These range from the widely accepted MnIV4 oxo‐hydroxo model, which presumes that O−O bond formation occurs in the ultimate transient intermediate (S4) of the catalytic cycle, to a MnIII2MnIV2 peroxo model representative of the contrasting “early‐onset” O−O bond formation hypothesis. Density functional theory energetics of suggested S3 redox isomers are inconclusive because of extreme functional dependence. Here, we use the power of the domain‐based local pair natural orbital approach to coupled cluster theory, DLPNO‐CCSD(T), to present the first correlated wave function theory calculations of relative stabilities for distinct redox‐isomeric forms of the S3 state. Our results enabled us to evaluate conflicting models for the S3 state of the oxygen‐evolving complex (OEC) and to quantify the accuracy of lower‐level theoretical approaches. Our assessment of the relevance of distinct redox‐isomeric forms for the mechanism of biological water oxidation strongly disfavors the scenario of early‐onset O−O formation advanced by literal interpretations of certain crystallographic models. This work serves as a case study in the application of modern coupled cluster implementations to redox isomerism problems in oligonuclear transition metal systems.The electronic and geometric structures of the water‐oxidizing complex of photosystem II in the steps of the catalytic cycle that precede dioxygen evolution remain hotly debated. Recent structural and spectroscopic investigations support contradictory redox formulations for the active‐site Mn The domain‐based local pair natural orbital–coupled cluster approach, DLPNO‐CCSD(T), was employed to obtain reliable relative energetics for distinct redox‐isomeric forms of the Mn4Ca cofactor of the oxygen‐evolving complex of photosystem II in its final metastable S3 state. It is shown that the MnIV4 oxo‐hydroxo model is significantly more stable than the MnIII2MnIV2 peroxo model, thus disfavoring the scenario of early‐onset O−O bond formation in biological water oxidation. The implications for the mechanism of water oxidation are enormous, because the two possibilities lead to profoundly different scenarios in terms of the distinct redox transformations implicated in overall water oxidation, the number of electrons and nature of intermediates involved in each elementary step, and the partitioning of the Si‐state cycle into storage versus catalytic transitions.The active site of the oxygen‐evolving complex (OEC) of photosystem II contains a cluster of four Mn and one Ca ions linked by oxo bridges, Mn Figure a.1] T T4CaOx0→S1, S1→S2, and S2→S3 transitions that continuously raise the metal oxidation states from MnIII3MnIV in S0 to MnIV4 in S3. The removal of three electrons from S0 to S3 is accompanied by alternate removal of two protons in the S0→S1 and S2→S3 transitions.3 state,3 state is well supported by spectroscopy[i states.[IV‐oxyl radical4] state that initiates O5−O6 bond formation through oxo‐oxyl coupling.A widely accepted representative of the first type defined above is the so‐called “oxo‐hydroxo” model Figure a. This iIII2MnIV2 peroxo model depicted in Figure 3 state.3 state merely completing dioxygen formation rather than triggering it. The peroxo model is weakly correlated with spectroscopic observations, in order to derive the complete set of spin eigenstates.For the calculation of exchange coupling constants This methodology has been detailed in several studies and used successfully for a large number of oligonuclear exchange‐coupled Mn systems.S=6 for 3OS and S=7 for 3PS). Two basis set combinations were used for the two‐point extrapolations (see Supporting Information for details); TZ/TZ: ZORA‐def2‐TZVP on all atoms, except for C and H where the ZORA‐def2‐SVP basis sets were used, and QZ/TZ: ZORA‐def2‐QZVPP on Mn ions, ZORA‐def2‐TZVP on N, O, Ca and Cl, and ZORA‐def2‐SVP for C and H.F defined as [Eq. Using a constant d as Eq. :(4)F=yEquation 2 can be expressed as Eq. :(5)E∞=F is 1.5, as it has been determined to give optimal results for large benchmark sets. Using three different TCutPNO thresholds at both NormalPNO and TightPNO settings, we plotted fits of the correlation energy as a function of x, based on Equation (2) . The determined F values are within the 1.51–1.66 range. Given that these values are close to the more widely benchmarked recommended value of 1.5, for the sake of standardization we used F=1.5 for the PNO space extrapolations.The recommended3 state of the OEC, computational models were constructed containing the complete first coordination sphere amino acids and water molecules (W1–W4) as well as selected second coordination sphere water molecules and the hydrogen‐bonding D1‐His337 in its protonated form.3‐state peroxo isomer and calculated an S=4 ground state, which does not agree with the S=3 species observed experimentally. The models used for the coupled‐cluster calculations are sufficiently large to be representative of the electronic structure of the OEC and to naturally adopt realistic protein‐like geometries upon optimization , whi3OS) or as peroxo (3PS) forms in their respective high‐spin configuration using the B3LYP functional. The most important calculated interatomic distances of the derived structures are shown in Table S1, where comparisons of key structural parameters with PDB IDs: 5WS6 ,III and Mn4III ions in the peroxo structure are collinear, as in one of the Jahn‐Teller isomeric forms of the resting S1 state.3PS isomer is in best agreement with the 5WS6 XFEL model, which has been directly interpreted as a peroxo form,3OS models show similar level of RMSD with respect to all XFEL models associated with the S3 state. In particular, the O5−O6 distance of 1.4 Å observed for 3PS is identical with the 5WS6 crystallographic model, whereas the O5−O6 distance of 2.5 Å calculated for 3OS is much larger than that observed in the XFEL structures. By contrast, optimized Mn−Mn distances of the 3OS isomer are more consistent with EXAFS spectroscopy than 3PS, due to the larger Mn3‐Mn4 distance in 3PS, consistently with previous observations.ijJ (Table S5) the dominant ground state spin configurations of 3OS and 3PS are αααβ and βααα, respectively, and both forms adopt S=3 ground states. Notably, geometric relaxation of 3OS and 3PS in their lowest‐energy broken‐symmetry states has negligible effect on the structural parameters and relative energies of the models. From the above results, we conclude that the small OEC models are adequate to describe the basic features of the S3 isomers under investigation.The structures were optimized either as oxo‐hydroxo  and S3‐peroxo (3PS) isomers, used throughout this work is defined as [Eq. Having established the respective structural models, we proceed with the primary objective of the present work, which is to perform DLPNO‐CCSD(T) calculations to extract reliable and reasonably converged relative energies for the two forms. The electronic energy difference, Δd as Eq. :(6)ΔEP0) and (T1) corrections as well as the contribution of each correlation method to the relative energy, ΔEPO are presented. Single‐point DLPNO‐CCSD(T) calculations were performed with different levels of approximation , in order to explore the convergence behavior of the method on the absolute energies of the OEC models and on their relative energy, ΔEPO (see Table S6 for a detailed presentation of the results).The results of the DLPNO‐CCSD(T) calculations are given in Table TCutPNO threshold, which is the most important threshold for the accuracy of the method. Additionally, we approached the basis set limit of the DLPNO‐CCSD(T) calculations with respect to the metal ions. Selective increase of the basis set size used to describe the metal ions significantly enhances the accuracy of the method,We investigated the dependence of the DLPNO‐CCSD(T) correlation energy on the dimension of the PNO space, described by the EHFCBSthe HF energy extrapolated to the CBS limit according to Equation (S1), ESD∞and ET1∞are the DLPNO‐CCSD and (T1) contributions to the correlation energy, respectively, extrapolated to the infinite PNO space limit according to Equation (5) and δSDCBSand δT1CBSare additive corrections for the incompleteness of the basis sets used, defined in equation S3.where 3OS and the 3PS structures, respectively. It is apparent that extensive retrieval of correlation energy is required in order to provide a reliable energy profile. Inclusion of the semicanonical triples, denoted (T0), recovers an additional 4.4 % of the correlation energy for 3PS, but 4.6 % for 3OS. The more accurate perturbative triples correction where the triples amplitudes are computed iteratively, denoted (T1), show additional stabilization for the 3OS form, leading to a final ΔEPO value of 38.5 kcal mol−1.In the extrapolated results using NormalPNO settings, inclusion of only singles and doubles excitations (DLPNO‐CCSD) is only able to recover 95.0 and 95.3 % of the total correlation energy including the perturbative triples correction, for the TCutPNO, TCutPairs and TCutDO thresholds get tighter, the SD, (T0) and (T1) retrieved correlation energies increase. The increase is larger for the low spin 3OS structure, which results in a larger ΔEPO value as the accuracy of the method is enhanced. The (T0) and (T1) contributions to the ΔEPO value consistently increase, starting from 32.5 and 36.8 kcal mol−1, respectively, with the LoosePNO settings (Table S6) and reaching 35.9 and 41.8 kcal mol−1, respectively, with the NormalPNO settings with TCutPNO 1×10−7 , “strong pairs” in Orca convention, are treated at the coupled cluster level. For the “weak pairs”, which are expected to contribute negligibly to the total correlation energy, the corresponding LMP2 energy is added to the CCSD correlation energy to give the final DLPNO‐CCSD energy (SD). As the TCutPNO threshold decreases, more electron pairs are treated as “strong pairs”, leading to increase in the CCSD correlation energy and decrease in the LMP2 energy attributed to reduced number of “weak pairs”. We observed that the SD=CCSD+LMP2 correlation energy is slightly decreased when TCutPNO threshold increases. This is possibly due to a slight overestimation of the LMP2 correlation energy, which however, compensates for the perturbative triples correlation energy. Even though the error is very small ∼0.001 %, it leads to non‐negligible error in the calculated ΔEPO values, by overstabilizing the 3OS form. Based on the above observations, the most reliable ΔEPO estimate is 37.6 kcal/mol, derived from the CCSD correlation energy contribution from the PNO space extrapolated DLPNO‐CCSD/TightPNO/Mn CBS limit calculation and the perturbative triples (T1) correlation energy contribution from the PNO space extrapolated DLPNO‐CCSD/NormalPNO/Mn CBS limit calculation, according to the equation [Eq. Upon inspection of the results