We demonstrate that efficient laccase reduction requires an electron relay like methyl viologen. In the presence of dioxygen, electrons transiently stored in superoxide ions are scavenged by laccase to form water instead of H2O2. The net result is the photo accumulation of highly oxidizing [Ru(bpy)3]3+. This study provides ground for the use of laccase in tandem with a light-driven oxidative process and O2 as one-electron transfer relay and as four-electron substrate to be a sustainable final electron acceptor in a photocatalytic process.Photobiocatalysis uses light to perform specific chemical transformations in a selective and efficient way. The intention is to couple a photoredox cycle with an enzyme performing multielectronic catalytic activities. Laccase, a robust multicopper oxidase, can be envisioned to use dioxygen as a clean electron sink when coupled to an oxidation photocatalyst. Here, we provide a detailed study of the coupling of a [Ru(bpy)  •An electron relay boosts photoreduction of laccase•2O2Superoxide is efficiently captured by laccase preventing formation of H•Light activation reveals information on elementary steps inside the enzyme•2 as terminal electron acceptor for oxidative photocatalysisLaccase enables O Chemistry ; Catalysis ; Biomolecules Electron transfer (ET) reactions are of fundamental importance in biological processes such as photosynthesis and respiration . Underst2 to water  ions widely used in photocatalytic multielectronic oxidative processes 3]2+ (600 ns) as compared with the zinc porphyrin triplet state (720 μs). However, other factors such as energy transfer quenching and back electron transfer might also hamper this bimolecular light-activation process. Such pathways can hardly be assessed from measurements of overall yields under continuous illumination. To improve the efficiency of the light-driven activation of laccase by [Ru(bpy)3]2+, we set to reinvestigate the photophysical events occurring upon excitation of a [Ru(bpy)3]2+ chromophore and its interaction with laccase. In the present study we show that the efficiency of light-induced electron transfer between our synthetic photosensitizer and the laccase enzyme can be strongly enhanced, from 1% to 20%, by the introduction of methyl viologen (MV2+) as an electron relay. We track the role of methyl viologen acting as an efficient oxidative quencher of the photosensitizer excited triplet state to form a methyl viologen radical, MV⋅+, which efficiently reduces the laccase. We furthermore delineate the reaction pathways under aerobic conditions, where MV⋅+ reacts with O2 to form superoxide radicals that are efficiently scavenged by the enzyme to perform the 4-electron reduction of O2 to H2O. As a result of these processes the powerful and long-lived oxidant [Ru(bpy)3]3+ with an oxidizing power of ca. 1300 mV versus NHE is generated. At all stages experimental data are confronted with kinetic simulations to arrive at a consistent description of the reaction sequence and the interactions at play between the different components.Laccases are robust enzymes used in many industrial and biotechnological processes . In natuto water . The catto water . At a tyreaction . The TNCreaction . Althougrocesses . From ourocesses . When a rocesses  but suff3]2+ photosensitizer and the laccase enzyme. Steady-state and time-resolved emission and absorption studies were performed on solutions containing [Ru(bpy)3]2+ and various concentrations of laccase (LAC3 from Trametes sp. C30) in de-aerated Britton-Robinson (B&R) buffer at pH 6. As illustrated in 3]2+ chromophore is characterized by its 450-nm metal-to-ligand charge-transfer (MLCT) band  (ε = 5600 M−1 cm−1) (We first reinvestigated the interaction between the [Ru(bpy)−1 cm−1)  with an −1 cm−1) . These s9·M−1s−1 (3]3+ and reduced LAC3 (CuI) of only 0.7%  = −0.84 V; E(CuII/CuI) = 0.68 V) (3]2+∗ emission spectrum and the absorption band of the CuII of the T1 site (R0 = 2.6 nm) calculated for this interaction implies that upon encounter, through-space resonance energy transfer will largely outcompete ET, the rate of which is seven orders of magnitude slower at that distance  . These f T1 site  3]2+ chromophore and LAC3 , present in equilibrium with the separated species before and directly after the excitation flash. Such association complexes between ruthenium polypyridine and other transition metal complexes with blue copper proteins have been reported before  are in association with a LAC3 