Structure Of Photosystem II Research Articles

Photosystem I: A Paradigm for Understanding Biological Environmental Adaptation Mechanisms in Cyanobacteria and Algae.

The process of oxygenic photosynthesis is primarily driven by two multiprotein complexes known as photosystem II (PSII) and photosystem I (PSI). PSII facilitates the light-induced reactions of water-splitting and plastoquinone reduction, while PSI functions as the light-driven plastocyanin-ferredoxin oxidoreductase. In contrast to the highly conserved structure of PSII among all oxygen-evolving photosynthetic organisms, the structures of PSI exhibit remarkable variations, especially for photosynthetic organisms that grow in special environments. In this review, we make a concise overview of the recent investigations of PSI from photosynthetic microorganisms including prokaryotic cyanobacteria and eukaryotic algae from the perspective of structural biology. All known PSI complexes contain a highly conserved heterodimeric core; however, their pigment compositions and peripheral light-harvesting proteins are substantially flexible. This structural plasticity of PSI reveals the dynamic adaptation to environmental changes for photosynthetic organisms.

Composition Heterogeneity of Metal Ions Bound at the Oxygen-Evolving Center of Photosystem II in Living Cells.

Recent resolution advancement of in situ cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) enables us to visualize large enzymes-in-action in atomic detail in their native environments inside living cells, such as photosystem II (PSII) and the ribosome. A variety of crystallographic and cryo-EM structures of PSII have been published for the purified PSII dimeric core complexes by itself, in supercomplexes with photosystem I (PSI) and light-harvesting complexes (LHC), and in megacomplexes with phycobilisome (PBS). In the latter case, two or five copies of asymmetric dimeric PSII molecules are present in highly asymmetric environments that differ from other 2-fold symmetric structures. Previous systematic analysis of X-ray free-electron laser (XFEL) crystal structures of PSII has shown different degrees of composition heterogeneity of metal ion cofactor bound at the oxygen-evolving center (OEC), including between two monomers of the same PSII dimer. This study analyzed the metal ions bound at four OECs in two asymmetric dimeric PSII molecules within in situ cryo-ET structures reported for an asymmetric PBS-PSII-PSI-LHC megacomplex determined in a living organism without purification and shows that composition heterogeneity with reduced metal ion occupancies at the OEC of PSII is a general phenomenon. This finding could have profound implications for spectroscopic interpretations of unpurified PSII samples.

Investigating the Balance between Structural Conservation and Functional Flexibility in Photosystem I.

Photosynthesis, as the primary source of energy for all life forms, plays a crucial role in maintaining the global balance of energy, entropy, and enthalpy in living organisms. Among its various building blocks, photosystem I (PSI) is responsible for light-driven electron transfer, crucial for generating cellular reducing power. PSI acts as a light-driven plastocyanin-ferredoxin oxidoreductase and is situated in the thylakoid membranes of cyanobacteria and the chloroplasts of eukaryotic photosynthetic organisms. Comprehending the structure and function of the photosynthetic machinery is essential for understanding its mode of action. New insights are offered into the structure and function of PSI and its associated light-harvesting proteins, with a specific focus on the remarkable structural conservation of the core complex and high plasticity of the peripheral light-harvesting complexes.

Effects of Cu2O nanoparticles on the kinetic characteristics of rapid chlorophyll fluorescence induction and related genes in wheat seedlings.