Tables  and S6, T0) and T retrieveTCutPNO threshold x=7 (that involves the lowest number of “weak” pairs) is used instead. In addition, the LMP2 correlation energy was not extrapolated to the CBS limit.The LMP2 correlation energy was not extrapolated with respect to the PNO space, because in the PNO space limit zero pairs will be characterized as “weak”. The TightPNO value of the calculation with the tighter EPO extrapolated to the CBS limit and to the PNO space limit are shown in Figure EPO values calculated at the DLPNO‐CCSD(T0)/TightPNO and DLPNO‐CCSD(T0)/NormalPNO levels of theory are almost identical . This is because the slight overestimation of the LMP2 correlation energy in the NormalPNO settings makes up for the correlation energy that is retrieved with tighter PNO cutoffs.The DLPNO‐CC derived values for Δ3OS and 3PS models, the ground state spin configurations are αααβ and βααα, respectively, and the ground state spin estimated by diagonalization of the spin Hamiltonian is S=3 for each model (Table S5). Therefore, the magnetic ground states of both systems are intrinsically multireference in character. This problem is circumvented by performing the DLPNO‐CCSD(T) calculations using the high‐spin determinant of each S3 isomer, that is, S=6 for 3OS and S=7 for 3PS. As expected for high‐valent manganese systems, both the T1 diagnostic and the largest excitation amplitudes show that none of the isomers has multireference character in the high‐spin states. The S=6 excited state for 3OS is 241 cm−1 higher than the ground state and the S=7 state for 3PS is 428 cm−1 higher, thus the energy difference between the ground state and the state of highest spin is only about 0.5 kcal mol−1 higher for 3PS than for 3OS. This means that the DLPNO‐CCSD(T) ΔEPO value computed for the respective high‐spin states is directly transferable to the two redox isomers in their S=3 ground states.A technical aspect related to the presently employed theoretical approach is the multireference nature of the wave function that describes the ground state spin configuration of the OEC models. Based on the calculated pairwise exchange coupling constants for 1) approach and the extrapolation to the complete PNO space mitigate some, but not all sources of error that were present in past studies. Nevertheless, even in the presence of such possible uncertainties, the DLPNO‐CC approach provides the maximum attainable accuracy for the system under consideration, while the magnitude of the energy difference offers strong confidence that the conclusions are qualitatively definitive. The net result is that 3OS is more stable than 3PS by 37.6 kcal mol−1 for the present models.At this point we should consider possible limitations of the methodology presented in this study. A possible source of error may relate to the extrapolation to the CBS limit are the size of the basis sets used, as well as the fact that CBS extrapolation was performed only with respect to the Mn ions. Additional errors may arise from the DLPNO approximation itself43 or frEPO calculated using B3LYP (20 % HF exchange) in the respective high spin configurations (S=6 for 3OS and S=7 for 3PS) is 21.4 kcal mol−1. Single point energy calculations of ΔEPO using the B3LYP and TPSSh functionals with different HF exchange percentages give a wide range of ΔEPO values, which are plotted in Figure EPO is −101 kcal mol−1, indicating a gross overstabilization of the peroxo form. This is consistent with the observation that E(HS)−E(LS) energy difference values generally increase with the degree of treatment of electronic correlation, because electron correlation is more important in lower‐spin states.To study the effect of Hartree‐Fock exchange in the calculated energy difference, a set of single point DFT calculations using functionals with varying contribution of exact exchange were performed. ΔEPO estimate to the DLPNO‐CCSD(T1) calculation  value, in favor of the 3PS  crystal structure. The structures were optimized in the lowest broken‐symmetry spin configurations using the TPSSh functional. TPSSh gives an energy difference ΔEPO of 18.7 kcal mol−1 and single point calculations using B3LYP with 10 % HF exchange gives ΔEPO of 17.6 kcal mol−1. Dispersion corrections with the D4 model at the TPSSh level used for geometry optimizations contribute to a small differential stabilization of 3PS by 1.1 kcal mol−1, thus we arrive at a combined best estimate of 16.4 kcal mol−1 for the energy difference ΔEPO. This result is close to the value of ∼15 kcal mol−1 reported by Isobe et al. using B3LYP with 10 % HF exchange for models of similar size.3PS with respect to 3OS, which can be attributed to the Jahn‐Teller distorted Mn1III and Mn4III ions of 3PS that need a larger and more flexible protein backbone to optimize Mn−O bond distances. Additional long‐range electrostatic protein matrix effects could be considered within a QM/MM framework, but past convergence studies suggested that their effect on the local electronic structure of the inorganic cluster is limited.After achieving convergence with respect to the accuracy of the wavefunction method used to calculate the respective correlation energies of the compact OEC models, we examined how the inclusion of additional, more remote second‐sphere elements of the protein matrix into the QM model might affect the energetics. We constructed 325 atom S3PS form, but this remains considerably higher in energy than the oxo‐hydroxo form 3OS.In conclusion, second‐shell effects selectively stabilize the 3 state requires the existence of an equilibrium between the oxo‐hydroxo and peroxo forms, achieved by intramolecular proton transfer from O6 to W2 concerted with two one‐electron transfer steps from O5 and O6 to Mn4 and Mn1, which leads to MnIV→MnIII reductions.[55Mn hyperfine coupling constants for a peroxo model do not agree with experiment, in contrast to the oxo‐peroxo form. Thus, assuming both forms were accessible, one would expect that the oxo‐hydroxo form must be lower in energy than the peroxo form, even though this cannot constrain the ΔEPO value, as EPR experiments were carried out at low temperatures. The calculated energy difference of 16.4 kcal mol−1 indicates that either the peroxo structure is not formed in the ground state of S3 or it exists in negligible quantities. The second hypothesis would be consistent with the suggestion3 can be preferentially oxidized by the transiently formed tyrosyl radical after the final light‐driven oxidation step of the cycle (S3YZ. state), so that the O−O bond is formed prior to formal S4‐state formation by a radical coupling mechanism, which potentially lowers the activation barrier. In either case, the peroxo isomer cannot be observable during experimental characterization of S3 in the ground state. Here lies the paradox with the current XFEL models; the disagreement between these models and the spectroscopic as well as computational results leads us to the conclusion that the available XFEL crystallographic models of the S3 state are far from definitive. Uncertainties in these structural studies with respect to the OEC structure in the S3 state are associated not simply with the low overall resolution, but also with the specific uncertainties in the positions of the light O atoms, the quantification of Si state conversion, ambiguities in analysis and interpretation of experimental data, the effect of dark adaptation of the samples, and the persisting possibility of partial reduction.[The proposed “early onset” O−O bond formation mechanism between O5 and O6 in the Sductions.14, 202‐dependent reactions,2O2n+ core in their active sites .2 activation is achieved through concerted electron flow between the M−O bonds of the cluster along with structural changes, therefore to understand the chemical reactivity of high‐valent metal‐oxo bonds, one needs to accurately describe their electronic structure.[O⇌P redox isomerism, that is, the equilibrium between peroxo with reduced metal ions and bis(oxo) with high‐valent metal ions, is a typically challenging process for DFT due to involvement of different spin states and configurations.2 antibonding orbitals to break the O−O bond. The Mn1‐O6‐O5‐Mn4 group in the S3‐state of the OEC resembles two mononuclear Mn complexes forming a possible O⇌P equilibrium, similar to other metalloenzymes. The results of the present work establish that this equilibrium, if at all relevant for the S3 state of the OEC, is very strongly shifted to the left.Formation of metal peroxo intermediates is a common feature of metalloenzymes that catalyze OIV4 oxo‐hydroxo form O should be a better representation of the S3 state and possibly the predominant species, which confirms the spectroscopic conclusion that the S2→S3 transition must involve Mn‐centered rather than ligand‐centered oxidation. This contradicts the literal interpretations of XFEL models that have been evolving in the last few years and evidently have plenty of room for further improvement and refinement. This conclusion stands independently of the likely equilibrium between various all‐MnIV forms comprising the heterogeneous S3 state, which might be affected among others by the extent of hydration4 state. More experimental information both on the S3 state and on the nature of the S3YZ. metalloradical intermediate will be required to address this point.This makes sense in the context of biological water oxidation because it enables the enzyme to avoid formation of undesirable, and possibly damaging, partially oxidized products. Therefore, the Mn3 state of the oxygen‐evolving complex. Within the DLPNO‐CCSD(T) framework, we investigated the convergence of the different correlation energies, that is, single and double excitations, SD, semicanonical perturbative triples, (T0), and iterative perturbative triples, (T1), with respect to the basis set and to the TCutPNO threshold, which is considered to be the most important for the accuracy of the method. Despite the “black box” character of the method in terms of performing the calculations, one should expend dedicated effort to carefully examine the convergence behavior of the method and evaluate the effect of the various cutoffs in order to reach the maximum attainable accuracy and efficiency. Nevertheless, this remains a promising approach for similar systems and problems, while additional algorithmic improvements or multilayer implementations can serve to expand the applicability of the method. We therefore expect further applications to the energetics of large biologically relevant molecules where species with different numbers of unpaired electrons are involved. Our evaluation of DFT against the coupled cluster results for the case of the S3 state isomers supports the use of hybrid functionals with low exact exchange for such systems. In terms of the chemical nature of the system under study, this work reaches the clear conclusion that the oxo‐hydroxo model is strongly stabilized energetically compared to the peroxo formulation for the final metastable S3 state of the OEC. This disfavors the possibility of Mn reduction in the S2→S3 transition and, by extension, the formation of an O−O bond to any considerable extent in the S3 state. Other proposed S3 redox isomers include ligand‐based radical forms such as the superoxo and the oxyl‐oxo formulation, which are not investigated in this work. Although the former is not a credible candidate for the S3 state, the latter is a possibility that should be considered more closely.[3 state. This serves as a warning against literal interpretations of the various geometric models inferred from XFEL crystallographic studies, particularly after the compelling demonstrations of crystallographically unresolved heterogeneity in the S3 state by EPR spectroscopy.In this work, we applied the DLPNO‐CCSD(T) method to derive accurate reference values for the relative energy of oxo‐hydroxo and peroxo forms of the SThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