protein.Interestingly, a careful comparison of the kinetics of the emission transient at 610 nm, expected to represent the decay of the 2+ excited state to the copper T1. We exclude formation of the doublet excited state of the T1 copper ion, as this state was shown to decay to the ground state in less than 1 ps, which would certainly not allow us to observe a bleaching of T1 absorption persisting for hundreds of ns (3]2+ complex (Dexter mechanism) could explain the transient bleaching of T1 absorption. However, both energy transfer processes should lead to a concomitant recovery of the [Ru(bpy)3]2+ ground state for which no indication is detectable in the transient absorption trace recorded at 450 nm. For this reason and to account for the formation of stable Ru3+ and reduced T1 (kcs describes charge separation from the excited state of the chromophore yielding the oxidized state of the chromophore (Ru3+) and reduced laccase (T1–), and kbr describes charge recombination within the complex. It should be noted that the transiently formed species Ru3+ is characterized by an extinction coefficient close to the one of the ∗Ru2+ excited state , the rate constants are surprisingly close. To obtain more information on the driving force dependance of these ET rates the same process was investigated using [Ru(bpz)3]2+ (bpz: bipyrazine), a ruthenium sensitizer with significantly higher Ru3+/Ru2+∗ and Ru3+/Ru2+ potentials (0.13 V and +2.23 V for [Ru(bpz)3]2+ compared with −0.84 V and +1.26 V/NHE for [Ru(bpy)3]2+ (3]2+ sensitizer the driving force for charge separation is about 1 eV lower than with [Ru(bpy)3]2+, whereas the driving force for charge recombination is increased by the same amount. Global kinetic simulation (3]2+ for LAC3 about 2 times lower than in the case of [Ru(bpy)3]2+ (Ka = 2.36 mM−1 versus 4.75 mM−1), implying a lower steady-state concentration of complexes in the case of [Ru(bpz)3]2+. The rate constants for the two ET reactions in the association complex between [Ru(bpz)3]2+ and LAC3 were determined as kcs = 6.6 × 106 s−1and kbr = 1.2 × 106 s−1, close to the values found in the case of [Ru(bpy)3]2+.The question that arises then is whether the observed bleaching of T1 absorption in the association complex should be attributed to an electron or energy transfer process from the [Ru(bpy)ds of ns . Formatiduced T1  we attriduced T1 :(Equatiobpy)3]2+ . Consequmulation  revealed2+/Ru3+ (or Ru2+∗/Ru3+) transition .With the known driving forces for the two reactions in eq (A) for the two sensitizers, the rates could be analyzed in the framework of Marcus theory permitting an estimation of the reorganization energy and distances for the ET reactions . A valueansition . Also, tulations . Interesulations  the dono3]2+ in a solution containing LAC3 are summarized in 3]2+ are shown in 3]2+∗ to LAC3 is short circuited by Förster energy transfer because the latter operates over longer distances and that only excitation of a sensitizer present in a Ru:LAC3 association complex at the time of photon absorption leads to electron transfer to the enzyme. The right-side mechanism in 3]3+:LAC3– complex described by the rate koff and charge recombination within the complex described by the rate kbr. The association constant for the charge separated state can be determined from the value of koff resulting from the global fit (koff = 1.2 × 105 s−1), yielding Kacs = 8.5 mM−1. The slightly increased affinity of the sensitizer for LAC3 in the charge separated state (Kacs = 8.5 mM−1 versus Ka = 4.75 mM−1) can be attributed to an increased electrostatic interaction between oxidized Ru3+ and reduced laccase. The ratio of koff/(koff + kbr) then gives a yield of 6% for the effective formation of Ru3+ and LAC3– from the charge separated state. With only 12.5% of the ruthenium sensitizers engaged in such a complex, the overall quantum yield of persistent, not only transient, laccase reduction (and Ru3+ formation) drops to 0.75%. Put together, these different phenomena explain well the observed low yield of photoreduction of laccase from the excited state of the ruthenium sensitizer.The flash-induced energy and electron transfer reactions occurring upon excitation of [Ru(bpy)3]2+∗ to LAC3 had to be considered in the analysis. In other words, it appears that in the complex the rate of energy transfer is slower than the rate of ET. This situation is reminiscent of a recent observation in a flexible BODIPY-C60 dyad where photoinduced ET occurs in the folded