Metal nanoparticles could be accumulated in soils, which threatens the ecological stability of crops. Investigating the effects of cuprous oxide nanoparticles (Cu2O-NPs) on photosystem Ⅱ (PSⅡ) of wheat seedling leaves holds considerable importance in comprehending the implications of Cu2O-NPs on crop photosynthesis. Following the hydroponic method, we investigated the effects of 0, 10, 50, 100, and 200 mg·L-1 Cu2O-NPs on chlorophyll fluorescence induction kinetics and photosynthetic-related genes in wheat seedlings of "Zhoumai 18". The results showed that, with the increases of Cu2O-NPs concentrations, chlorophyll contents in wheat leaves decreased, and the standardization of the OJIP curve showed a clearly K-phase (ΔK＞0). Cu2O-NPs stress increased the parameters of active PSⅡ reaction centers, including the absorption flux per active RC (ABS/RC), the trapping flux per active RC (TRo/RC), the electron transport flux per active RC (ETo/RC), and the dissipation flux per active RC (DIo/RC). Cu2O-NPs stress decreased the parameters of PSⅡ energy distribution ratio including the maximum quantum yield of PSⅡ (φPo), the quantum yield of electron transport from QA (φEo), and the probability that a trapped exciton moved an electron further than QA (Ψo), while increased the quantum ratio for heat dissipation (φDo). Moreover, there was a decrease in photosynthetic quantum yield Y(Ⅱ), photochemical quenching coefficient (qP), net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of leaves with the increases of Cu2O-NPs concentration. Under Cu2O-NPs stress, the expression levels of genes which included PSⅡ genes (PsbD, PsbP, Lhcb1), Rubisco large subunit genes (RbcL), cytochrome b6/f complex genes (PetD, Rieske), and ATP synthase genes (AtpA, AtpB, AtpE, AtpI) were downregulated. These results indicated that Cu2O-NPs stress altered the activity and structure of PSⅡ in wheat seedlings, affected the activity of PSⅡ reaction centers, performance parameters of PSⅡ donor and acceptor sides. PSⅡ related genes were downregulated and exhibited significant concentration effects.

Theoretical examination of nuclear spin diffusion in light-induced spin coherences in photosystem I

Microsecond coherence times are predicted in spin dynamics calculations of a donor–acceptor electron spin pair P+A1A− created upon light excitation of photosystem I (PSI). The effects of nuclear spin diffusion (NSD) due to the anisotropic protein environment on the predicted coherence times TM are studied. Closely positioned pairs and triples of protons located 5–8 Å from the electron spin are shown to largely control TM. Knowledge of the crystal structure of PSI allows for spin dynamics calculations where specific cofactors and amino acid residues are removed or replaced and the identification of anisotropic environmental features controlling electronic decoherence. Finally, we show that NSD alone cannot explain the >3× shorter experimentally observed coherence times and suggest that methyl groups at key protein sites may explain this discrepancy.

Immunophilin FKB20-2 participates in oligomerization of Photosystem I in Chlamydomonas.

PSI is a sophisticated photosynthesis protein complex that fuels the light reaction of photosynthesis in algae and vascular plants. While the structure and function of PSI have been studied extensively, the dynamic regulation on PSI oligomerization and high light response is less understood. In this work, we characterized a high light-responsive immunophilin gene FKB20-2 (FK506-binding protein 20-2) required for PSI oligomerization and high light tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Biochemical assays and 77-K fluorescence measurement showed that loss of FKB20-2 led to the reduced accumulation of PSI core subunits and abnormal oligomerization of PSI complexes and, particularly, reduced PSI intermediate complexes in fkb20-2. It is noteworthy that the abnormal PSI oligomerization was observed in fkb20-2 even under dark and dim light growth conditions. Coimmunoprecipitation, MS, and yeast 2-hybrid assay revealed that FKB20-2 directly interacted with the low molecular weight PSI subunit PsaG, which might be involved in the dynamic regulation of PSI-light-harvesting complex I supercomplexes. Moreover, abnormal PSI oligomerization caused accelerated photodamage to PSII in fkb20-2 under high light stress. Together, we demonstrated that immunophilin FKB20-2 affects PSI oligomerization probably by interacting with PsaG and plays pivotal roles during Chlamydomonas tolerance to high light.

Redox Potentials of Iron-Sulfur Clusters in Type I Photosynthetic Reaction Centers.