We found that bisulfate ion could efficiently stabilize all the three conformers of H2CO3 and reduce the energy differences of isomers with H2CO3 in three different conformations compared to the isolated H2CO3 molecule. Calculated isomerization pathways and ab initio molecular dynamics simulations suggest that all the optimized isomers of the complex have good thermal stability and could exist at finite temperatures. We also explored the hydrogen bonding properties in this interesting complex and simulated their harmonic infrared spectra to aid future infrared spectroscopic experiments. This work could be potentially important to understand the fate of carbonic acid in certain complex environments, such as in environments where both sulfuric acid (or rather bisulfate ion) and carbonic acid (or rather carbonic dioxide and water) exist.Carbonic acid is an important species in a variety of fields and has long been regarded to be non-existing in isolated state, as it is thermodynamically favorable to decompose into water and carbon dioxide. In this work, we systematically studied a novel ionic complex [H Specifically, M06-2X functional and aug-cc-pVTZ basis set were employed, as previous studies showed that this combination could provide reliable results for hydrogen bonded, bisulfate ion-containing complexes [−4 eV. Quantum theory of atoms in molecules (QTAIM) [2CO3·HSO4]−.Geometry optimizations of all minima and transition states on the potential energy surface of the anionic molecular complex −. To better understand the changes of each component in [H2CO3·HSO4]− upon complexation, we provide the optimized three conformers of carbonic acid, bisulfate ion, and sulfuric acid in 2CO3·HSO4]−, we have obtained six stable isomers, among which isomers 2a and 2b could be considered as enantiomers, and isomers 3a and 3b could also be considered as enantiomers. While isomers 1, 3, and 4 may be regarded as HSO4–·H2CO3, isomer 2 is better recognized as H2SO4·HCO3−, which is only about 2 kJ/mol higher in energy than isomer 1. Isomer 2 is unexpected solely from gas-phase proton affinity prediction and its stability can be attributed to the formation of two strong hydrogen bonds and highly delocalized extra electrons according to previous findings by Hou et al. − and the energy barriers needed to be overcome. The calculations show that the isomerization between isomers 2a and 2b, isomers 3a and 3b involves the rotation of −OH group in bicarbonate or carbonic acid, and the barriers are almost equal to be 45.4 and 46.4 kJ/mol, respectively. Such relatively high energy barriers may preclude the isomerization between the enantiomers of 2 and 3, and they may all exist at finite temperatures. Isomerization between isomers 2a and 3a, isomers 2b and 3b has low barriers of only about 8 kJ/mol, indicating that the proton translocation between the two negatively charged oxygen atoms should not be difficult, as found in HCOO−·H+·HSO4– −, we performed AIMD simulations for isomers 1 and 2a, as presented in 1 and isomer 2a are only about 0.5 eV, confirming their high-temperature stability −, as presented in Temperature-controlled infrared spectroscopy is a useful experimental approach to probe the proton translocation dynamics as demonstrated by Johnson and co-workers ,54 and v−1 corresponding to the O–H stretching of the HSO4– moiety, and the strong vibration at ~3180 cm−1 is the symmetric double O–H stretching of the H2CO3 moiety. The spectra of isomers 2a and 2b are almost identical, as they are enantiomers; the vibrations at ~2230 and 2410 cm−1 are the asymmetric and symmetric stretching of the two shared H+ between SO42– and HCO3– moieties. Similarly, the spectra of isomers 3a and 3b are almost identical; the weak mode at ~3700 cm−1 is the free O–H stretching of the H2CO3 moiety, and the two strong modes at ~1810 and 3270 cm−1 are the shared H+ translocation and hydrogen bonded O–H stretching of the HSO4– moiety. In the simulated spectrum of isomer 4, the vibrations at ~2130, 3590, and 3700 cm−1 are the shared H+ translocation, hydrogen bonded O–H stretching of the HSO4– moiety, and the free O–H stretching of the H2CO3 moiety. Those vibrational vectors are included in In isomer 1, there is a very weak vibrational mode at ~3680 cm2CO3·HSO4]− complex to investigate if bisulfate ion is capable to stabilize the exotic conformers of carbonic acid. Our results showed that compared to the bare H2CO3 molecule, the energy differences between the different isomers of [H2CO3·HSO4]− complex with H2CO3 moiety in the conformation of trans-trans, cis-trans, and cis-cis become smaller, suggesting that bisulfate ion can efficiently stabilize the three conformers of H2CO3. Notably, for bare H2CO3, the cis-cis conformer is the most stable, while in [H2CO3·HSO4]− the isomer with H2CO3 in trans-trans becomes the most stable due to the formation of two strong hydrogen bonds. Combining quantum theory of atoms in molecules topological analysis and a fitted empirical equation, we estimated the strengths of the hydrogen bonds. The calculated isomerization pathways between the different isomers of [H2CO3·HSO4]− and also ab initio molecular dynamics simulations of the first two low-lying isomers revealed that these isomers could coexist at finite temperatures. We further simulated their harmonic infrared spectra to facilitate future infrared spectroscopic experiments to fully understand the proton translocation dynamics in this interesting complex.In summary, we theoretically explored the potential energy surface of the anionic [H


Our results show that different concentration of the leaching solutions can significantly influence the final product of the recycling process. However, it was possible to recover relatively pure La2O3 and ZrO2 to re‐synthesize the cubic LLZO phase, whose high purity was confirmed by XRD measurements.All‐solid‐state lithium ion batteries (ASS‐LIBs) are promising due to their safety and higher energy density as compared to that of conventional LIBs. Over the next few decades, tremendous amounts of spent ASS‐LIBs will reach the end of their cycle life and would require recycling in order to address the waste management issue along with reduced exploitation of rare elements. So far, only very limited studies have been conducted on recycling of ASS‐LIBS. Herein, we investigate the recycling of the Li Recycling of solid‐state batteries, which are expected to become extremely important in the energy industry over the next decade, is often very complex due to the solid electrolyte. Here, recycling of all‐solid‐state lithium ion batteries has been approached by a combination of mechanical/hydrometallurgical methods. Promising results confirm the recovering of the solid‐electrolyte. In this respect, we are on track to produce millions of tons of spent LIBs that are on the verge of their cycle life. By recycling the spent LIBs, we can manage the battery waste as well as preventing/reducing damages to the environment due to extraction of precious elements used to manufacture LIBs. Obviously, a battery has a perfect elemental composition to refabricate the same battery again. As yet, less than 5 % of LIBs are recycled,6PS5Cl|LiCoO2)7La3Zr2O12)It is expected that in the near future, all‐solid‐state lithium ion batteries (ASS‐LIBs) will enter the market of the energy industry since they offer higher safety and energy density−3 S cm−1 at room temperature.Oxide‐type Li‐ion conductors have extensively been studied in the context of solid‐electrolytes for LIBs due to their safety (non‐flammable and non‐toxic) and stability .6b, 102CO3 to substitute Na and Li and form Li2CO3) at sufficiently high pH levels.Theoretically, recycling of LLZO by a hydrometallurgical approach appears simple: the garnet‐type LLZO is somehow dissolved, then different elements will be extracted upon alkali precipitation at different pH levels, and finally Li could be recovered by a substitution reaction  and Li4Ti5O12 (LTO) as cathode and anode materials, respectively. The main focus of the study is to recycle the LLZO solid electrolyte within such a complex system by recovering La and Zr, preferably in form of pure La2O3 and ZrO2. Therefore, several leaching/precipitation scenarios have been investigated for the recycling processes. This study should help to improve our understanding on the dissolution/precipitation behavior of the different components of the system, which can also be adapted to other similar cases.In this work, LLZO is recycled by hydrometallurgical methods within a cell consisting of LiFePOTo understand the effect of the concentration of the acidic solution, the (selective) leaching processes of the LFP/LLZO/LTO mixture have been performed at three different conditions according to the concentration of the HCl solution (from very high to low concentrations). A summary of the leaching/precipitation processes and the obtained products in each case will be discussed in the following. It should be taken into consideration that the electrochemical cells were made without any housing, casing and current collectors to reduce the complexity of the system. However, in real‐case scenarios, the issue of impurities arising from battery casing and current collectors is a challenge. In this respect, two strategies are usually followed for the recycling of conventional LIBs with liquid electrolyte.2O2 was used to oxidize Fe2+ to Fe3+ and assist to form the precipitate. The final products after the acid leaching/alkali precipitation are shown in Figures In the first leaching/precipitation process, the LFP/LLZO/LTO mixture has been subjected to a selective leaching in HCl solution with pH<0.2. Figure 2O2) to the filtered solution (previously leached at pH<0.2), the