conformer (R = 8.8 Å), whereas energy transfer is observed in extended conformers (R = 17–20 Å)  is expected to lead to a crossing of the k(r) curves with the rate of electron transfer becoming faster than the rate of energy transfer for short distances, in our case this crossing occurs at too short distance to provide a simple explanation for the dominance of ET over energy transfer (R0 (dashed green line in ∗Ru2+ triplet state and a doublet ground state of the copper T1. Although examples of triplet-singlet and triplet-doublet energy transfer reactions have been described (R0) where the rate of electron transfer is slow but could be crucial at short distances where the rate of electron transfer is fast enough to compete (Although the simple reaction sequence proposed to occur in the association complexes (right panel of 17–20 Å) . Similar17–20 Å) . Our restransfer . It mighescribed , the selescribed . In our  compete . It woul2+) as an electron relay between [Ru(bpy)3]2+∗ and LAC3. Systems combining [Ru(bpy)3]2+ and MV2+ are probably the most extensively studied photoredox systems 3]2+∗ was quenched by the addition of 10 mM MV2+ with a shortening of the lifetime of the excited state from 600 ns to 110 ns. The classic oxidative quenching of the [Ru(bpy)3]2+∗ by MV2+ involves a diffusion-limited encounter followed by an intermolecular electron transfer process (3]3+ - MV⋅+ formed by dissociation of the encounter complex in competition with the back ET is described by a cage escape yield of about 20% (3]2+/MV2+ mixture did not result in a significant change of the [Ru(bpy)3]2+∗ excited state lifetime. This experimental observation suggests that the dominating deactivation pathway of the excited triplet state [Ru(bpy)3]2+∗ in the presence of both MV2+ and LAC3 is an ET process to MV2+, which is faster than energy transfer to the laccase because of the much higher concentration of MV2+ compared with LAC3. Transient absorption spectra were recorded at different time delays after the laser flash  were observed indicating the formation of about 1 μM of the [Ru(bpy)3]3+ - MV⋅+ CSS by the oxidative quenching mechanism discussed above. Spectral evolution up to 500 μs shows that the signature of reduced methyl viologen disappears completely followed by a bleaching around 610 nm, which denotes the consumption of the MV⋅+ radical and the loss of oxidized T1 in LAC3. These findings can be assigned to the charge shift from MV⋅+ to the T1 locus of the laccase while the bleaching due to the [Ru(bpy)3]3+ species partially persists  should have little absorption in the visible to avoid absorption of excitation light, and, most importantly, the reduced molecule must be able to act as a reductant for the enzyme. In contrast to many other small redox molecules investigated as mediator, MV process . The chabout 20% . The adder flash . At 1 μs3]2+/MV2+/LAC3 system, excitation of [Ru(bpy)3]2+ leads to the generation of 7.8 μM [Ru(bpy)3]2+∗ triplet excited state as deduced from the maximum bleaching observed at 450 nm immediately after the laser flash (see legend of 3]2+∗ transfers an electron to the MV2+ leading to the formation of 1.52 μM [Ru(bpy)3]3+ detected by the absorption bleach at 450 nm after decay of the excited state, and the same amount of MV⋅+ radical, observed by the absorption increase at 610 nm at early times after the flash (εMV+⋅605 = 13700 M−1 cm−1)  due to charge recombination with [Ru(bpy)3]3+ yielding mirror-like absorption transients at 450 and 610 nm  on the other hand slows down in the presence of laccase, and the lifetime of both species, reduced T1 and oxidized chromophore, extends into the ms time range. Using the extinction coefficients at 610 nm εT1610 ≈ 5600 M−1 cm−1 and εRu3+610 ≈ 2000 M−1 cm−1 (⋅+ produced. This clearly illustrates that MV⋅+ can reduce the laccase (ΔG = −1.13 eV) with diffusion-limited kinetics (k2 = 3.8 × 108 M−1 s−1). Importantly, the quantum yield of formation of [Ru(bpy)3]3+ and [CuI] depends only on the competition between the forward ET from MV⋅+ to LAC3 and the charge recombination of the [Ru(bpy)3]3+ - MV⋅+ CSS. This competition is in favor of laccase reduction even for low concentrations of LAC3 because the flash-induced concentration of MV⋅+ (and [Ru(bpy)3]3+, around 1 μM) is much smaller than the LAC3 concentration. It is important to recall that in this system, optimization of laccase reduction is equivalent to optimization of formation of the oxidized form of the ruthenium