The electron transfer pathways in type I photosynthetic reaction centers, such as photosystem I (PSI) and reaction centers from green sulfur bacteria (GsbRC), are terminated by two Fe4S4 clusters, FA and FB. The protein structures are the basis of understanding how the protein electrostatic environment interacts with the Fe4S4 clusters and facilitates electron transfer. Using the protein structures, we calculated the redox potential (Em) values for FA and FB in PSI and GsbRC, solving the linear Poisson-Boltzmann equation. The FA-to-FB electron transfer is energetically downhill in the cyanobacterial PSI structure, while it is isoenergetic in the plant PSI structure. The discrepancy arises from differences in the electrostatic influences of conserved residues, including PsaC-Lys51 and PsaC-Arg52, located near FA. The FA-to-FB electron transfer is slightly downhill in the GsbRC structure. Em(FA) and Em(FB) exhibit similar levels upon isolation of the membrane-extrinsic PsaC and PscB subunits from the PSI and GsbRC reaction centers, respectively. The binding of the membrane-extrinsic subunit at the heterodimeric/homodimeric reaction center plays a key role in tuning Em(FA) and Em(FB).

Structural insights into the action mechanisms of artificial electron acceptors in photosystem II

Photosystem II (PSII) utilizes light energy to split water, and the electrons extracted from water are transferred to QB, a plastoquinone molecule bound to the D1 subunit of PSII. Many artificial electron acceptors (AEAs) with molecular structures similar to that of plastoquinone can accept electrons from PSII. However, the molecular mechanism by which AEAs act on PSII is unclear. Here, we solved the crystal structure of PSII treated with three different AEAs, 2,5-dibromo-1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2-phenyl-1,4-benzoquinone, at 1.95 to 2.10Å resolution. Our results show that all AEAs substitute for QB and are bound to the QB-binding site (QB site) to receive electrons, but their binding strengths are different, resulting in differences in their efficiencies to accept electrons. The acceptor 2-phenyl-1,4-benzoquinone binds most weakly to the QB site and showed the highest oxygen-evolving activity, implying a reverse relationship between the binding strength and oxygen-evolving activity. In addition, a novel quinone-binding site, designated the QD site, was discovered, which is located in the vicinity of QB site and close to QC site, a binding site reported previously. This QD site is expected to play a role as a channel or a storage site for quinones to be transported to the QB site. These results provide the structural basis for elucidating the actions of AEAs and exchange mechanism of QB in PSII and also provide information for the design of more efficient electron acceptors.

Solar water splitting Pt-nanoparticle photosystem I thylakoid systems: Catalyst identification, location and oligomeric structure

Photosynthetic conversion of light energy into chemical energy occurs in sheet-like membrane-bound compartments called thylakoids and is mediated by large integral membrane protein-pigment complexes called reaction centers (RCs). Oxygenic photosynthesis of higher plants, cyanobacteria and algae requires the symbiotic linking of two RCs, photosystem II (PSII) and photosystem I (PSI), to split water and assimilate carbon dioxide. Worldwide there is a large research investment in developing RC-based hybrids that utilize the highly evolved solar energy conversion capabilities of RCs to power catalytic reactions for solar fuel generation. Of particular interest is the solar-powered production of H2, a clean and renewable energy source that can replace carbon-based fossil fuels and help provide for ever-increasing global energy demands. Recently, we developed thylakoid membrane hybrids with abiotic catalysts and demonstrated that photosynthetic Z-scheme electron flow from the light-driven water oxidation at PSII can drive H2 production from PSI. One of these hybrid systems was created by self-assembling Pt-nanoparticles (PtNPs) with the stromal subunits of PSI that extend beyond the membrane plane in both spinach and cyanobacterial thylakoids. Using PtNPs as site-specific probe molecules, we report the electron microscopic (EM) imaging of oligomeric structure, location and organization of PSI in thylakoid membranes and provide the first direct visualization of photosynthetic Z-scheme solar water-splitting biohybrids for clean H2 production.

