contribution at 434 nm was shifted to 431 nm, while the contribution at 446 nm was hardly affected. In Synechocystis sp. PCC 6803, by modifying the PD2 axial ligand with the H197A mutation in the D2 protein, the contribution at 446 nm was downshifted by approx. 3 nm to approx. 443 nm, while the main contribution at 432 nm was only slightly shifted upwards to 433 nm. This result suggests that the bleaching seen at 446 nm involves PD2. This could reflects a change in the [PD1+PD2]/[PD1PD2+] equilibrium or a more complex mechanism.
... Photosystem II (PSII) produces dioxygen by extracting electrons and protons from water, which takes place at the oxygen-evolving complex (OEC), an oxo-bridged Mn 4 CaO 5 cluster with a shape that resembles a distorted chair 2,3,8 . The Mn atoms in the OEC accumulate oxidative power through a four-step cycle of S i states (i = 0-4) that is initiated by the light-driven excitation of P680, a reaction centre that is a complex of chlorophyll a molecules 1 (Extended Data Fig. 1a) 1 . This is followed by a rapid charge separation that produces a pair of positive and negative charges on P680 •+ /pheophytin •− (Pheo •− ) on a picosecond timescale 9,10 . ...
Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0–4) at the Mn4CaO5 cluster1–3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4–7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca²⁺ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O–O bond formation.
... However, what remains elusive is the existence of a highvalent-high-spin species in a multinuclear manganese complex, which is an issue closely intertwined with important oxidation reactions such as the Kok cycle. 66 We hope that this study will encourage future studies into such systems. ...
The spin state of metal centers in many catalytic reactions has been demonstrated to be a rate limiting factor when high-valent metal centers such as manganese are involved. Although numerous manganese(V) complexes have been identified, thus far only one, [MnVH3buea(O)], has been directly confirmed to exist in a high spin state. Such a high-spin manganese(V) center may play a crucial role in the oxygen formation process in the elusive S4 state of the Kok cycle in Photosystem II. In this study, we provide direct experimental evidence, using X-ray magnetic circular dichroism (XMCD) and X-ray absorption spectroscopy (XAS), of gas phase [OMnO]+ as the second known high-spin manganese(V) oxo complex. We conclusively assign the ground state as 3B1 (C2v). Additionally, we provide fingerprint spectra not only for [OMnVO]+, but also for the high-spin hydroxydooxidomanganese(IV) ion [OMnIVOH]+ in its 4A'' (Cs) ground state that is expected to exhibit similar XAS and XMCD spectral signatures as neutral dioxidomanganese(IV).
... In the presence of HCO 3 -, the O 2 evolution is elevated by ~15 fold, and there is a 4-5-fold increase in ferricyanide reduction in maize chloroplasts. The Sstate kinetic model for oxygen evolution by Kok et al. (1970) was considered to support this result of Stemler and Govindjee (1974c), as HCO 3 was suggested to maintain a high oxidation state of the primary electron donor of PS II. However, the observations of Wydrzynski and Govindjee (1975), mentioned above, initiated the idea of the acceptor side effect of HCO 3 -, which was confirmed with many subsequent experiments (see the section above). ...
... The reaction takes place in photosystem II (PS II; Fig. 1), where the Mn 4 CaO 5 cluster stores four oxidizing equivalents while cycling through its S 0 to S 4 intermediate states [Kok cycle;Fig. 1(c)] (Joliot et al., 1969;Kok et al., 1970;. This cycle is driven by sequentially generated ultra-fast one-electron photochemical charge-separation reactions at the reaction center chlorophylls. ...
The water oxidation reaction in photosystem II (PS II) produces most of the molecular oxygen in the atmosphere, which sustains life on Earth, and in this process releases four electrons and four protons that drive the downstream process of CO2 fixation in the photosynthetic apparatus. The catalytic center of PS II is an oxygen-bridged Mn4Ca complex (Mn4CaO5) which is progressively oxidized upon the absorption of light by the chlorophyll of the PS II reaction center, and the accumulation of four oxidative equivalents in the catalytic center results in the oxidation of two waters to dioxygen in the last step. The recent emergence of X-ray free-electron lasers (XFELs) with intense femtosecond X-ray pulses has opened up opportunities to visualize this reaction in PS II as it proceeds through the catalytic cycle. In this review, we summarize our recent studies of the catalytic reaction in PS II by following the structural changes along the reaction pathway via room-temperature X-ray crystallography using XFELs. The evolution of the electron density changes at the Mn complex reveals notable structural changes, including the insertion of OX from a new water molecule, which disappears on completion of the reaction, implicating it in the O—O bond formation reaction. We were also able to follow the structural dynamics of the protein coordinating with the catalytic complex and of channels within the protein that are important for substrate and product transport, revealing well orchestrated conformational changes in response to the electronic changes at the Mn4Ca cluster.
... (Lubitz et al. 2019, Shevela et al. 2023) for some reviews. After four charge separations, the Mn4CaO5 cluster accumulates four oxidizing equivalents and thus cycles through five redox states denoted S0 to S4. Upon formation of the S4-state, two molecules of water are oxidized, the S0-state is regenerated and O2 is released (Joliot et al. 1969, Kok et al. 1970). ...
Flash-induced absorption changes in the Soret region arising from the (PD1PD2)+ state, the chlorophyll cation radical formed upon light excitation of Photosystem II (PSII), were measured in Mn-depleted PSII cores at pH 8.6. Under these conditions, TyrD is i) reduced before the first flash, and ii) oxidized before subsequent flashes. In wild-type PSII, when TyrDox is present, an additional signal in the (PD1PD2)+-minus-(PD1PD2) difference spectrum was observed when compared to the first flash when TyrD is not oxidized. The additional feature was “W-shaped” with troughs at 434 nm and 446 nm. This feature was absent when TyrD was reduced, but was present i) when TyrD was physically absent (and replaced by phenylalanine) or ii) when its H-bonding histidine (D2-His189) was physically absent (replaced by a Leucine). Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, seemed to require the native structural environment around the reduced TyrD and its H bonding partners to be present. We found no evidence of involvement of PD1, ChlD1, PheD1, PheD2, TyrZ, and the Cytb559 heme in the W-shaped difference spectrum. However, the use of a mutant of the PD2 axial His ligand, the D2-His197Ala, shows that the PD2 environment seems involved in the formation of “W-shaped” signal.