chromophore. The energetics and kinetics of the reactions occurring in the [Ru(bpy)3]2+/MV2+/LAC3 system are summarized in The absorption transients at specific wavelengths  allowed −1 cm−1) . From th complex . In the M−1 cm−1 , a conce3]2+/MV2+/LAC3 mixture. The [Ru(bpy)3]3+ state is the only species able to oxidize EDTA, regenerating the light-active [Ru(bpy)3]2+ state that can engage in subsequent ET reactions until the full four-electron reduction of laccase is achieved.Time-resolved laser flash photolysis studies such as those presented above are an important tool to investigate the sequence of light-induced reaction steps and determine the relevant rate constants. On the other hand, photocatalytic processes employ continuous irradiation where reactions are governed by steady state concentrations significantly different from those encountered in synchronized flash excitation studies. To study the role of the electron mediator between the chromophore and LAC3 in the photoreduction process under continuous illumination, EDTA was added as an electron donor to the 2+∗ or MV⋅+ occurs via the T1 site . Furthermore, we neglect the details of reactions occurring within association complexes between [Ru(bpy)3]2+ and LAC3 and modeled laccase reduction from the sensitizer's excited state by a low-yield process. With these simplifications and because many rate constants are reasonably well known from kinetic flash photolysis measurements and are independent of MV2+ concentration, a reasonable fit to the experimental data could be obtained, which reproduces the major features of the photoreduction assay with a common set of parameters  to the fully reduced form . The initial absorption decrease at 610 nm describes the loss of absorption from oxidized T1 and corresponds to the consumption of three electrons, leading to reduced T1 and doubly reduced TNC  meaning that T1 stays almost fully reduced during the process although T1 is the entrance side for the electron. In fact, the kinetic parameters determined from the global fit clearly show that the third reduction of the TNC from T1red is significantly endergonic , in contrast to the first two IET steps that are close to equilibrium 3]2+∗ excited state. In contrast, the MV⋅+ species is long-lived and can progressively reduce the state T1oxTNC3 present at low concentration in the above-mentioned equilibrium with T1redTNC2 to form T1redTNC3. Of note, in the presence of dioxygen where all internal electron transfer steps are nearly isoenergetic, the complete, four-electron reduction of laccase via [Ru(bpy)3]2+∗ is possible.We use the nomenclature of Sekretaryova et al.  to descris study . The lagilibrium . Becauseibrium 3]2+∗ but the dissociation rate for MV⋅+ or Ru3+ from the Ru3+:MV⋅+:LAC3 complex might be too low to avoid charge recombination between MV⋅+ and Ru3+.Finally, to obtain a satisfactory global fit e enzyme A, we hade 2+ see , corresp2 competes with the laccase for the electron from the MV⋅+ species as it is well known that in aqueous solution MV⋅+ reacts rapidly with O2 to form O2⋅– (or O2H⋅ at our operating pH)  of MV2+. In the absence of MV2+, the rate increases linearly with the concentration of LAC3, a behavior easily explained by an increased reduction of laccase from the excited state of the chromophore due to increased formation of association complexes (see above). When MV2+ is present, but no laccase, a fast dioxygen consumption rate is observed, which corresponds to the two-electron reduction of O2 to H2O2 via dismutation of O2⋅–  leads to a very significant reduction of the dioxygen consumption rate  . This cots of O2 . Surpris2 is performed by the enzyme (eqs. C and F) in addition to the two-electron/two-proton partial direct reduction of O2 to H2O2 (eqs. B and D). A substantial addition of an enzyme consuming half as much O2 for the same concentration of photogenerated MV⋅+ could thus show up in the dioxygen consumption curve as a decrease in the dioxygen consumption rate compared with MV2+ alone. Therefore, the pattern in 2 is being progressively substituted by LAC3 as electron recipient from MV⋅+ because the two paths distinctively produce end products, either H2O2 or H2O  is surprisingly pronounced. This suggests that laccase reduction is more efficient than expected from a simple competition between laccase and O2 for electrons from MV⋅+ as observed in ⋅+ but also and quite significantly by the O2⋅– formed via electron transfer from MV⋅+ to O2 . Analysis of the O2 consumption kinetics in the absence and presence of laccase . The tweriments . The rea2⋅– can replace MV⋅+ for reduction of LAC3 as suggested by the O2 consumption experiments, then the question arises why is the bleaching of T1 absorption, as detected in the laser flash experiment , about two orders of magnitude slower than the rate of laccase reduction by MV+⋅ 3]3+ (1300 mV versus NHE), is photo produced, ready to drive the oxidation of more tenacious organic substrates .Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Winfried Leibl (This study did not generate new unique reagents.The published article includes all datasets/code generated or analyzed during this study.All methods can be found in the accompanying


N‐alkyl‐N‐alkyl pyrrolidinium‐based ionic liquids (ILs) are promising candidates as non‐flammable plasticizers for lowering the operation temperature of poly(ethylene oxide) (PEO)‐based solid polymer electrolytes (SPEs), but they present limitations in terms of lithium‐ion transport, such as a much lower lithium transference number. Thus, a pyrrolidinium cation was prepared with an oligo(ethylene oxide) substituent with seven repeating units. We show, by a combination of experimental characterizations and simulations, that the cation's solvating properties allow faster lithium‐ion transport than alkyl‐substituted analogues when incorporated in SPEs. This proceeds not only by accelerating the conduction modes of PEO, but also by enabling new conduction modes linked to the solvation of lithium by a single IL cation. This, combined with favorable interfacial properties versus lithium metal, leads to significantly improved performance on lithium‐metal polymer batteries. Ionic liquids (ILs) allow improvement of the ionic conductivity of ternary PEO‐based solid polymer electrolytes. However, the lack of Li‐ion coordination of these plasticizers and the addition of extra ions results in a low Li‐ion conductivity. An oligo(ethylene oxide)‐based IL was synthesized to overcome these limitations and enable additional transport modes, resulting in a high Li‐ion conductivity. Li+ ion‐transport modes can be differentiated between “structural” transport, such as that occurring along the PEO chain by exchanges of Li+ ion solvating units in its dynamic solvation sphere, and “vehicular” transport, corresponding to the transport of the Li+ ion with its solvation sphere before any exchange occurs  liquid electrolytes).+ ions since the anion is far less coordinating than PEO. As a result, PEO/Li salts conductive paths are “diluted” by the IL addition and the Li‐PEO interaction is in some cases increased, slowing down Li+ ion transport.[+ ion transport, results in a reduced lithium transference number (tLi+), which limits the potential performance gains when employing these electrolytes in LMBs.The current electrification of transport and decarbonizing of electricity production pushes battery researchers to explore new battery systems beyond the currently and dominant lithium‐ion technology.ransport.14, 15+ ion transport, at least partially, from the segmental mobility of PEO chains, solvating ILs are a priori more suitable for enabling further transport modes as illustrated in Figure + ion solvation spheres). In particular, the presence of solvating cations could advantageously accelerate Li+ ion transport via the formation of complexes with two positive charges overall. We reported previously on improved interaction of Li+ ion with short oligo(ethylene oxide) chains‐substituted pyrrolidinium‐based ILs in liquid electrolytes. The data suggested that the first oxygen from the nitrogen center is not interacting with lithium and that longer oligo(ethylene oxide) chains are necessary for reaching the full solvation of Li+ ions by a single cation considering that Li+ ion complexes have a preferred coordination number of 6.