Structure of Photosystem I Supercomplex Isolated from a Chlamydomonas reinhardtii Cytochrome b6f Temperature-Sensitive Mutant

The unicellular green alga, Chlamydomonas reinhardtii, has been widely used as a model system to study photosynthesis. Its possibility to generate and analyze specific mutants has made it an excellent tool for mechanistic and biogenesis studies. Using negative selection of ultraviolet (UV) irradiation-mutated cells, we isolated a mutant (TSP9) with a single amino acid mutation in the Rieske protein of the cytochrome b6f complex. The W143R mutation in the petC gene resulted in total loss of cytochrome b6f complex function at the non-permissive temperature of 37 °C and recovery at the permissive temperature of 25 °C. We then isolated photosystem I (PSI) and photosystem II (PSII) supercomplexes from cells grown at the non-permissive temperature and determined the PSI structure with high-resolution cryogenic electron microscopy. There were several structural alterations compared with the structures obtained from wild-type cells. Our structural data suggest that the mutant responded by excluding the Lhca2, Lhca9, PsaL, and PsaH subunits. This structural alteration prevents state two transition, where LHCII migrates from PSII to bind to the PSI complex. We propose this as a possible response mechanism triggered by the TSP9 phenotype at the non-permissive temperature.

Structure of Dunaliella photosystem II reveals conformational flexibility of stacked and unstacked supercomplexes.

Photosystem II (PSII) generates an oxidant whose redox potential is high enough to enable water oxidation , a substrate so abundant that it assures a practically unlimited electron source for life on earth . Our knowledge on the mechanism of water photooxidation was greatly advanced by high-resolution structures of prokaryotic PSII . Here, we show high-resolution cryogenic electron microscopy (cryo-EM) structures of eukaryotic PSII from the green alga Dunaliella salina at two distinct conformations. The conformers are also present in stacked PSII, exhibiting flexibility that may be relevant to the grana formation in chloroplasts of the green lineage. CP29, one of PSII associated light-harvesting antennae, plays a major role in distinguishing the two conformations of the supercomplex. We also show that the stacked PSII dimer, a form suggested to support the organisation of thylakoid membranes , can appear in many different orientations providing a flexible stacking mechanism for the arrangement of grana stacks in thylakoids. Our findings provide a structural basis for the heterogenous nature of the eukaryotic PSII on multiple levels.

A novel protein domain is important for photosystem II complex assembly and photoautotrophic growth in angiosperms

Photosystem II (PSII) is a multi-subunit protein complex of the photosynthetic electron transport chain that is vital to photosynthesis. Although the structure, composition, and function of PSII have been extensively studied, its biogenesis mechanism remains less understood. Thylakoid rhodanese-like (TROL) provides an anchor for leaf-type ferredoxin:NADP+ oxidoreductase. Here, we report the chacterizaton of a second type of TROL protein, TROL2, encoded by seed plant genomes whose function has not previously been reported. We show that TROL2 is a PSII assembly cofactor with essential roles in the establishment of photoautotrophy. TROL2 contains a 45-amino-acid domain, termed the chlorotic lethal seedling (CLS) domain, that is both necessary and sufficient for TROL2 function in PSII assembly and photoautotrophic growth. Phylogenetic analyses suggest that TROL2 may have arisen from ancestral TROL1 via gene duplication before the emergence of seed plants and acquired the CLS domain via evolution of the sequence encoding its N-terminal portion. We further reveal that TROL2 (or CLS) forms an assembly cofactor complex with the intrinsic thylakoid membrane protein LOW PSII ACCUMULATION2 and interacts with small PSII subunits to facilitate PSII complex assembly. Collectively, our study not only shows that TROL2 (CLS) is essential for photoautotrophy in angiosperms but also reveals its mechanistic role in PSII complex assembly, shedding light on the molecular and evolutionary mechanisms of photosynthetic complex assemblyin angiosperms.

Functional Water Networks in Fully Hydrated Photosystem II.

Water channels and networks within photosystem II (PSII) of oxygenic photosynthesis are critical for enzyme structure and function. They control substrate delivery to the oxygen-evolving center and mediate proton transfer at both the oxidative and reductive endpoints. Current views on PSII hydration are derived from protein crystallography, but structural information may be compromised by sample dehydration and technical limitations. Here, we simulate the physiological hydration structure of a cyanobacterial PSII model following a thorough hydration procedure and large-scale unconstrained all-atom molecular dynamics enabled by massively parallel simulations. We show that crystallographic models of PSII are moderately to severely dehydrated and that this problem is particularly acute for models derived from X-ray free electron laser (XFEL) serial femtosecond crystallography. We present a fully hydrated representation of cyanobacterial PSII and map all water channels, both static and dynamic, associated with the electron donor and acceptor sides. Among them, we describe a series of transient channels and the attendant conformational gating role of protein components. On the acceptor side, we characterize a channel system that is absent from existing crystallographic models but is likely functionally important for the reduction of the terminal electron acceptor plastoquinone QB. The results of the present work build a foundation for properly (re)evaluating crystallographic models and for eliciting new insights into PSII structure and function.