... Not only does the water oxidation release the molecular oxygen to refresh the atmosphere vitally in the industrial age, but provides the logistic supply of 4 electrons and 4 protons in a closed circle to convert carbon dioxide into the organic molecules. The catalytic pathway of the OEC is known as the Kok's cycle [2] that circulates through five redox states (S i , i = 0, · · · 4,), driven by the carousel of photoelectron in the Photosystem II (PSII) reaction centre, P 680 . In the past, great endeavor both in experiments and simulations has been made to identify the structures of OEC states S i=0,··· ,3 prior to the dioxygen generation at S 4 whose structure was reported most recently [3]. ...
Water oxidation in the oxygen evolving complex (OEC) of the photosystem II is catalyzed by the core cluster CaMn$_4$O$_5$ which was projected to experience five intermediate states S($\rm_i$) in the Kok's cycle since 1970's. However, the detailed dynamics of state evolutions still remains unclear, albeit with the general fact that the process is initiated by the transfer of photoelectrons with the steady electron donors of the water molecules. Based on density functional simulations, we find that the spin flips of Manganese atoms between the consecutive states in the electric polarization field can be used as a marker to uncover the intricate dynamic evolutions and the underlying dynamics. The dynamic electron and proton transfers and water insertion and dissociation are traced to reveal the evolution pathways of S$_0$$\sim$S$_3$ with commensurate spin flips towards the exact spin configuration of the next state. In particular, the various water insertions and dissociations at coordination sites of the S$_2$ open and closed cubane isomers are predicted with constraints on the necessary spin flips. Our study lays a solid ground for getting through the whole Kok's cycle via the pending S$_4$ state that is crucial for dioxygen generation.
... At these rather low currents a catalytic cycle has evolved that is made possible only by the high electronic and structural control of PSII as a supramolecular system. 6,15 Several technological approaches based on water splitting have been or are currently developed, with very different requirements for the working conditions in which the required OER catalysts must persist. [16][17][18][19] For instance, moderate currents in the range of 10 − 20 mA/cm 2 would be appropriate for solar water splitting applications, which however in most cases disallow extreme pH values and require specific optical or morphological properties. ...
Inspired by photosystem II (PS II), Mn oxide based electrocatalysts have been repeatedly investigated as catalysts for the electrochemical oxygen evolution reaction (OER), the anodic reaction in water electrolysis. However, a comparison of the conditions in biological OER catalysed by the water splitting complex CaMn4Ox with the requirements for an electrocatalyst for industrially relevant applications reveals fundamental differences. Thus, a systematic development of artificial Mn-based OER catalysts requires both a fundamental understanding of the catalytic mechanisms as well as an evaluation of the practicality of the system for industrial scale applications. Experimentally, both aspects can be approached using in situ and operando methods including spectroscopy. This paper highlights some of the major challenges common to different operando investigation methods and recent insights gained with them. To this end, vibrational spectroscopy, especially Raman spectroscopy, absorption techniques in the bandgap region and operando X-ray spectroelectrochemistry (SEC), both in the hard and soft X-ray regime are particularly focused on here. Technical challenges specific to each method are discussed first, followed by challenges that are specific to Mn oxide based systems. Finally, recent in situ and operando studies are reviewed. This analysis shows that despite the technical and Mn specific challenges, three specific key features are common to most of the studied systems with significant OER activity: structural disorder, Mn oxidation states between III and IV, and the appearance of layered birnessite phases in the active regime.
... (Lubitz et al. 2019) for a review. After four charge separations, the Mn4CaO5 cluster accumulates four oxidizing equivalents and thus cycles through five redox states denoted S0 to S4. Upon formation of the S4-state, two molecules of water are oxidized, the S0-state is regenerated and O2 is released (Joliot et al. 1969, Kok et al. 1970). ...
Flash-induced absorption changes in the Soret region arising from the [P D1 P D2 ] ⁺ state, the chlorophyll cation radical formed upon excitation of Photosystem II (PSII), were obtained using Mn-depleted PSII cores at pH 8.6. Under these conditions, Tyr D is reduced before the first flash but oxidised before subsequent flashes. When Tyr D • is present, an additional signal in the [P D1 P D2 ] ⁺ - minus -[P D1 P D2 ] difference spectrum was observed when compared to the first flash. The additional feature was W-shaped with troughs at 434 nm and 446 nm. This feature was absent when Tyr D was reduced, but was present when Tyr D was physically absent (and replaced by phenylalanine) or when its H-bonding histidine (D2-His190) was physically absent (replaced by a Leucine),. Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, required the native structural environment around the reduced Tyr D and its H bonding partners to be present. A range of PSII variants were surveyed, and we found no evidence of involvement of P D1 , Chl D1 , Phe D1 , Phe D2 , Tyr Z , and the Cyt b 559 heme in difference spectrum. Direct data ruling out the participation of P D2 is lacking. It seems possible that the specific H-bonding environment of around reduced Tyr D allows a more homogenous electrostatic environment for [P D1 P D2 ] ⁺ . A role for P D2 in the double-trough Soret signal may be tested using mutants of P D2 axial His ligand D2-His197.
... This necessitates four sequential oxidation events catalyzed by the Mn cluster that cycles through different states, denoted as S i states (i 5 0À4). The ability to span such a wide range of redox states makes Mn an ideal element for building the prosthetic group of OEC, with five intermediate states needed to fully oxidize water molecules into molecular oxygen (Kok et al., 1970;McEvoy and Brudvig, 2006). ...
... Each sample was pre-illuminated with 25 flashes, followed by a 5 min dark adaptation. The calculation of the oxygen flash yields was performed according to the non-cooperative Kok's model of oxygen evolution [63]. The kinetic parameters of the initial oxygen burst under continuous illumination, amplitudes and time constants (A 1 , A 2 , t 1 and t 2 ) were evaluated by the fitting of the oxygen burst decay curve with two exponential components. ...