first precipitate formed at a pH level of 4.0. According to Table 2.By adding NaOH +HO2 to the2 . However, the co‐existence of Ti and Fe in the recovered ZrO2 is not desirable for the purpose of recycling of LLZO. Nevertheless, it is worth noting that most of the extractable Ti was also extracted in this step .The obtained zirconium oxide is a mixture of monoclinic and tetragonal modifications of ZrO2 Table . The oth2O3 (≈75 wt %), while the pyrochlore phase of La2Zr2O7 has also been detected with a weight percent of ≈14 %. This phase is not problematic during the recycling of LLZO, since it can easily be converted to LLZO at temperatures higher than 1000 °C.2O3 and La2Zr2O7, some amount of Fe in form of LaFeO3 can be observed 2) and LiZr2(PO4)3 could be detected after calcination  during this step. The results obtained here reveal that leaching the LFP/LLZO/LTO mixture in a solution with pH<0.2 does not serve to recover the desired precursor materials such as ZrO2 or La2O3 with acceptable purity . For this reason, it was not used further in the recycling of LLZO.By leaching the mixture using such highly concentrated HCl, only a small amount of the mixture sample was left undissolved . Then, the undissolved powders were leached once more using a more concentrated HCl solution , 25 wt % perovskite LaFeO3 and 18 wt % LaPO4 phases formed after calcination  did not serve to remove LaPO4. EDX measurements . EDX results in Table The next fraction of the precipitates was observed after increasing the pH of the solution from 6 up to 11 Figure a. XRD me2 (≈55 wt %), but also LiZr2(PO4)3 (≈25 wt %) and ZrO2 (≈21 wt %)  Å. The closest match to this phase was found to be cubic ZrO2. However, cubic ZrO2 can normally be observed below its melting point (2800 K) up to 2640 K.2 found in the literature (4.861(1) Å (Table 2 and stabilized the structure to make a fluorite‐type (ZrTi)O4 phase. This could explain the reduction of the lattice parameter of this cubic phase compared to cubic ZrO2.The rest of the powders which were not dissolved in the leaching solution (pH=1.0) was subjected to the second leaching step Figure a in a coo LiZr2PO3  formed together with LaFeO3  Å; however, two minor fractions of LaFeO3 could also be detected in the XRD patterns with different lattice parameters of 3.871(1) Å and 3.835(1) Å, respectively (see Table 3/Fe2O3 product (P03_01) amounts to only 6 wt % of the total obtained products, while ≈51 wt % belong to La2O3 , co‐precipitation of Fe and La is indicated from the formation of perovskite LaFeO2O3 has a very high purity (weight fraction of La ≈99 %).However, the EDX analysis presented in Table Following this strategy, all of the extractable lanthanum and most of iron could be extracted. The next step was to try to extract Zr. Therefore, the undissolved powders were leached once again in a stronger acid with a pH level of 1.2. After leaching the mixture, the filtered solution was subjected to alkali precipitation by adding sufficient amount of NaOH to increase the pH up to 5.0.2 (about 83 wt %). In addition, a zirconium phosphate phase (≈10 wt %) and Fe2TiO5 (≈7 wt %) could also be detected by XRD  is considerable (≈83 wt %). Nevertheless, the whole products at this stage was determined to be only 28 wt % of the total products that formed in the whole acid leaching/alkali precipitation process  after calcination of the precipitates include mostly ZrO Figures . The pur2 in rutile and anatase modifications 3 and 17 wt % of ZrO2  phases, as shown in Figures Finally, the undissolved powder after the second leaching step has been calcined, which has a portion of ≈14 wt % . Furthermore, carbon (added for improving the electronic conductivity) does not significantly dissolve in the acidic solutions due to its hydrophobic nature.Here we will briefly compare the previously stated leaching experiments 2) and LiZr2(PO4)3 during calcination, while no other phosphate‐related phase(s) could be detected after the precipitation step of the filtered solution , in which different elements may affect each other's solubility through co‐precipitation. Nevertheless, a phosphate (Zr2+x(PO4)3) phase could be formed once the undissolved powders were further leached for the second time by a solution with a pH of around 1.0 . As can be seen in Table Table 2O3 and ZrO2 , loss during weighing steps, and, finally, incomplete precipitation from the solution. Nevertheless, since all the three leaching/precipitation processes have been performed as similar as possible, the different amounts of loss in the different processes are assumed to be arising mainly from un‐precipitated particles (left dissolved in the solution).From the data in Table 2O3 and ZrO2 from the complex mixture of LFP/LLZO/LTO. However, the purity of the recovered compounds was different at various leaching procedures. Nevertheless, we used the recovered products of La2O3 and ZrO2 to re‐form the LLZO phase according to the following Equation (1): In the previous sections, we have shown that it is in principle possible to extract La2) were mixed with P01_02, P02_02 and P3_02 (as a source for La2O3), respectively, to form LLZO. Since this article aims to address the recovery of the heavy elements instead of Li, which has been described in literature before,2CO3 has been added from an external source. Further, extraction of such low amount of Li in form of Li2CO3 (corresponds to ≈0.26–0.52 g) as a common method Finally, an unidentified phase was also observed  and ZrO2 (P03_03) at pH=2.0 described in Figure 2Zr2O7 phase as depicted in Figure (1−x/3)Fe1−xTixO3 Fe1−xTixO3 phase can be found in Table S4 (a=5.5811(4) Å, b=5.5572(5) Å, c=7.8545(7) Å, Pbnm), showing that the composition is more close to LaFeO3, while some B‐sites may have been occupied by Ti atoms since the lattice parameters slightly differ from LaFeO3 found in the literature.I4/mmm. From the overall scattered intensity, it is estimated that this phase has a maximum relative weight fraction of ≈7 % . The formation of this phase, which can result in a reduction of the ionic conductivity,[2O3 sources and high enough sintering temperatures.2CO3 content (a sufficient amount of La2O3 was added according to the stoichiometry of LLZO in each case), showing that increasing the amount of excess Li2CO3 results in reduction of the relative amount of the La2Zr2O7 phase. However, adding too big an excess of Li2CO3  leads to a reaction of Li with the crucible and formation of LiAlO2 phase . A marginal ≈2 wt % La2Li0.5Al0.5O4 could also be detected in each case (see Table −1).So far, we have shown that LLZO can be recycled; however, the best results with respect to purity of the LLZO were obtained for the case that the precursors were recovered from leaching a mixture of ball‐milled LFP/LLZO/LTO in HCl solution with a pH level of 1.0 Figures  and 8. Auctivity,11, 37e Figure a. The hi2O3 and ZrO2 as pure as possible and, by that, reproducing cubic garnet‐type LLZO solid electrolyte from the recovered materials. The results reveal that LLZO and LFP are not stable even in weakly concentrated HCl solution (pH=2.0), while LTO is quite stable in more concentrated HCl solutions (pH=1.0) and can be  dissolved only in very concentrated HCl solutions (pH<0.2). Furthermore, in such a complex system, co‐precipitation of the compounds, which contain different elements, is a challenge to overcome in order to recover desired compositions with high purity. For instance, iron tends to co‐precipitate with lanthanum upon leaching in weakly concentrated HCl solutions, which results in formation of perovskite LaFeO3 after calcination. The other example is co‐precipitation of zirconium, lanthanum, iron and phosphate at the same time when leaching the mixture at pH=1.0, leading to formation of ZrO2 with impurities of LaFeO3 and LaPO4. It is worth noting that the LaFeO3 phase can easily be removed by a further acid‐washing step. Therefore, these steps may have to be repeated further to improve the element separation. However, separation of these phases is not always that simple and usually requires further treatments. Nevertheless, upon leaching the mixture at pH≥1.0, high‐purity La2O3 could be obtained. Furthermore, using an HCl solution with a pH level of 1.0 seems to be the most suitable condition to obtain La2O3 and ZrO2 with a purity high enough to recover a high purity re cubic LLZO phase.In this study, we investigated the recycling of LLZO by hydrometallurgical approaches in the context of a complex cell system of LFP/LLZO/LTO. The main focus of the study lies on the recovering of LaIn summary, the hydrometallurgical method is a strong and cost‐efficient technique to recycle LLZO even in a complex system. It should be taken into great consideration, that one important target of ASS‐LIBs is to use Li‐metal as the negative electrode and by that increasing the potential window. Therefore, it is less likely to have such a complex oxide system (LFP/LLZO/LTO) in practical cases. However, the aim of the present study was to improve our understanding of the system from a recycling point of view in order to adapt the findings to other ASS‐LIB systems. This might be of special importance for all‐solid‐state batteries, when it comes to cradle‐to‐cradle design through using the battery waste as the feed for production of new batteries.LLZO Electrolyte: Cubic LLZO solid electrolyte has been synthesized by solid‐state reactions adopted from the literature.