Exogenous trehalose protects photosystem II in heat-stressed wheat

Background: Photosystem II (PSII) is susceptible to heat stress. Plants naturally accumulate trehalose to improve stress tolerance. However, the mechanism by which trehalose affects PSII during heat stress is still unknown.
 Questions: How does trehalose affect PSII during heat stress?
 Studied species: Triticum aestivum L.
 Study site and dates: Shanghai, China. 2019-2021.
 Methods: Trehalose -pretreated wheat seedlings suffered from heat stress and their photosynthetic parameters were measured.
 Results: Heat stress caused a reduction in the photochemical efficiency of PSII, the electron transfer rate (ETR(II)), the quantum yield of regulated energy dissipationY(NPQ) and the coefficient of photochemical quenching (qP), but increased the quantum yield of non-regulated energy dissipation of PSII (Y[NO]). The shape of the fast chlorophyll fluorescence induction kinetics (OJIP) curve in the heat-stressed wheat was altered and the primary photochemistry maximum yield of PSII (Fv/Fo) and the PSII performance indicator PIabs were reduced. Accordingly, the activities of PSII and electron transport chain, the amount of ordered α-helix structures and the content of D1 protein also decreased. However, in trehalose-pretreated wheat, D1 protein and protein secondary structures of PSII were both protected, the electron transport activities of PSII and the whole chain were improved and greater fluorescence parameters values were maintained. Lower Y(NO) and more stable OJIP were obtained.
 Conclusions: Exogenous trehalose acted a vital role in the protection of the function of PSII, resulting in higherphotosynthetic capacity under heat stress.

Deacylation of galactolipids decomposes photosystem II dimers to enhance degradation of damaged D1 protein.

Photosystem II (PSII) contains many lipid molecules that are essential for the function and maintenance of PSII. Under strong light conditions, PSII complexes are dynamically modified during the repair process; however, the molecular mechanism of the dynamic changes in the PSII structure is still unclear. In the present study, we investigated the role of a lipase in the repair of PSII in Synechocystis sp. PCC 6803. We identified a protein encoded by the sll1969 gene, previously named lipase A (lipA), in the Synechocystis sp. PCC 6803 genome as a candidate for the lipase involved in PSII repair. Recombinant protein expressed in Escherichia coli cells hydrolyzed fatty acids at the sn-1 position of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol as well as triacylglycerol esterified with stearic acids. PSII repair in a disrupted mutant of the lipA gene was suppressed by the slow degradation of damaged D1 protein under strong light. The level of the PSII dimer remained higher in lipA mutant cells than wild-type (WT) cells under strong light. LipA protein was associated with the PSII dimer in vivo, and recombinant LipA protein decomposed PSII dimers purified from WT cells to monomers by reducing MGDG content in the PSII complex. These results indicate that LipA reacts with PSII dimers, dissociates them into monomers by digesting MGDG, and enhances D1 degradation during PSII repair.

Release of Electrons and Protons from Substrate Water Molecules at the Oxygen-Evolving Complex in Photosystem II

Oxygen evolution proceeds at the oxygen-evolving complex (OEC), the Mn4CaO5 cluster, in photosystem II (PSII), releasing protons and electrons from substrate water molecules. However, most mechanisms proposed for oxygen evolution is based on theoretical studies, where the deprotonation and oxidation sites of the Mn4CaO5 cluster are pre-assumed without analyzing the redox active site (e.g., redox potential) or the H-bond (e.g., pKa) for the release of the electrons or protons, respectively. Here, we summarize the characteristics of the OEC, focusing on the energetics of the electron-releasing and proton-releasing sites based on the PSII structure.