Salinity is one of the most extreme abiotic stress factors that negatively affect the development and productivity of plants. The salt-induced injuries depend on the salt tolerance of the plant species, salt concentration, time of exposure and developmental stage. Here, we report on the response of pea plants (Pisum sativum L. cv Ran 1) to exposure to increasing salt concentrations (100, 150 and 200 mM NaCl) for a short time period (5 days) and the ability of the plants to recover after the removal of salt. The water content, membrane integrity, lipid peroxidation, pigment content and net photosynthetic rate were determined for the pea leaves of the control, treated and recovered plants. Salt-induced alterations in the primary photosynthetic reactions and energy transfer between the main pigment–protein complexes in isolated thylakoid membranes were evaluated. The pea plants were able to recover from the treatment with 100 mM NaCl, while at higher concentrations, concentration-dependent water loss, the disturbance of the membrane integrity, lipid peroxidation and an increase in the pigment content were detected. The net photosynthetic rate, electron transport through the reaction centers of PSII and PSII, activity of PSIIα centers and energy transfer between the pigment–protein complexes were negatively affected and were not restored after the removal of NaCl.
... Green plants contain two sets of photosynthetic systems, photosystem I (PS I) and photosystem II (PS II), each of which consists of a reaction center, a core antenna complex and a peripheral antenna complex. The function of PS II is light harvesting and splitting water into molecular oxygen, electrons and protons [26]. Structural observation showed that each PS II complex was composed of over 20 subunits and 77 cofactors [27]. ...
Sugar metabolism influences the quality of sweet corn (Zea mays var. saccharate Sturt) kernels, which is a major goal for maize breeding. In this study, the genome-wide transcriptomes from two supersweet corn cultivars (cv. Xuetian 7401 and Zhetian 11) with a nearly two-fold difference in kernel sugar content were carried out to explore the genes related to kernel sugar metabolism. In total, 45,748 differentially expressed genes (DEGs) in kernels and 596 DEGs in leaves were identified. PsbS, photosynthetic system II subunit S, showed two isoforms with different expression levels in leaf tissue between two cultivars, indicating that this gene might influence sugar accumulation in the kernel. On the other hand, hexokinases and beta-glucosidase genes involved in glycolysis, starch and sucrose metabolism were found in developing kernels with a genome-wide transcriptome analysis of developing kernels, which might contribute to the overaccumulation of water-soluble polysaccharides and an increase in the sweetness in the kernels of Xuetian 7401. These results indicated that kernel sugar accumulation in sweet corn might be influenced by both photosynthesis efficiency and the sugar metabolism rate. Our study supplied a new insight for breeding new cultivars with high sugar content and laid the foundation for exploring the regulatory mechanisms of kernel sugar content in corn.
... After the third flash, O 2 was released at most. After the fourth flash, the evolution of O 2 was the second highest, followed by a gradual decrease to a constant value (Allen and Franck 1955;Kok et al. 1970;Joliot 2003). The above flash-induced photosynthetic O 2 has also been experimentally proven that it came only from water photolysis. ...
Carbon neutrality is widely concerned and highly valued by many countries. Biosphere has always maintained the balance between oxidized organic substances and assimilated organic matter, resulting in net-zero carbon dioxide (CO2) emissions and maintaining its own carbon neutrality. Nature has set a good example for human beings to coordinate oxygen (O2) balance and CO2 balance, and achieve carbon neutrality. How does photosynthetic oxygen evolution initiate carbon and water neutrality? My synthesis shows that photosystem II functions as carbonic anhydrase to catalyze the reaction of CO2 hydration under physiological conditions, and CO2 hydration coupled with chemical equilibrium, H++HCO3−→1/2 O2 + 2e−+2 H++CO2, occurs in a photosystem II core-complex. Meanwhile, I focused on the revisiting of four classical heavy oxygen (O18) labeling experiments and found that bicarbonate can promote photosynthetic oxygen evolution, and that photosynthetic oxygen evolution can alternately come from bicarbonate and water, not only water. Bicarbonate photolysis and water photolysis account for half of the photosynthetic oxygen evolution respectively, which can well explain the bicarbonate effect, Dole effect and plants’ environmental adaptability. Photosynthetic oxygen evolution initiated the journey of water metabolism and carbon metabolism in nature, which led to the coupling as 1:1 (mol/mol) stoichiometric relationship between the reduction of CO2 and oxidation of organic carbon, coordinated the evolution of the atmosphere, hydrosphere, lithosphere and biosphere, and realized “carbon neutrality” in the whole Earth system.
Link https://link.springer.com/article/10.1007/s11631-022-00580-9
... It is now widely accepted that the water-splitting complex of PSII consists of four manganese (Mn) ions organized into a metal cluster situated within or close to the reaction center heterodimer that is formed by the core D1 and D2 polypeptides (Nixon and Diner, 1994). This concept has evolved from the initial studies examining the flash-induced oscillatory patterns of oxygen evolution that gave rise to the S-state clock model (Kok et al., 1970). The requirement for the accumulation of four oxidizing equivalents prior to the oxidation of two water molecules, is compatible with the presence of four manganese atoms that are thought to undergo consecutive valency changes. ...
One of nature’s most fundamental biological processes is the light-driven oxidation of water during higher plant photosynthesis. It is the photosystem II (PSII) enzyme complex of the photosynthetic apparatus which utilizes light energy to oxidize water into oxygen and liberates the protons and electrons necessary for CO2 reduction to synthesize the organic molecules essential for plant growth. Under normal conditions water is oxidized to produce the relatively non-reactive dioxygen species, which is released into the atmosphere and used by both plants and animals for respiration. On a global basis, this water oxidation process is enormous, generating approximately 10 000 tons of oxygen per second. Hence, an understanding the mechanisms involved in water oxidation is an intense area of biological research. For example, accumulating evidence indicates now that even minor perturbations to the protein structure of PSII caused by environmental stresses such as atmospheric changes in temperature, UV-irradiation, pollutants etc., alters the water oxidation chemistry such that reactive oxygen species are produced rather than O2. This is an important observation since it is known that the more reactive oxygen species facilitate the breakdown of the protein structure, which can in turn induce the further production of active oxygen. Factors such as these interact synergistically to progressively decrease the overall efficiency and stability of photosynthesis. By understanding the nature of water oxidation in the PSII complex, it may become possible to engineer plants with greater resistant to photosynthetic stresses. This goal becomes ever more important in the future as science aims to maintain the agricultural output needed to sustain a burgeoning population in a world environment that is increasingly being disturbed. Ultimately, an understanding of the nature of water oxidation may have even more widespread implications in future technologies which aim to design synthetic analogues of the PSII reaction process for the manufacture of solar cells as both an alternative energy source to fossil fuels and perhaps for the production artificial oxygen-evolving atmospheres.