[6.25Al0.25La3Zr2O12.2CO3 (Alfa Aesar 99 %), La2O3 (Alfa Aesar 99.9 %), ZrO2 (Alfa Aesar 99 %) and Al2O3 (Alfa Aesar 99.9 %) were mixed and ball milled in presence of isopropanol for 18 h at a rotational speed of 250 RPM. Note that Li2CO3 has been used 20 % in excess. Moreover, La2O3 and Al2O3 were dried at 800 °C for 12 h and then weighted inside an Ar‐filled glovebox (99.999 %) prior to mixing. In the next step, the mixture has been calcined at 900 °C for 12 h under air followed by an intermediate milling under Ar at 500 rpm for 3 h without any intermediate breaking. Finally, the powders were sintered at 1100 °C for 10 h.LFP Cathode Material: Carbon‐coated LFP (LiFePO4/C) was synthesized by solid‐state reactions according to the literature.2CO3 (Alfa Aesar 99 %), iron(III) citrate hydrate  and ammonium dihydrogen phosphate  were mixed by ball milling (for 18 h at 150 rpm and no intermediate breaking) using acetone as a dispersant agent. The mixture was then heated at 300 °C for 3 h under air followed by heating at 750 °C for 12 h under Ar (flow rate 0.5 SLM) with intermediate hand grinding. The obtained carbon‐coated LFP was then mixed with PVDF , carbon black (dried at 190 °C for 48 h) and as‐synthesized LLZO to make the LFP cathode composite material. The weight ratios considered to be LFP/C: 40 %, LLZO: 40 %, PVDF: 10 % and C: 10 %. The mixture was then milled for 2 h at a rotational speed of 120 rpm under an Ar atmosphere .LTO Anode Material: LTO (Li4Ti5O12) was prepared by solid‐state synthesis based on the literature:2CO3 (Alfa Aesar 99 %) and TiO2 (Alfa Aesar 99.9 %) were mixed in stoichiometric ratios (Li2CO3 was used in an excess of 10 %) by ball milling (500 rpm for 12 h using isopropanol as a dispersing agent and without any intermediate breaking). The mixture was then calcined at 800 °C for 8 h under air. To prepare the anode composite material, as‐synthesized LTO was mixed with as‐synthesized LLZO, PVDF and C in a weight ratio of LTO: 40 %, LLZO: 40 %, PVDF: 10 % and C: 10 %. Finally, the mixture was milled for 2 h under Ar  at a rotational speed of 120 rpm.LFP/LLZO/LTO Mixture: To make the desired mixture, first a large pellet  made from three powder layers of LFP/LLZO/LTO was uniaxially pressed together using a pressure of 15 tons for 10 min followed by an isostatic pressing by 700 kN for 90 s. The total weight of the pellet was measured to be ≈10 g; however, the LLZO solid electrolyte was determined to amount to almost 80 % of the total mass of the sample  was leached in hydrochloric acid at different pH levels according to Figures m to 8 m NaOH solution (supplied by Merck) at room temperature under permanent rotation of a magnetic stirring bar. Normally, 1 to 2 mL of hydrogen peroxide (H2O2 35 % supplied by Roth) was added to the solution during alkali precipitation. The pH of the solution was increased until formation of a stable precipitate and then the solution was filtered. After filtration, the pH level of the solution was further increased by further alkali precipitation to form the next stable precipitate. This process was repeated to reach a pH level of 11. In each case, the precipitates were calcined mainly at 1000 °C for 12 h under air. This helps to reform oxides of each element  well crystallized and free of carbonates, making the obtained products suitable for reproducing the all‐oxide starting materials. It is worth noting that, prior to each calcination, the intermediate products were washed with distilled water to remove any traces of probable NaCl (expected to be formed during HCl leaching/NaOH precipitation processes) followed by washing with acetone.After each leaching step, the solution was filtered using VWR qualitative filter paper 303 (particle retention 5–13 μm). The leftover powder on the filter paper (undissolved) was then further leached or calcined according to the flow charts depicted in Figures The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Clinical Science (2015) 129(4), 331–344; DOI: https://doi.org/10.1042/CS20140789. The ischemia reperfusion model was ‘ischemia for 30 minutes and reperfusion for 120 minutes’. However Figure 3C in the original publication was incorrectly labelled as ‘reperfusion for 30 minutes’. Therefore, the labelling for Figure 3C should be the same as in Figure 3D to reflect that R(min) is 120.The authors would like to address an incorrect labelling in Figure 3C in the original publication of this paper in


This article has been corrected: The authors replaced SA-β-Gal staining panels for Control and TGFβ1 in Figure 6A. Originally the same Control image was used in Figure 6A and Supplementary Figure S2A, and the same TGFβ1 image was used in Figure 6A and Figure 3C. The replacement was done with the representative images from the original set of experiments. This alteration does not affect the results or conclusions of this work.Figure 6 is presented below.New


One group was left unmodified, serving as the control group. Water sorption and solubility were measured by weighing the specimens before and after immersion in distilled water and desiccation. The data were analyzed by using one‐way ANOVA and Tukey's post hoc test (α = .05).Fifty disc‐shaped specimens (10 × 2 mm) of heat‐cured PMMA were prepared and divided into five groups (p = .016), but did not change the water solubility (p = .222) compared with the control group. The 7.5 wt.% CuO and both concentration of TiO2 nanoparticles did not change the water sorption, but significantly increased the solubility of heat‐cured PMMA (p ≤ .05).The 2.5 wt.% CuO nanoparticles significantly decreased the water sorption (2 and 7.5 wt.% CuO do not affect the water sorption, but increase the water solubility of heat‐cured PMMA.Adding 2.5 wt.% CuO nanoparticles to heat‐cured PMMA decreases the water sorption; although, it has no significant effect on the solubility. Likewise, 2.5 and 7.5 wt.% TiOReinforcing the heat‐cured PMMA denture base resin materials with the right concentration and type of nanoparticles can decrease the water sorption of resin base materials, and consequently can influence the durability of dentures. This material has desirable characteristics such as ease of processing and pigmentation, low cost, light weight, stability in the oral cavity, acceptable esthetics, cost‐effectiveness, and minor toxicity  into acrylic resin improved the abrasive wear resistance, tensile and fatigue strength, while decreasing the water sorption, solubility and porosity of heat‐cured denture base resin  to PMMA improved the flexural strength of acrylic resin, and decreased the thermal expansion coefficient, water sorption and solubility. Another study showed that adding ZrO2 significantly decreased the water sorption and solubility of PMMA  are among the biocompatible nontoxic materials. These nanoparticles have chemical stability, resistance to corrosion, and high refractive index  is another nanoparticle with antimicrobial effects against a wide range of pathogenic bacteria  to be incorporated with 2.5 or 7.5 wt.% of either TiO2 or CuO nanoparticles . One group was left unmodified, serving as the control group. The concentrations of both nanoparticles were selected according to the previous studies . The specimens were divided into five groups  and CuO nanoparticles  were weighed by using an electronic balance  with accuracy of 0.0001 g, for concentrations of 2.5 and 7.5 wt.%. The powders were mixed with PMMA monomer in aseptic conditions. The suspensions were stirred with an ultrasonic homogenizer to disperse the nanoparticles in the MMA monomer  were invested with dental stone . When the stone set, the flasks  of wax patterns were opened and dewaxed in boiling water for 5 min. The appropriate mass of TiOAcrylic was packed into the mold spaces. The flask halves were placed into water bath curing unit and processed by being heated to 74°C for 90 min and then to 100°C for 30 min according to the manufacturer's instructions. Once the polymerization process was finished, all the flasks were left to cool down in water, and the specimens were removed after deflasking. Finishing of the specimens was done by using silicon carbide discs (grit 600) and a polishing machine  at 250 rpm, and polishing was done by a cloth wheel and a 0.5‐μm diamond suspension.2.21) was obtained after 3 days (mass variation was less than ±0.001 mg).The specimens were dried in desiccator (Isolab Laborgeräte GmbH) containing freshly dried silica gel (Sigma‐Aldrich), stored in an incubator at 37 ± 2°C. After 24 h, the specimens were removed and weighed to an accuracy of 0.0001 g by using an analytical scale . The 24‐h desiccation cycle was repeated until a constant mass (M3 according to the following formula: V = π(d/2)2h, where d is the diameter and h is the thickness of the specimens.The diameter and thickness of all the specimens were measured by using a digital caliper (Mitutoyo Corp) with an accuracy up to 0.1 mm. The mean diameter was calculated by measuring the diameter of each specimen at two points; these diameters were at right angles to each other. The mean thickness was calculated by measuring the thickness at five equally spaced points on the circumference of the specimen. The volume (V) of each specimen was calculated in mm2) after 7 days. Every 24 h, the specimens were removed from the solution, gently wiped with a soft paper towel to remove excess solution, weighed and immediately returned into the solution. At the end of immersion period, the specimens were desiccated as previously mentioned, until the specimens reached the constant mass (M3). Water sorption and solubility (µg/mm3) were calculated for each of the specimens through the following formulas .Data were analyzed by using SPSS software . Descriptive data were presented as means and SDs, explored for normality using Shapiro Wilk test. One‐way ANOVA was used to compare water sorption and water solubility among the tested groups. Tukey's post hoc test was used to compare the water sorption and solubility between the groups (3p < .001) and water solubility (p < .001)  Table  were cal3.1p > .05), except for the 2.5 wt.% CuO, which had significantly lower water sorption than the control group (p = .016). The findings also indicated that water sorption was concentration‐dependent, as the water sorption of 7.5 wt.% TiO2 was significantly higher than that of the 2.5 wt.% TiO2 group (p = .004), and the water sorption of 7.5 wt.% CuO was significantly higher than that of the 2.5 wt.% CuO group (p = .011). No significant difference was detected between CuO and TiO2 in neither 7.5 wt.% (p = .105) nor 2.5 wt.% concentration (p = .236).Tukey's post hoc test showed no significant difference between the experimental groups and the control group (3.22 (p < .001) and 7.5 wt.% CuO (p < .001) were significantly higher than that of the control group. However, the difference was not significant between 2.5 and 7.5 wt.% of TiO2 (p = .697), nor was it significant between 2.5 and 7.5 wt.% of CuO (p = .256). The water solubility of 7.5 wt.% CuO was significantly lower than that of 7.5 wt.% TiO2 (p = .013) and the water solubility of 2.5 wt.% CuO was significantly lower than that of 2.5 wt.% TiO2 (p = .001).The water solubility of both concentrations of TiO42 and CuO nanoparticles affected both water sorption and solubility of PMMA denture base resin. These two features of denture base materials can be affected by several factors like the type of material, amount of plasticizer or filler content, and the solution in which they are immersed  The 2.5 wt.% CuO significantly decreased the water sorption and did not change the solubility of heat‐cured PMMA.(2)2 and 7.5 wt.% CuO nanoparticles significantly increased the water solubility but did not change the water sorption of heat‐cured PMMA than the control group without any nanoparticle.Both concentrations of TiOThe authors declare that there are no conflict of interests.Rashin Giti and Elham Ansarifard conceived the idea. Neda Zare Khafri and Maryam Firouzmandi conducted the literature review. Neda Zare Khafri and Rashin Giti performed the experiments. Neda Zare Khafri and Elham Ansarifard collected and analyzed the data. Maryam Firouzmandi and Neda Zare Khafri drafted the paper. Rashin Giti led the writing.