Investigation of Photosystem II Functional Size in Higher Plants under Physiological and Stress Conditions Using Radiation Target Analysis and Sucrose Gradient Ultracentrifugation.

The photosystem II (PSII) reaction centre is the critical supramolecular pigment–protein complex in the chloroplast which catalyses the light-induced transfer of electrons from water to plastoquinone. Structural studies have demonstrated the existence of an oligomeric PSII. We carried out radiation inactivation target analysis (RTA), together with sucrose gradient ultracentrifugation (SGU) of PSII, to study the functional size of PSII in diverse plant species under physiological and stress conditions. Two PSII populations, made of dimeric and monomeric core particles, were revealed in Pisum sativum, Spinacea oleracea, Phaseulus vulgaris, Medicago sativa, Zea mais and Triticum durum. However, this core pattern was not ubiquitous in the higher plants since we found one monomeric core population in Vicia faba and a dimeric core in the Triticum durum yellow-green strain, respectively. The PSII functional sizes measured in the plant seedlings in vivo, as a decay of the maximum quantum yield of PSII for primary photochemistry, were in the range of 75–101 ± 18 kDa, 2 to 3 times lower than those determined in vitro. Two abiotic stresses, heat and drought, imposed individually on Pisum sativum, increased the content of the dimeric core in SGU and the minimum functional size determined by RTA in vivo. These data suggest that PSII can also function as a monomer in vivo, while under heat and drought stress conditions, the dimeric PSII structure is predominant.

Effects of mutations of D1-R323, D1-N322, D1-D319, D1-H304 on the functioning of photosystem II in Thermosynechococcus vulcanus.

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.

Structure of a tetrameric photosystem I from a glaucophyte alga Cyanophora paradoxa

Photosystem I (PSI) is one of the two photosystems functioning in light-energy harvesting, transfer, and electron transfer in photosynthesis. However, the oligomerization state of PSI is variable among photosynthetic organisms. We present a 3.8-Å resolution cryo-electron microscopic structure of tetrameric PSI isolated from the glaucophyte alga Cyanophora paradoxa, which reveals differences with PSI from other organisms in subunit composition and organization. The PSI tetramer is organized in a dimer of dimers with a C2 symmetry. Unlike cyanobacterial PSI tetramers, two of the four monomers are rotated around 90°, resulting in a completely different pattern of monomer-monomer interactions. Excitation-energy transfer among chlorophylls differs significantly between Cyanophora and cyanobacterial PSI tetramers. These structural and spectroscopic features reveal characteristic interactions and excitation-energy transfer in the Cyanophora PSI tetramer, suggesting that the Cyanophora PSI could represent a turning point in the evolution of PSI from prokaryotes to eukaryotes.

Glycerol binding at the narrow channel of photosystem II stabilizes the low-spin S2 state of the oxygen-evolving complex.

The oxygen-evolving complex (OEC) of photosystem II (PSII) cycles through redox intermediate states Si (i = 0-4) during the photochemical oxidation of water. The S2 state involves an equilibrium of two isomers including the low-spin S2 (LS-S2) state with its characteristic electron paramagnetic resonance (EPR) multiline signal centered at g = 2.0, and a high-spin S2 (HS-S2) state with its g = 4.1 EPR signal. The relative intensities of the two EPR signals change under experimental conditions that shift the HS-S2/LS-S2 state equilibrium. Here, we analyze the effect of glycerol on the relative stability of the LS-S2 and HS-S2 states when bound at the narrow channel of PSII, as reported in an X-ray crystal structure of cyanobacterial PSII. Our quantum mechanics/molecular mechanics (QM/MM) hybrid models of cyanobacterial PSII show that the glycerol molecule perturbs the hydrogen-bond network in the narrow channel, increasing the pKa of D1-Asp61 and stabilizing the LS-S2 state relative to the HS-S2 state. The reported results are consistent with the absence of the HS-S2 state EPR signal in native cyanobacterial PSII EPR spectra and suggest that the narrow water channel hydrogen-bond network regulates the relative stability of OEC catalytic intermediates during water oxidation.