... Quantum chemical methods have been successfully applied to understand the catalytic pathways of the oxygen evolution process like the Kok cycle 34,35 for the OEC of photosystem-II. The density functional theory (DFT) is one of the efficient tools of the computational method [36][37][38] for mechanistic studies. ...
In this study, we explore the water oxidation process with the help of density functional theory. The formation of an oxygen-oxygen bond is crucial in the water oxidation process. Here, we report the formation of the oxygen-oxygen bond by the N5-coordinate oxoiron species with a higher oxidation state of FeIV and FeV. This bond formation is studied through the nucleophilic addition of water molecules and the transfer of the oxygen atom from meta-chloroperbenzoic acid (mCPBA). Our study reveals that the oxygen-oxygen bond formation by reacting mCPBA with FeVO requires less activation barrier (13.7 kcal mol-1) than the other three pathways. This bond formation by the oxygen atom transfer (OAT) pathway is more favorable than that achieved by the hydrogen atom transfer (HAT) pathway. In both cases, the oxygen-oxygen bond formation occurs by interacting the σ*dz2-2pz molecular orbital of the iron-oxo intermediate with the 2px orbital of the oxygen atom. From this study, we understand that the oxygen-oxygen bond formation by FeIVO with the OAT process is also feasible (16 kcal mol-1), suggesting that FeVO may not always be required for the water oxidation process by non-heme N5-oxoiron. After the oxygen-oxygen bond formation, the release of the dioxygen molecule occurs with the addition of the water molecule. The release of dioxygen requires a barrier of 7.0 kcal mol-1. The oxygen-oxygen bond formation is found to be the rate-determining step.
Identifying the two substrate water sites of nature’s water-splitting cofactor (Mn 4 CaO 5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S 1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved ¹⁷ O-Electron-electron Double resonance detected NMR (TR- ¹⁷ O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H 2 ¹⁸ O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn 4 CaO 5 cofactor after mixing with H 2 ¹⁷ O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (W S ) in the TR-MIMS experiments and ii) that the exchange rates of O5 and W S are both enhanced by Ca ²⁺ →Sr ²⁺ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being W S . This strongly restricts options for the mechanism of water oxidation.
D1-Tyr161 (TyrZ) forms a low-barrier H-bond with D1-His190 and functions as a redox-active group in photosystem II. When oxidized to the radical form (TyrZ-O•), it accepts an electron from the oxygen-evolving Mn4CaO5 cluster, facilitating an increase in the oxidation state (Sn; n = 0–3). Here we investigated the mechanism of how TyrZ-O• drives proton-coupled electron transfer during the S2 to S3 transition using a quantum mechanical/molecular mechanical approach. In response to TyrZ-O• formation and subsequent loss of the low-barrier H-bond, the ligand water molecule at the Ca2+ site (W4) reorients away from TyrZ and donates an H-bond to D1-Glu189 at Mn4 of Mn4CaO5 together with an adjacent water molecule. The H-bond donation to the Mn4CaO5 cluster triggers the release of the proton from the lowest pKa site (W1 at Mn4) along the W1…D1-Asp61 low-barrier H-bond, leading to protonation of D1-Asp61. The interplay of the two low-barrier H-bonds, involving the Ca2+ interface and forming the extended Grotthuss-like network [TyrZ…D1-His190]-[Mn4CaO5]-[W1…D1-Asp61], rather than the direct electrostatic interaction, is likely a basis of the apparent long-distance interaction (11.4 Å) between TyrZ-O• formation and D1-Asp61 protonation.
Flash-induced absorption changes in the Soret region arising from the [PD1PD2]+ state, the chlorophyll cation radical formed upon light excitation of Photosystem II (PSII), were measured in Mn-depleted PSII cores at pH 8.6. Under these conditions, TyrD is i) reduced before the first flash, and ii) oxidized before subsequent flashes. In wild-type PSII, when TyrD● is present, an additional signal in the [PD1PD2]+-minus-[PD1PD2] difference spectrum was observed when compared to the first flash when TyrD is not oxidized. The additional feature was “W-shaped” with troughs at 434 nm and 446 nm. This feature was absent when TyrD was reduced, but was present (i) when TyrD was physically absent (and replaced by phenylalanine) or (ii) when its H-bonding histidine (D2-His189) was physically absent (replaced by a Leucine). Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, seemed to require the native structural environment around the reduced TyrD and its H bonding partners to be present. We found no evidence of involvement of PD1, ChlD1, PheD1, PheD2, TyrZ, and the Cytb559 heme in the W-shaped difference spectrum. However, the use of a mutant of the PD2 axial His ligand, the D2-His197Ala, shows that the PD2 environment seems involved in the formation of “W-shaped” signal.
Chloride (Cl-) is essential for O2 evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P+QA-), rates of water oxidation steps (S-states), rate of proton evolution, and flash O2 yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O2 evolution rate (-20 %) and proton release (-36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S2 → S3 → S0') slow with increasing light intensity and during O2/water exchange (S0' → S0 → S1), while the non-protonogenic S1 → S2 transition is kinetically unaffected. As flash rate increases in Cl- cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br-. The Cl- advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H3O+Cl- in dry water channels vs. separated Br- and H+ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br- cultures for both proton evolution and less PSII charge recombination. In Br- cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br- with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.