This bistability is implicated in gating the final catalytic states preceding O−O bond formation, but it is unknown how the biological system enables its emergence and controls its effect. Here we show that the Mn4CaO5 cluster in the resting (dark‐stable) S1 state adopts orientational Jahn–Teller isomeric forms arising from a directional change in electronic configuration of the “dangler” MnIII ion. The isomers are consistent with available structural data and explain previously unresolved electron paramagnetic resonance spectroscopic observations on the S1 state. This unique isomerism in the resting state is shown to be the electronic origin of valence isomerism in the S2 state, establishing a functional role of orientational Jahn–Teller isomerism unprecedented in biological or artificial catalysis.The tetramanganese–calcium cluster of the oxygen‐evolving complex of photosystem II adopts electronically and magnetically distinct but interconvertible valence isomeric forms in its first light‐driven oxidized catalytic state, S III orientational Jahn–Teller isomerism that is identified as the electronic origin of subsequent valence isomerism in the catalytic cycle of water oxidation.The dark‐stable resting state of the biological oxygen‐evolving complex is shown to accommodate a rare type of functionally important Mn Several explanations for this phenomenon have been considered (see Supporting Information), but the most well‐supported scenario suggests that the signals arise from two valence isomeric forms (referred to as 2SA and 2SB) that differ in the position of the unique MnIII ion, Mn1 in 2SA and Mn4 in 2SB, respectively  z‐axis along the Mn4‐W1 vector.The oxygen‐evolving complex (OEC) is a unique system common to all oxygenic photosynthetic organisms that catalyzes the four‐electron oxidation of water, powered by light‐driven charge separation at the reaction center of photosystem II (PSII). The Mne Figure . The esshe enzyme8, 9 y2 state may be crucial for controlling access to the last experimentally observable S3 state.[2SA) is more easily deprotonated yet the minority form (2SB) can be more easily oxidized to an all‐MnIV species before binding an additional water molecule.3 state  species with high effective anisotropy,3 intermediates that have intermediate‐spin S=3 ground states.[2‐S3 transition and the precise composition of the heterogeneous S3 state are subjects of intense current research efforts because of their direct implications for the mechanism of the final unresolved steps of O2 formation and evolution. On the other hand, despite the functional relevance of the S2 state valence isomerism for catalytic progression, the fundamental question of how the enzyme enables and controls its emergence remains entirely open. Although structural heterogeneity in the resting S1 state of the OEC has been previously considered,[1 state, by establishing the existence and demonstrating the functional role of orientational Jahn–Teller isomerism in the OEC.The emergence of valence isomers in the SS3 state.26, 27e Figure  was rece2 state,[1 state. Geometry optimizations were carried out with density functional theory for various spin configurations, with the Mn4 ligand W2 in both the hydroxo and aquo forms, and the O5 bridge in both the oxo and hydroxo forms (Table S1). Two different motifs of the inorganic core within the same coordination environment and valence distribution III‐IV‐IV‐III for the Mn1‐Mn2‐Mn3‐Mn4 cluster were identified  ion with its (pseudo‐)Jahn–Teller axis of tetragonal elongation oriented along Asp342‐O5, but differ in the Jahn–Teller distortion of the Mn4(III) ion. In the form labeled 1SA the Jahn–Teller elongation axis of Mn4 lies along the Asp170‐Glu333 vector, perpendicular to the Jahn–Teller axis of Mn1, whereas in the 1SB isomer the Jahn–Teller elongation axis of Mn4 is collinear to that of Mn1 and oriented along the W1‐O5 vector. The axial Mn‐O elongations are so prominent that the orientations of Jahn–Teller axes can be read directly from the geometric parameters, but the electronic structure also perfectly mirrors the above description. The Kohn–Sham molecular orbitals  in the space defined by the tetragonal and orthorhombic distortion coordinates Qθ and Qϵ has three minima at the level of quadratic interaction corresponding to the three directions of tetragonal distortions  Jahn–Teller axis along a single “privileged” direction. In the present case the 1SA and 1SB are assigned as local Jahn–Teller minima with respect to the Mn4 ion, whereas a third isomer, 1SC, was also located and assigned as the transition state that connects them . In 1SC the Mn4 ion has a singly occupied dx2-y2orbital and a Jahn–Teller compression axis along the Mn4‐W2 bond, similar to a model previously discussed by Paul et al.The adiabatic potential energy surface for an octahedral homoleptic Mn1SA) or W1‐O5 (1SB). A factor enabling this type of isomerism may be the unusual ligation mode of D1‐Asp170, a residue that bridges Mn4 and Ca but interacts with the latter in a lopsided fashion that allows bifurcated H‐bonding interaction with CP43‐Arg357  of less than 0.17 Å with respect to the highest‐resolution crystallographic model of Photosystem II (Table S1). 1SA Mn‐Mn distances are closer to the corresponding crystallographic and X‐ray absorption spectroscopy (EXAFS) derived values than 1SB, which is attributed to the elongation of Mn3‐Mn4 distance and decrease of the Mn1‐Mn3 distance that take place as the Mn4‐O5 distance increases . The isomers reported here resemble forms of the cluster reported by Narzi et al.combination of the 1SA and 1SB isomeric forms provides the best agreement with EXAFS and X‐ray crystallography structural data . Given that 1SC has intermediate values between 1SA and 1SB it would also appear in better agreement with structural data than each of the minima individually, but the energetics and nature of the stationary points support 1SA/1SB heterogeneity rather than predominance of the 1SC form. S1 state heterogeneity combined with a percentage of the S0 state,III compared to the S1 state,[All S1SA and 1SB in the S1 state provides a unique yet straightforward interpretation of Fourier transform infrared (FTIR) spectroscopic studies that documented spontaneous changes in carboxylate vibrational modes during dark adaptation of the S1 state of the OEC.1 state. The isomerism proposed here accounts for the FTIR observations because rotation of the Jahn–Teller axis of Mn4 directly affects the carboxylate Asp170 and Glu333 ligands by toggling the antibonding character of the respective Mn4−O bonds.The existence of the orientational Jahn–Teller isomers 1SA and 1SB  were computed with broken‐symmetry density functional theory using TPSSh, an established functional for magnetic properties of manganese systems.[1SA is diamagnetic with a low lying triplet first excited state, whereas 1SB has an S=3 ground state with a quartet first excited state. Differences between the S1 models arise from J34, which is strongly antiferromagnetic when the Mn4‐O5 distance is short, but weakens rapidly as the Mn4‐O5 distance increases .Pairwise exchange coupling constants for structures 1 state is typically discussed as a single entity in structural studies, but observations from EPR spectroscopy have always pointed toward heterogeneity. The first reported EPR signal of the S1 state was a featureless signal around g=4.8,[g≈12 with resolved Mn hyperfine interactions was later reported for cyanobacterial OEC.1 state is sensitive to small structural perturbations. Dexheimer and Klein suggested that the state giving rise to the S1g=4.8 signal converts to the S2g=2 multiline state but does not correlate with the S2g=4.1 signal,2g=4.1 signal preferentially decays to the S1 multiline centered at g≈12.1YZ. component with the characteristic EPR signal at g=2.035 advances to the S2 multiline, while a 26 G wide signal advances to the g=4.1 conformation.g=2.035 component of the S1YZ. at X‐band and W‐band frequencies revealed that the major features of the spectra are due to the interaction of the S=1 state of the Mn4 cluster with the S=1/2 spin of YZ..1 state consists of two isomers that are the predecessors of the g=2 or g=4.1 forms of the S2 state. The two EPR signals described above remain without explicit atomistic explanation and even their common origin has been questioned.1 state and have a one‐to‐one correspondence with the Jahn–Teller isomers described above.Most compelling is the connection of the present models with EPR spectroscopy, which provides rich and selective information on the electronic structure of the OEC. The Snd g=4.8,53, 541SA/1SB Jahn–Teller isomerism and available EPR data, we calculated local zero field splitting tensors (ZFS) of Mn1(III) and Mn4(III) ions using the multireference local complete active space configuration interaction (L‐CASCI) approach.III is connected to a pronounced Jahn–Teller distortion that produces sizable site anisotropy, contrary to the small site anisotropy (on the order of 0.3 cm−1) of MnIV ions. Consequently, Mn1(III) and Mn4(III) largely determine the total anisotropy of the complex. Calculated on‐site axial and rhombic anisotropy parameters are presented in Table S2 and orientations of the principal axes of the onsite DSOC for each MnIII ion are depicted in Figure D| values of all MnIII ions are ca. 3 cm−1, which indicates that perturbations on octahedral symmetry have limited impact on the axial part of the local anisotropy. Differences between the coordination environment of Mn1 and Mn4 are reflected in the rhombic ZFS values (E/D), as Mn1 has lower rhombicity than Mn4 due to higher asymmetry of the Mn4 coordination sphere. Additionally, the Dzicomponent of each MnIII ion is collinear with its Jahn–Teller axis.To examine possible connections between the 1SA is diamagnetic and the observed signal derives from a low‐lying magnetically excited state with S=1, while 1SB has an S=3 ground state and the first excited state is likely not populated at low temperatures. Based on the computed local ZFS tensors and the ground state spin configurations predicted by the pairwise exchange coupling constants, we calculated total ZFS tensors for structures 1SA and 1SB (see Supporting Information) and further examined whether the aforementioned spin configurations of the S1 isomeric forms and their ZFS parameters reproduce the experimental X‐band EPR spectra in parallel mode. The S1 EPR spectrum at geff≈4.8 is indeed reproduced for S=1 , whereas oxidation of 1SB favors oxidation of Mn1, leading to the 2SB model with a pendant Mn4(III) ion that retains its Jahn–Teller axis oriented towards O5.The most important implication of Jahn–Teller isomerism in the S1 as a function of Mn1‐O5 bond length. The Mn4 spin population (orange line) reflects the aforementioned Mn4 dz2stabilization as O5 moves toward Mn1, while the Mn1 line (blue) shows the concurrent Mn1 dz2destabilization. According to the calculated spin populations, removal of an electron from 1SA indeed takes place from Mn4, directly connecting the oxidized forms of 1SA with 2SA. One‐electron oxidized 1SB has a spin population of 3.67 on Mn1 and of 3.53 on Mn4. As shown in Figure 1 occurs at a Mn1‐O5 distance of 2.2 Å, which thus connects the oxidized form of 1SB with 2SB. In conclusion, the emergence of valence isomers in the S2 state is a direct consequence of orientational Jahn–Teller isomerism in the dark‐stable S1 state of the enzyme. The diagram in Figure 1 and S2 states of the OEC.Figure 1 state offers a new perspective for the analysis of substrate exchange kinetics.1SA but at non‐bonding distance to either of the terminal Mn ions in 1SΒ. If O5 is a substrate,[1SΒ model, positioned along the Jahn–Teller axes of both Mn1 and Mn4, as opposed to the 1SA model, where it is more strongly bound to Mn4.In addition to structural and spectroscopic implications, orientational Jahn–Teller isomerism in the S1 state, the OEC of PSII accommodates stabilization of two distinct minima that correspond to different orientations of the Mn4(III) Jahn–Teller axis of tetragonal elongation. The two isomers provide a structure‐based interpretation of spectroscopic observations relating to the known EPR heterogeneity of the S1 state and of the S1‐S2 intermediates, because they fully rationalize the combined effect of local anisotropies in producing distinct EPR signals. Crucially, the locus of oxidation is different for each Jahn–Teller minimum and hence oxidation of each S1 isomer leads to a distinct oxidation state distribution in the S2 state. This explains how valence isomerism arises in the S2 state, a phenomenon implicated in control of substrate access in the later stages of the catalytic cycle. The connection of orientational Jahn–Teller isomerism with redox isomerism suggests that the natural system may have leveraged a unique electronic feature of the MnIII ion to evolve an unprecedented mechanism for enabling and utilizing electronic structure fluxionality as an element of catalytic control.Despite the asymmetric coordination sphere of the pendant manganese ion in the dark‐stable SThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Gas‐electrolyte composition has a significant influence on formation, coulombic and energy efficiencies, C‐rate capability, and aging. Particularly, CO2 and O2 showed a higher C‐rate capability and a decrease in irreversible capacity loss during the first cycle compared to Ar. Similar discharge capacities and aging behaviors are observed for CO, C2H4 and CH4. Acetylene showed a large decrease in performance and cycle stability. Furthermore, electrochemical impedance spectroscopy revealed that the gases mainly contribute to changes in charge transfer processes, whereas the effects on resistance and solid electrolyte interphase performance were minor. Compared to all other gas–electrolyte mixtures, the use of CO2 saturated electrolyte showed a remarkable increase in all performance parameters including lifetime.Traces of species in batteries are known to impact battery performance. The effects of gas species, although often reported in the electrolyte and evolving during operation, have not been systematically studied to date and are therefore barely understood. This study reveals and compares the effects of different gases on the charge‐discharge characteristics, cycling stability and impedances of lithium‐ion batteries. All investigated gases have been previously reported in lithium‐ion batteries and are thus worth investigating: Ar, CO Gas saturation matters: Saturation of lithium‐ion battery electrolytes with various gases is systematically investigated. Significant differences in cell performance, C‐rate capability and charge transfer processes are identified between different gases. Depending on the gas and operating conditions, a trade‐off between performance increase or loss and aging behavior is observed. The best result is obtained for CO2‐saturated electrolytes. Beside these components, additives such as vinylene carbonate (VC) or cyclohexyl benzene (CHB) are added to provide enhanced performance or to increase safety..[2 may evolve [Eq. (5)], and when using a binder such as PVdF, H2 may evolve , which might lead to an insufficiently conductive SEI.An explanation for such significant differences in discharge capacity and lifetime might be that the different gases promote different SEI formation reactions [Eqs. (1)–(4)], which thus result in a different composition and SEI performance. In addition to the aforementioned frequently reported SEI reactions, COTo better understand the possible effects and to narrow down their causes and location, we analyzed the negative electrodes′ discharge and charge behavior including the potential progression and the impedance spectra.+,To clarify whether the above observed gas impact is caused by a difference in SEI formation, the focus is now turned onto the impact of gas saturation on the first charge and discharge during formation. The respective potential curves provide valuable information about the onset of the initial reactions taking place, as well as side reactions such as lithium loss. The potentials of the negative electrodes during the first formation step of the eight different dissolved gases are shown in Figure −1) were found for Ar, CO2, C2H4, H2, CH4 and O2, whereas CO saturated electrolyte consumed about 3 mAh g−1 more before a first intercalation took place. The largest amount of electric charge, roughly 40 % more than for Ar, was needed for C2H2, with about 28 mAh g−1. The different fractions in electric charges above 0.25 V vs. Li/Li+ indicate for CO and C2H2 that the gases trigger more (or different) reactions which take place during initial SEI formation.Similar consumptions of electric charges  and energy efficiencies (EE) were calculated.The SEI potential curve was again similar for all gases, excluding C2, H2 and O2 with high CE (>84 %); 2) CO, C2H4 and CH4 with slightly lower CE (ca. 83 %); and 3) C2H2 (with 77 % CE). The differences in CE need to be attributed to different reduction reactions, which change the composition or thicknesses of the surface films leading to different performances.In Figure 2 shows the highest efficiency (83 %). For Ar, H2 and O2 the EE are similar (ca. 82 %) for the first cycle, due to very similar progression of voltage during the discharge and charge process. In contrast, lower EE were found for CO, C2H4, CH4 (ca. 80 %) and C2H2 (74 %). Among the electrolytes, CO2 showed the highest coulombic and energy efficiency from the first charge‐discharge cycle, followed by O2, Ar and H2, whereas C2H2 showed the lowest performance. This compares very well with the different capacities and the stabilities observed in the following section.Likewise, the energy efficiencies (EE) follow a similar trend to the CE of the gases, whereby CO2 and C2H2 significantly alter the formation step and therefore the resulting SEI and its performance; CO2 has a positive impact and C2H2 a negative one.Thus, the crucial impact of gases on performance already occurs in the first charge‐discharge cycle. It must be noted that CO2 and O2. Furthermore, for CO2 an increase of 8 % at 2C, 24 % at 3C and even 48 % at 5C was achieved, compared to Ar. Moreover, O2 showed a significant increase in C‐rate capability: At 2C it had 4 %, at 3C 16 %, and at 5C 28 % more capacity than Ar. Nearly the same discharge capacities at 0.5/1/2C as those for Ar were found for CO, C2H4, H2 and CH4. Again, C2H2 showed the worst performance. In contrast to the lower C‐rates, where CO2 and O2 are the best, we can also see at 3C and 5C that CO (13 or 35 %) and H2 (12 or 25 %) have improved C‐rate capabilities. Notably, the gases, even if they have only a modest solubility ,C in a CC step and charged with 1C in a CC/CV step. Then the cells were aged by carrying out two sets of cycling procedures, each with 50 cycles at 1C, separated by an impedance measurement (EIS). Charging was performed with CC/CV and discharging with CC, followed by 10 min rest time. EIS was measured at a state of charge (SOC) of 50 % after formation, just after the 65th cycle (includes C‐rate test) and at the end after the 116th cycle. The EIS‐protocol was identical for all experiments and is listed in Table The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


To clarify the regulatory effect of Calcyclin (S100A6) on chondrocytes apoptosis and its relationship with progression of osteoarthritis in an effort to explore potential therapeutic targets for osteoarthritis.in vitro, in order to regulate the S100A6 expression. The influence of S100A6 on inflammatory reaction of osteoarthritis was detected by RT‐PCR. Also, Caspase‐3 activity assay and TUNEL assay were performed to evaluate the apoptosis changes. In addition, RT‐PCR and western blots were performed to verify that S100A6 mediated the PI3K/AKT signaling pathway. Through the usage of pathway regulator, we detected S100A6 produced the effect by mediating the PI3K/AKT pathway.Immunofluorescence assay was produced to identify the rat chondrocyte sample and western blots assay was detected the expression changes of S100A6 between control group and osteoarthritis model which induced by interleukin‐1β. Adenovirus were transfected into the chondrocytes We determined the expression of S100A6 decreased in osteoarthritis model, the relative expression level in osteoarthritis model was about 0.5 fold compared with control group. Through adenovirus transfection we revealed that the inflammatory factors of osteoarthritis  showed a negative correlation with the S100A6 expression. The relative expression level of interleukin‐6 and matrix metalloproteinase‐13 were 1.534 and 1.259 when S100A6 was up‐regulated and the values were up to 2.445 and 2.074, respectively, when S100A6 was down‐regulated. Also, the data verified the apoptosis could be reduced when the S100A6 was up‐regulated and be activated when the S100A6 was down‐regulated, the Caspase‐3 activity was 16.512 U/μg and 24.45 U/μg respectively. Similar results were shown in TUNEL assay, the apoptosis index was 4.46% and 31.44%, respectively. Additionally, the results of polymerase chain reaction and western blots both demonstrated that the expression level of PI3K and AKT were increased when S100A6 was up‐regulated, conversely the expression level of those two signal modules were reduced if the S100A6 was down‐regulated. More importantly, the apoptosis triggered by S100A6 can be offset by the PI3K/AKT pathway inhibitor and activator (LY294002 and IGF‐1), the values of Caspase‐3 activity and apoptosis index became close to the untreated osteoarthritis group. The experimental results in this study were statistically significant.We investigated that Calcyclin (S100A6) relieved the inflammation and mediated the chondrocyte apoptosis through PI3K/AKT pathway and we confirmed that S100A6 might be an attractive therapeutic target. S100A6 attenuates inflammatory response and mediates apoptosis of chondrocytes via the PI3K/AKT pathway. S100A6 plays a significant role in the progression of osteoarthritis. Osteoarthritis (OA) is one of the serious age‐related diseases, which has a high disability rate. In the environment of the aging society, the number of patients with this disease is increasing year by year. According to statistics, the prevalence rates of men and women are 10% and 18% respectively in people over 60 years old 454H736N120O138S3. S100A6 has many secondary structures, most of which are helix, especially it has two EF hand structures at the carboxyl end and the amino terminal respectively, which can bind to calcium ions. Then the conformation of the protein changes after combination, exposing the binding site for target protein to exert biological functionsin