Manganese (Mn) serves as the catalytic center for water splitting in photosystem II (PSII), despite the abundance of iron (Fe) on earth. As a first step toward why Mn and not Fe is employed by Nature in the water oxidation catalyst, we investigated the Fe4CaO5 cluster in the PSII protein environment using a quantum mechanical/molecular mechanical (QM/MM) approach, assuming an equivalence between Mn(III/IV) and Fe(II/III). Substituting Mn with Fe resulted in the protonation of μ-oxo bridges at sites O2 and O3 by Arg357 and D1-His337, respectively. While the Mn4CaO5 cluster exhibits distinct open- and closed-cubane S2 conformations, the Fe4CaO5 cluster lacks this variability due to an equal spin distribution over sites Fe1 and Fe4. The absence of a low-barrier H-bond between a ligand water molecule (W1) and D1-Asp61 in the Fe4CaO5 cluster may underlie its incapability for ligand water deprotonation, highlighting the relevance of Mn in natural water splitting.
Structural dynamics of water and its hydrogen-bonding networks play an important role in enzyme function via the transport of protons, ions, and substrates. To gain insights into these mechanisms in the water oxidation reaction in Photosystem II (PS II), we have performed crystalline molecular dynamics (MD) simulations of the dark-stable S1 state. Our MD model consists of a full unit cell with 8 PS II monomers in explicit solvent (861 894 atoms), enabling us to compute the simulated crystalline electron density and to compare it directly with the experimental density from serial femtosecond X-ray crystallography under physiological temperature collected at X-ray free electron lasers (XFELs). The MD density reproduced the experimental density and water positions with high fidelity. The detailed dynamics in the simulations provided insights into the mobility of water molecules in the channels beyond what can be interpreted from experimental B-factors and electron densities alone. In particular, the simulations revealed fast, coordinated exchange of waters at sites where the density is strong, and water transport across the bottleneck region of the channels where the density is weak. By computing MD hydrogen and oxygen maps separately, we developed a novel Map-based Acceptor-Donor Identification (MADI) technique that yields information which helps to infer hydrogen-bond directionality and strength. The MADI analysis revealed a series of hydrogen-bond wires emanating from the Mn cluster through the Cl1 and O4 channels; such wires might provide pathways for proton transfer during the reaction cycle of PS II. Our simulations provide an atomistic picture of the dynamics of water and hydrogen-bonding networks in PS II, with implications for the specific role of each channel in the water oxidation reaction.
Recent advances in serial femtosecond crystallography (SFX) of photosystem II (PSII), enabled by X-ray free electron lasers (XFEL), provided the first geometric models of distinct intermediates in the catalytic S-state cycle of the oxygen-evolving complex (OEC). These models are obtained by flash-advancing the OEC from the dark-stable state (S1) to more oxidized intermediates (S2 and S3), eventually cycling back to the most reduced S0. However, the interpretation of these models is controversial because geometric parameters within the Mn4CaO5 cluster of the OEC do not exactly match those expected from coordination chemistry for the spectroscopically verified manganese oxidation states of the distinct S-state intermediates. Here we focus on the first catalytic transition, S1 → S2, which represents a one-electron oxidation of the OEC. Combining geometric and electronic structure criteria, including a novel effective oxidation state approach, we analyze existing 1-flash (1F) SFX-XFEL crystallographic models that should depict the S2 state of the OEC. We show that the 1F/S2 equivalence is not obvious, because the Mn oxidation states and total unpaired electron counts encoded in these models are not fully consistent with those of a pure S2 state and with the nature of the S1 → S2 transition. Furthermore, the oxidation state definition in two-flashed (2F) structural models is practically impossible to elucidate. Our results advise caution in the extraction of electronic structure information solely from the literal interpretation of crystallographic models and call for re-evaluation of structural and mechanistic interpretations that presume exact correspondence of such models to specific catalytic intermediates of the OEC.
In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry1–3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4–6, disappears or relocates in parallel with Yz reduction starting at approximately 700 μs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.
Photosynthesis fuels life on Earth by storing solar energy in chemical form. Today’s oxygen-rich atmosphere has resulted from the splitting of water at the protein-bound manganese cluster of photosystem II during photosynthesis. Formation of molecular oxygen starts from a state with four accumulated electron holes, the S4 state—which was postulated half a century ago¹ and remains largely uncharacterized. Here we resolve this key stage of photosynthetic O2 formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combining these results with computational chemistry reveals that a crucial proton vacancy is initally created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in a single-electron, multi-proton transfer event. This is the slowest step in photosynthetic O2 formation, with a moderate energetic barrier and marked entropic slowdown. We identify the S4 state as the oxygen-radical state; its formation is followed by fast O–O bonding and O2 release. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O2 formation emerges. Our results provide insights into a biological process that is likely to have occurred unchanged for the past three billion years, which we expect to support the knowledge-based design of artificial water-splitting systems.
Mixed‐valence complexes, especially those consisting of multinuclear transition metal complexes or clusters with different oxidation states, play critical roles in a variety of metabolic pathways in biological systems involving electron‐transfer reactions. Several of these complexes or clusters in proteins are indispensable bioactive cofactors in metalloenzymes, and some of them act as electron transporters in a variety of essential processes in cells, including photosynthesis, respiration, nitrogen fixation, navigation, etc. This chapter summarizes the structures of mixed‐valence compounds or clusters found in bioactive proteins or enzymes, as well as their artificial bio‐mimic systems, which are synthesized as models of their natural cofactors to study the fundamental mechanisms and functions within corresponding proteins or enzymes.
The catalytic function of the water-splitting complex of photosynthesis II is still a scientific riddle. The center of the water-splitting complex is the oxygen-evolving center described as an heterometallic-oxide Mn4O5Ca Cluster with a “cube with backrest” stereometric structure. The correlation of the oxygen-evolving center with the five step KOK-JOLIOT cycle is unclear. Calcium is a solitary element, and an indispensable component of this “cube with backrest” Mn4O5Ca Cluster; however, Strontium is able to substitute Calcium in biological experiments.
We present a “20Ca-38Sr-d-orbitals related theory of electron storage and electron transfer” which allows to correlate the cube with backrest Mn4O5Ca Cluster to the five-state KOK-JOLIOT cycle. The five atomic orbitals of the M-3d type allow to store the 4 electrons of the KOK-JOLIOT cycle as well as an interference wave function of these four electrons; furthermore these five atomic orbitals of the M-3d type are the nearest filled orbitals of the Ca2+ ion to the TyrosinZ (TyrZ) electron bridge of the ligand DI protein. The same considerations hold for the Strontium N-4d orbitals.
We hope this quantum-biological, quantum-chemical insight helps to improve bionic respective artificial photosynthesis, helps to decarbonize economic sectors of interest, helps to empower the hydrogen economy, and helps to provide sustainable energy.
Cyanobacteria, the evolutionary originators of oxygenic photosynthesis, have the capability to convert CO2, water, and minerals into biomass using solar energy. This process is driven by intricate bioenergetic mechanisms that consist of interconnected photosynthetic and respiratory electron transport chains coupled. Over the last few decades, advances in physiochemical analysis, molecular genetics, and structural analysis have enabled us to gain a more comprehensive understanding of cyanobacterial bioenergetics. This includes the molecular understanding of the primary energy conversion mechanisms as well as photoprotective and other dissipative mechanisms that prevent photodamage when the rates of photosynthetic output, primarily in the form of ATP and NADPH, exceed the rates that cellular assimilatory processes consume these photosynthetic outputs. Despite this progress, there is still much to learn about the systems integration and the regulatory circuits that control expression levels for optimal cellular abundance and activity of the photosynthetic complexes and the cellular components that convert their products into biomass. With an improved understanding of these regulatory principles and mechanisms, it should be possible to optimally modify cyanobacteria for enhanced biotechnological purposes.
Ultrapurified Photosystem II complexes crystalize as uniform microcrystals (PSIIX) of unprecedented homogeneity that allow observation of details previously unachievable, including the longest sustained oscillations of flash-induced O2 yield over > 200 flashes and a novel period-4.7 water oxidation cycle. We provide new evidence for a molecular-based mechanism for PSII-cyclic electron flow that accounts for switching from linear to cyclic electron flow within PSII as the downstream PQ/PQH2 pool reduces in response to metabolic needs and environmental input. The model is supported by flash oximetry of PSIIX as the LEF/CEF switch occurs, Fourier analysis of O2 flash yields, and Joliot-Kok modeling. The LEF/CEF switch rebalances the ratio of reductant energy (PQH2) to proton gradient energy (H⁺o/H⁺i) created by PSII photochemistry. Central to this model is the requirement for a regulatory site (QC) with two redox states in equilibrium with the dissociable secondary electron carrier site QB. Both sites are controlled by electrons and protons. Our evidence fits historical LEF models wherein light-driven water oxidation delivers electrons (from QA⁻) and stromal protons through QB to generate plastoquinol, the terminal product of PSII-LEF in vivo. The new insight is the essential regulatory role of QC. This site senses both the proton gradient (H⁺o/H⁺i) and the PQ pool redox poise via e⁻/H⁺ equilibration with QB. This information directs switching to CEF upon population of the protonated semiquinone in the Qc site (Q⁻H⁺)C, while the WOC is in the reducible S2 or S3 states. Subsequent photochemical primary charge separation (P⁺QA⁻) forms no (QH2)B, but instead undergoes two-electron backward transition in which the QC protons are pumped into the lumen, while the electrons return to the WOC forming (S1/S2). PSII-CEF enables production of additional ATP needed to power cellular processes including the terminal carboxylation reaction and in some cases PSI-dependent CEF.
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). 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. Light‐induced water oxidation and dioxygen release in photosynthesis is catalyzed by a paramagnetic μ‐oxo‐bridged Mn4Ca cofactor. It passes through five metastable states (S0 ‐ S4) whose structure is described focusing on the essential electronic structure obtained from spectroscopy, especially EPR techniques, supported by quantum chemistry. A catalytic cycle is presented which describe structural isomers of key S‐state intermediates facilitating substrate binding and cofactor activation.
The open-access journal Microorganisms is pleased to publish this book, which reprints papers that appeared in a Special Issue on “Phototrophic Bacteria”, with Guest Editors Robert Blankenship and Matthew Sattley. This Special Issue included research on all types of phototrophic bacteria, including both anoxygenic and oxygenic forms. Research on these bacterial organisms has greatly advanced our understanding of the basic principles that underlie the energy storage that takes place in all types of photosynthetic organisms, including both bacterial and eukaryotic forms. Topics of interest include: microbial physiology, microbial ecology, microbial genetics, evolutionary microbiology, systems microbiology, agricultural microbiology, microbial biotechnology, and environmental microbiology, as all are related to phototrophic bacteria.
Robert Blankenship and Matthew Sattley
Editors
Three psbA genes (psbA1, psbA2, and psbA3) that encode for a key photosystem II (PSII) subunit are present in the thermophilic cyanobacterium Thermosynechococcus elongatus, and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen-bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to the change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen-bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, and we found the channel to be narrower, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen-bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, which may explain the oxygen evolution enhancement in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.
Die vorliegende Arbeit beschäftigt sich mit der Synthese und Charakterisierung von Manganoxiden als Katalysatoren für die Wasseroxidation. Um die Katalyse durch Manganoxide (vor allem durch Schichtoxide, sogenannte Birnessite) besser zu verstehen und die katalytische Aktivität der Oxide zu optimieren, werden in dieser Arbeit Studien zur Variation der Oxidsynthese vorgestellt. So wurden Manganoxide unterschiedlichen Strukturtyps, Morphologie, Partikelgröße und mit verschiedenarti-gen eingelagerten Kationen hergestellt. Auch die Reaktion des in den Wasseroxidations-Screenings üblichen Oxidationsmittels Ce4+ mit den Oxiden, die Vergrößerung des Reaktionsmaßstabs der Birnessitsynthese und die Darstellung von Manganoxidelektroden durch physikalische Gasphasenabscheidung wurden untersucht. Alle erhaltenen Oxide wurden gründlich chemisch und morphologisch charakterisiert und als Katalysatoren in der Wasseroxidation getestet.
In Summe erlauben die Ergebnisse die Identifizierung von Struktur-Eigenschafts-Beziehungen. In dieser Arbeit wurde eine optimale mittlere Manganoxidationsstufe zwischen Mn+3.5 und Mn+3.9 gefunden. Kationen und Wasser in den Zwischenschichten stellten sich als förderlich für die Wasseroxidationskatalyse heraus. Weiterhin ist die katalytische Rate pro Manganzentrum dann besonders hoch, wenn die Strukturen (wie in amorphen Oxiden) flexible Mn-O-Bindungen enthalten. Auch eine große Oberfläche fördert die Aktivität in der Katalyse der Wasseroxidation.