vitroCalcyclin, which is also called S100 calcium binding protein A6 (S100A6), is a member of S100 protein family and localized in cytoplasm and nucleus in many different kinds of cellsin vitro. Though the pathological mechanism of OA is not fully understood, inflammatory responses are shown as a vital factor in the progress of OA. Interleukin‐1β (IL‐1β), as a promoter of many inflammatory factors, could be activated many inflammatory pathways to disturb the cartilage metabolism and was always used to create an OA model in vitroAccording to recent research, there have been no studies to clarify the mechanism and function of S100A6 on osteoarthritis at a cellular level. Interestingly, we found that the expression of S100A6 was dramatically decreased in OA in our preliminary test. In order to further explore its mechanism in this study, we decided to create OA cell model 2. The cells were harvested when they had nearly reached confluence. While the OA model chondrocytes which were treated with IL‐1β (10 ng/mL) for 24 h were harvested for subsequent processes. PI3K/AKT pathway inhibitor LY294002 (10 μM) and pathway activator IGF‐1 (100 ng/mL) were incubated 24 h with some group of cells for relevant testing.The rat cartilage chondrocytes  were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 2% antibiotics . The cells were first revived from freezer stocks and then passaged in T175 flasks. The flasks were kept in an incubator at 37°C with 5% COAdenoviral vectors were designed and synthesized by Shanghai GenePharm Co., Ltd. The most efficient interference sequence was selected from three target sequences . The small interference RNA was packaged in plasmid. The chondrocytes with good growth condition were inoculated and mixed with Lipofectamine™ 2000  following the protocol. Cells were incubated for 24 h for the satisfactory expression of over‐expressed and down‐regulated S100A6 gene and cells transfected with empty vector were used as controls.The expression of type II collagen was analyzed by immunofluorescence microscopy for identification of chondrocyte. The cells were fixed with 4% paraformaldehyde for 10 min after washing three times with PBS. Cells were subsequently blocked with goat blocking serum for 30 min. Then the samples were incubated with rabbit anti‐collagen II polyclonal antibody  overnight at 4°C. After washing cells with PBST, the secondary antibody which was CY3 Conjugated AffiniPure goat Anti‐rabbit IgG  was added. Following incubation for 1 h at 37°C, the samples was counterstained with 4′,6‐diamidino‐2‐phenylindole  and finally observed with a fluorescence microscope . Because collagen II is a characteristic protein produced by chondrocytes, the positive results were used to identify rat chondrocyte samples.Total proteins were prepared using TRIzol reagent following the protocol. Protein concentration was determined using a BCA Protein Assay Kit . Protein extracts were denatured in sample loading buffer and resolved by 12% SDS‐PAGE gel, and then the proteins were migrated from the gel onto the PVDF membrane  which was pre‐soaked in methanol and transfer buffer. The membranes were blocked with 5% nonfat dried milk in PBS for 1 h at room temperature and then incubated in blocking solution at 4°C overnight with primary antibodies, which included rabbit anti‐PI3K polyclonal antibody, rabbit anti‐AKT1 monoclonal antibody  and rabbit anti‐S100 alpha 6 monoclonal antibody . Membranes were incubated with a secondary antibody, which was HRP Conjugated AffiniPure goat Anti‐rabbit IgG  in blocking solution at room temperature for 1 h. Chemiluminescence was used to detect the target protein, the membranes were covered with substrate solution and incubated. The immunoreactive bands were quantified by using Image J software and GADPH was used as internal reference protein for quantitative analysis.‐ΔΔCt method. All following primer sequences were used: GAPDH, 5′‐ACAGCAACAGGGTGGTGGAC‐3′ (forward), 5′‐ TTTGAGGGTGCAGCGAACTT‐3′ (reverse); AKT, 5′‐GCTCTTCTTCCACCTGTCTCG‐3′(forward), 5′‐ CACAGCCCGAAGTCCGTTA‐3′ (reverse); PI3K, 5′‐ GCAACAAGTCCTCTGCCAAA‐3′ (forward), 5′‐ ACGTAATAGAGGAGCTGGGC‐3′ (reverse); IL‐6, 5′‐ GTTGCCTTCTTGGGACTGATG‐3′ (forward), 5′‐ TACTGGTCTGTTGTGGGTGGT‐3′ (reverse); MMP‐13; 5′‐ ATACGAGCATCCATCCCGAG‐3′ (forward), 5′‐ CGTGTCCTCAAAGTGAACCG‐3′ (reverse); S100A6, 5′‐ CCCCCAGGAAGGCAACATAC‐3′ (forward), 5′‐ TGGCAAGGAGGGTGACAAGC‐3′ (reverse).Total RNA from chondrocytes were extracted using TRIzol reagent  and measured the absorbance at 260 and 280 nm for quantify. RT reactions were performed according to the protocol. RT‐PCR test was performed using SYBR Green PCR instrument on QuantStudio™ 6 Flex Real‐time PCR system . The cycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C, and 60 s at 60°C for annealing elongation. GADPH was used as the internal reference. The expression data were calculated using the 26 cells) for 15 min on ice and centrifugation at 12000 rpm for 10 min. The equal amount of protein mixed with Ac‐DEVD‐pNA substrate were incubated at 37°C for 4 h. The values measured by colorimetric measurement of pNA at an absorbance of 405 nm. Through testing the amount of enzyme of caspase‐3 by cleavage of 1 nmol Ac‐DEVD‐pNA to produce 1 nmol pNA per hour, the caspase‐3 activity can be calculated according to the formula.The cells were harvested and assessed using Caspase‐3 activity assay kit  following the standard protocol. The samples were lysed with lysis buffer . According to the protocol, cells were fixed with 4% formaldehyde for 25 min and mixed with proteinase K solution for 20 min. Proper concentration of equilibration buffer was added in the samples within time. Then the samples were incubated with reaction mixture for 60 min and cell nuclei were stained with DAPI . After the samples were washed with PBS, the slides were observed under a fluorescence microscope using × 200 and × 400 times magnification. In order to analyze the data more intuitively, the apoptosis index which means the percentage of positive staining cells in the total number of cells in the microscopic field was calculated in each group.RT‐PCR showed the relative expression of mRNA. Compared with the reference gene, the calculated value showed the relative expression of target protein mRNA. Also, western blotting bands reflect the expression of the target protein, the color and width of the bands can represent the protein content. The expression of target molecular S100A6 were detected through transcription and translation levels separately, which illustrated its changes in osteoarthritis.As the characteristic inflammatory molecules, IL‐6 and MMP‐13 were closely related to the destruction of chondrocyte and matrix, RT‐PCR was used to detect the expression of IL‐6 and MMP‐13, the values were proportional to the severity of osteoarthritis.Caspase‐3 is the initiation molecule of caspase apoptosis pathway and its activity reflects the active degree of early apoptosis. The Caspase‐3 activity can be determined by pNA which is produced by a Caspase‐3 catalyzed substrate called Ac‐DEVD‐pNA . The pNA could be quantified by measuring the optical density. Thus, the activity of Caspase‐3 can be calculated by the amount of pNA produced per unit time.In the late stage of apoptosis, DNA occurs fragmentation and exposes 3′‐OH terminal which can bind to fluorescein‐dUTP under the catalysis of terminal deoxynucleotidyl transferase (TdT). Fragmented DNA in the nucleus, which also represents apoptotic activity, can be observed by fluorescence staining. More intuitively, the intensity of apoptotic activity can be quantified by the percentage of positive staining cells in the total number of cells, which calculated as apoptosis index. The percentage was proportional to the activity of apoptosis.PI3K and AKT are key node molecules which can reflect the status of PI3K/AKT signaling pathway, so their expression levels were detected by RT‐PCR and western blot and the value explained the up or down regulation of signaling pathway.P < 0.05 was considered statistically significant.All data were analyzed with GraphPad Prism 8.0 software , and RStudio. The results were shown as the mean ± standard deviation. Student's t‐test and One‐way analysis of variance (ANOVA) were separately used to assess the comparison of two groups and three or more groups. A value of Immunofluorescence assay was performed to test type II collagen, which is produced by condrocyte and is one of the main components of cartilage. Under the fluorescence microscope (× 400 magnification), type II collagen were labeled red fluorescence and the nuclei of chondrocytes were labeled blue fluorescence Fig. . We deteTo investigate the influence of S100A6 to inflammation, the mRNA level of matrix metalloproteinase‐13 (MMP‐13) and interleukin‐6 (IL‐6) were tested by RT‐PCR Fig. . FollowiIn order to examine the effect of S100A6, a part of the OA chondrocytes was treated by adenovirus containing the S100A6 gene (AdS100A6) to up‐regulate the expression and some OA chondrocytes were transfected by adenovirus containing the S100A6 short interfering gene (AdsiS100A6) to down‐regulate the expression. First of all, the caspase‐3 activity assay shows the value of the OA model was nearly 2 fold compared with normal cells, the data were 20.549 U/μg and 9.881 U/μg. As presented in Fig. To explore whether the PI3K/AKT pathway was changed by S100A6, the recombinant adenovirus was used to modify the expression of S100A6. Rat S100A6 gene (AdS100A6) and S100A6 short interfering RNA gene (AdsiS100A6) were transfected into the OA model chondrocytes respectively. RT‐PCR tests were performed to detect the mRNA level of PI3K and its downstream signal molecule AKT Fig. . The expOsteoarthritis is characterized by the degeneration of cartilage, which is manifested as the abnormal apoptosis of chondrocyte and the loss of cartilage. At present, non‐surgical treatments of osteoarthritis include non‐drug therapy, such as exercise and weight control, medications like NSAIDsWe detected the expression of S100A6 was decreased in OA model samples. To investigate the role of S100A6, small interference RNA was transfected into chondrocyte to modulate the expression of S100A6. Previous studies showed inflammatory reaction is associated closely with OA pathogenesis and symptemsAdditionally, metabolic molecules associated apoptosis were mitochondrial apoptotic pathway which represented with Bcl family and Caspase pathway. Caspase‐3 was an activator of the Caspase family, which initiated caspase apoptosis cascadeA number of studies have showed the PI3K/AKT pathway is involved in cell proliferation, apoptosis, or even autophagyin vivo researches are required to verify such issues. Also, more details on metabolic mechanism, take the downstream branch of PI3K/AKT pathway for example, are needed to be further studied. As a summary of this research, S100A6 may applied as a novel biomarker or medicine target in clinic.In conclusion, this study showed the S100A6 was related to the inflammation of osteoarthritis. More importantly, this protein serves a key role in apoptosis of chondracytes by activated or impressed PI3K/AKT signaling pathway. So, these results improve the understanding of the pathophysiological process of osteoarthritis. And S100A6 may be used as a potential treatment target for patients. However, limitations still exist in this study. First, more