Neben mechanistischen Erkennissen konnte auch eine wesentliche Verbesserung der katalytischen Wasseroxidation mit Ce4+ als Oxidationsmittel erreicht werden. Birnessite wurden dabei pro Manganzentrum als aktivste Katalysatoren gefunden. Die Aktivität in der Wasseroxidationskatalyse der bisher bekannten Birnessitsysteme konnte durch einen Temperschritt bei 400°C oder die Herstellung von Eisenoxid-Birnessit Core-Shell-Partikeln nochmals verdoppelt werden.
Somit ließen sich wichtige Materialparameter für eine effiziente Wasseroxidationskatalyse durch Manganoxide identifizieren: Geschichtete Manganoxide, die Ca2+ in den Zwischenschichten enthalten und eine große Oberfläche besitzen, sind die klaren „Gewinner“ dieser Arbeit. Zukünftige Arbeiten sollten sich besonders dem Studium der elektrokatalytischen Eigenschaften der Oxide und dem Aufbau von Elektroden oder Halbleiterstrukturen zur Wasserspaltung beschäftigen.
Serial Femtosecond Crystallography at the X-ray Free Electron Laser (XFEL) sources enabled the imaging of the catalytic intermediates of the oxygen evolution reaction of Photosystem II (PSII). However, due to the incoherent transition of the S-states, the resolved structures are a convolution from different catalytic states. Here, we train Decision Tree Classifier and K-means clustering models on Mn compounds obtained from the Cambridge Crystallographic Database to predict the S-state of the X-ray, XFEL, and CryoEM structures by predicting the Mn’s oxidation states in the oxygen-evolving complex. The model agrees mostly with the XFEL structures in the dark S1 state. However, significant discrepancies are observed for the excited XFEL states (S2, S3, and S0) and the dark states of the X-ray and CryoEM structures. Furthermore, there is a mismatch between the predicted S-states within the two monomers of the same dimer, mainly in the excited states. We validated our model against other metalloenzymes, the valence bond model and the Mn spin densities calculated using density functional theory for two of the mismatched predictions of PSII. The model suggests designing a more optimized sample delivery and illumiation systems are crucial to precisely resolve the geometry of the advanced S-states to overcome the noncoherent S-state transition. In addition, significant radiation damage is observed in X-ray and CryoEM structures, particularly at the dangler Mn center (Mn4). Our model represents a valuable tool for investigating the electronic structure of the catalytic metal cluster of PSII to understand the water splitting mechanism.
Photosystem II (PSII) is a multi-subunit membrane protein complex that catalyzes light-driven oxidation of water to molecular oxygen. The chloride ion (Cl−) has long been known as an essential cofactor for oxygen evolution by PSII, and two Cl− ions (Cl-1 and Cl-2) have been found to specifically bind near the Mn4CaO5 cluster within the oxygen-evolving center (OEC). However, despite intensive studies on these Cl− ions, little is known about the function of Cl-2, the Cl− ion that is associated with the backbone nitrogens of D1-Asn338, D1-Phe339, and CP43-Glu354. In green plant PSII, the membrane extrinsic subunits—PsbP and PsbQ—are responsible for Cl− retention within the OEC. The Loop 4 region of PsbP, consisting of highly conserved residues Thr135–Gly142, is inserted close to Cl-2, but its importance has not been examined to date. Here, we investigated the importance of PsbP-Loop 4 using spinach PSII membranes reconstituted with spinach PsbP proteins harboring mutations in this region. Mutations in PsbP-Loop 4 had remarkable effects on the rate of oxygen evolution by PSII. Moreover, we found that a specific mutation, PsbP-D139N, significantly enhanced the oxygen-evolving activity in the absence of PsbQ, but not significantly in its presence. The D139N mutation increased the Cl− retention ability of PsbP and induced a unique structural change in the OEC, as indicated by light-induced Fourier transform infrared (FTIR) difference spectroscopy and theoretical calculations. Our findings provide insight into the functional significance of Cl-2 in the water-oxidizing reaction of PSII.
The oxygen-evolving center (OEC) in photosystem II (PSII) of oxygenic photosynthetic organisms is a unique heterometallic-oxide Mn4CaO5-cluster that catalyzes water splitting into electrons, protons, and molecular oxygen through a five-state cycle (Sn, n = 0 ~ 4). It serves as the blueprint for the developing of the man-made water-splitting catalysts to generate solar fuel in artificial photosynthesis. Understanding the structure–function relationship of this natural catalyst is a great challenge and a long-standing issue, which is severely restricted by the lack of a precise chemical model for this heterometallic-oxide cluster. However, it is a great challenge for chemists to precisely mimic the OEC in a laboratory. Recently, significant advances have been achieved and a series of artificial Mn4XO4-clusters (X = Ca/Y/Gd) have been reported, which closely mimic both the geometric structure and the electronic structure, as well as the redox property of the OEC. These new advances provide a structurally well-defined molecular platform to study the structure–function relationship of the OEC and shed new light on the design of efficient catalysts for the water-splitting reaction in artificial photosynthesis.
Photosynthetic water oxidation is catalyzed by a manganese-calcium oxide cluster, which experiences five "S-states" during a light-driven reaction cycle. The unique "distorted chair"-like geometry of the Mn4CaO5(6) cluster shows structural flexibility that has been frequently proposed to involve "open" and "closed"-cubane forms from the S1 to S3 states. The isomers are interconvertible in the S1 and S2 states, while in the S3 state, the open-cubane structure is observed to dominate inThermosynechococcus elongatus (cyanobacteria) samples. In this work, using density functional theory calculations, we go beyond the S3+Yz state to the S3nYz• → S4+Yz step, and report for the first time that the reversible isomerism, which is suppressed in the S3+Yz state, is fully recovered in the ensuing S3nYz• state due to the proton release from a manganese-bound water ligand. The altered coordination strength of the manganese-ligand facilitates formation of the closed-cubane form, in a dynamic equilibrium with the open-cubane form. This tautomerism immediately preceding dioxygen formation may constitute the rate limiting step for O2 formation, and exert a significant influence on the water oxidation mechanism in photosystem II.
Light-driven water oxidation in photosynthesis occurs at the oxygen-evolving center (OEC) of photosystem II (PSII). Chloride ions (Cl⁻) are essential for oxygen evolution by PSII, and two Cl⁻ ions have been found to specifically bind near the Mn4CaO5 cluster in the OEC. The retention of these Cl⁻ ions within the OEC is critically supported by some of the membrane-extrinsic subunits of PSII. The functions of these two Cl⁻ ions and the mechanisms of their retention both remain to be fully elucidated. However, intensive studies performed recently have advanced our understanding of the functions of these Cl⁻ ions, and PSII structures from various species have been reported, aiding the interpretation of previous findings regarding Cl⁻ retention by extrinsic subunits. In this review, we summarize the findings to date on the roles of the two Cl⁻ ions bound within the OEC. Additionally, together with a short summary of the functions of PSII membrane-extrinsic subunits, we discuss the mechanisms of Cl⁻ retention by these extrinsic subunits.
An attempt is made to reconcile the apparently contradictory observations in flash photosynthesis using short flashes (ca. 10−5 sec.) and long flashes (ca. 10−3 sec.). It is shown that the introduction of a new period of ca. 10−4 sec. not only obviates these previous difficulties, but also is not in contradiction with any existing observations. In addition, a mechanism for the primary photochemical process, suggested by one of us (J. F.) and discussed in detail elsewhere, is presented.Using reversible phosphorescence quenching as a means of measuring very small concentrations of oxygen, we have also studied the evolution of oxygen under anaerobic conditions by single flashes of light. The biological material was the algae Scenedesmus obliquus in the presence of either carbon dioxide or the Hill reagent quinone.It has been observed that while a single, intense, half-millisecond flash evolves oxygen in the Hill reaction, it does not evolve oxygen in photosynthesis. A flash 50 times as long and of much lower intensity results in the evolution of oxygen in both systems. Further, in the Hill reaction, the yield from an intense half-millisecond flash is increased by a previous similar flash preceding it by one full second. This observation suggests that certain photoproducts are capable of surviving longer than the generally assumed 10−2 sec. Various other observations on the effect of overlapping short and long flashes, and some plausible explanations, are described.
The Mn content of spinach chloroplasts has been decreased by growth deficiency, extraction and by ageing at 35°. We studied the effect of subnormal Mn content upon the chloroplasts capacity to evolve O2 and to photooxidize electron donors other than water via Photosystem II. We observed the following: 1.1. In fresh chloroplasts ascorbate and other reducing agents, if present in sufficient concentration, fully replace water as the System II oxidant and can sustain maximum rates of 1000–1200 equiv/chlorophyll per h.2.2. None of the studied donors proved entirely specific for System II; to a variable extent all could react with the oxidant of System I. We therefore considered only the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(DCMU)-sensitive fraction of the observed rates as pertinent.3.3. Normal fresh chloroplasts contained chlorophyllsII and showed flash yields of approx. chlorophylls. This indicates that each System II trapping and O2-evolving center contains three Mn atoms.4.4. O2 evolution capacity is abolished when about of the total Mn pool is removed by way of Tris or hydroxylamine extraction, i.e. upon removal of two of the three Mn atoms normally present per reaction center. Between the limits of 1 Mn per trap and 3 Mn per trap O2 evolution capacity is linear with Mn content.5.5. Mn removal affects the rates of O2 evolution in strong light and in weak light (quantum yield) in the same fashion. This indicates that complete O2 reaction centers are inactivated.6.6. With Mn removal the capacity for donor (ascorbate or p-phenylenediamine) photooxidation in strong light declines in a very similar fashion as the O2 evolving capacity. However, after removal of of the Mn pool (by Tris or hydroxylamine extraction) 15–20% of the maximum rate remains (100–250 equiv/chlorophyll per h) as previously noticed by other workers. Secondly, the rate in weak light (quantum yield) of these photooxidations remains unaffected by Mn removal. This shows that for donor photooxidation the larger of the two Mn pools is not essential.7.7. Complete removal of Mn ( chlorophylls) led to 90–95% loss of donor photooxidation in strong light.8.8. Removal of of the Mn left a low fluorescence yield (variable fraction = 0) which could be fully restored by adding DCMU. After complete removal of Mn ( chlorophylls) DCMU enhanced the yield of the variable fluorescence to only the maximum level but the full maximum could be restored by chemical reduction. This indicates that fluorescence quencher of System II, Q, is not affected by Mn removal.9.9. Of the three Mn associated with each trapping center, one is linked more closely to the center than the other two. While all three are essential for O2 evolution, artificial donors can enter with various rate constants at several loci on the oxidant side of System II.
In preceding papers2,3 a new amperometric method was described for the detection of photosynthetic oxygen production by unicellular algae. The algae are illuminated by a modulated light and only the modulated component of the amperometric current is detected.This apparatus has now been modified to allow the study of isolated chloroplasts in the presence of electron acceptors and donors. The time resolution of the method has been improved and reaches 5 msec under the best conditions. The method can be used to measure, in relative units, the rate of reduction of electro-active Hill reagents such as quinone, ferricyanide or methyl viologen. It is also possible to measure the relative amounts of oxygen evolved or Hill reagent reduced, by a single saturating flash of short duration. The latter determinations can be performed simultaneously with the modulated rate measurements.
Abstract— It appears that even if some kinetic aspects of the activation and stimulation phenomena of photosystem II remain not understood. the only interpretation we can propose involves the addition of the effect of two photoreactions on the same photochemical center. The main arguments we developed in favour of this hypothesis are:
(1) the formation of one atom of oxygen requires the addition of the effect of two photo-reactions (one photoreaction per electron).
(2) after a dark period two quanta must be absorbed by the same photochemical center in order to produce oxygen.
(3) the existence of a form of the photochemical substrate in which energy is stored is proved by the fact that. after a preillumination by two flashes, the quantum requirement for the production of one molecule of oxygen can reach a value almost two times lower than the quantum requirement measured in stationary conditions.