[+ ion solvation by a single pyrrolidinium cation and thus enabling vehicular transport by the formation of charged complex with two positive charges, we report here on the synthesis of N‐methyl‐N‐oligo(ethylene oxide) pyrrolidinium TFSI with a median oligo(ethylene oxide) chain length of seven repeating units 7TFSI; Supporting Information). We compared the designed IL with Pyr1,4TFSI in terms of physiochemical properties and Li+ ion‐IL interaction in binary liquid electrolyte as well as in TSPEs. Lithium mobility and its conduction mode in TSPEs were analyzed by electrochemical measurements, pulsed field gradient nuclear magnetic resonance (PFG‐NMR) spectroscopy and MD simulation. Practical improvements were then verified by testing the rate performance and cycling stability of Li∥Li and LiFePO4 (LFP)∥Li cells.Therefore, to decouple Liber of 6.18, 191,(2O)7TFSI was characterized as liquid binary electrolyte LiTFSI:IL (mol:mol) prior to membrane preparation and characterization.For the fundamental understanding of the coordination process and its evidence, Pyr1,(2O)7TFSI is compared with that of Pyr1,4TFSI 7TFSI leads expectedly to a higher viscosity and consequently to a lower ionic conductivity than Pyr1,4TFSI.The ionic conductivity and the viscosity of the pure Pyr1,4TFSI complex exhibits two melting transitions corresponding to crystalline phases.Tg), although it increases with the salt content as a result of increased interactions and decreased ion mobility, the 1:1 mixture still exhibits a Tg of −48 °C that is remarkably low for such a high salt concentration and hinting at a well‐preserved ion mobility as compared with the Pyr1,4TFSI complex that exhibits a 12 °C higher Tg, even though Pyr1,4TFSI has a Tg of −86 °C1,(2O)7TFSI). At high temperature, TGA results show a slightly earlier onset of Pyr1,(2O)7TFSI weight loss. The TSPE 20:2:1O7, cross‐linked or linear, exhibits a thermal stability very close to that of Pyr1,4TFSI TSPEs .Interestingly though, when mixed with LiTFSI at high mole fractions, the binary mixtures are all amorphous Figure , whereas+ ion solvation in TFSI‐based electrolytes, since the TFSI− bands are sensitive tools for analyzing the ionic coordination. The band at 742–744 cm−1 is attributed to “free” (uncoordinated) TFSI− whereas a band at 747–750 cm−1 is associated with coordinated TFSI.− coordinated to Li+ 7TFSI mixtures up to 1:1. In contrast, the Pyr1,4TFSI electrolytes show a linear increase of coordinated TFSI− from 1:10 to 1:2  where 60 % of TFSI− anions are coordinated to Li+ ions. In terms of TFSI− per Li+ ions coordination shell, the Pyr1,4TFSI electrolytes shows approximately two TFSI− per Li+, similarly to previous reports,−/Li+) for the 1:2 electrolyte. For Pyr1,(2O)7TFSI, on the other hand, even at a very high 1:1 mole ratio we calculate only 0.24 TFSI− per Li+, which shows that most Li+ do not have any TFSI− in their solvation spheres (only 12 % coordinated TFSI−) and are thus mostly coordinated by the cations. The number of TFSI− per solvation sphere only significantly increases above 1:1 molar ratio (i.e. above the saturation of the oligo(ethylene oxide) solvating sites) with a rather linear trend, corroborating our initial hypothesis that seven repeating units are required for the pyrrolidinium side chain to fully solvating one Li+ ion.Raman spectroscopy allows deriving the Li+ Figure  and deril Figure . For LiT1,(2O)7TFSI cationic solvation on the physicochemical properties of TSPE membranes was then investigated. The preparation of the TSPEs is described in the Supporting Information. In the following, the TSPEs are labeled using their PEO:LiTFSI:IL molar stoichiometry (the number for PEO corresponds to the number of repeating ‐(CH2)2O‐ units) and the IL used is indicated as subscript 7TFSI and 1,4 for Py1,4TFSI). The prefix (cl‐) is used for TSPEs that have been cross‐linked by UV irradiation. Cross‐linking is typically introduced used to improve the mechanical properties of “dry” and plasticized polymer electrolytes, preventing creeping under pressure, while also hindering membrane crystallization.Tg steps). In fact, only the membrane with the highest IL content (cl‐20:2:4) shows a transition in the ionic conductivity curves  below the melting transition). Cross‐linking allows increasing the IL content in the TSPEs to lift the ionic conductivities even higher . Even though higher ionic conductivities can be reached for the cl‐20:2:4O7 membrane and IL phase separation might be favorable to improve the performance and wettingThe effect of PyrThe mobility of each ionic species based on the self‐diffusion coefficients derived from PFG‐NMR experiments Figure .1,4TFSI to Pyr1,(2O)7TFSI in 20:2:1 TSPEs results in a moderate relative increase of TFSI− mobility, with a significantly higher relative increase of lithium diffusion coefficient in (i.e. a 98 % increase from 0.292×10−12 to 0.578×10−12 m2 s−1), whereas the ion mobility of the larger pyrrolidinium cation is lower. This shows that the interaction of Li+ with the immobile PEO matrix is lowered and suggests that in TSPEs, IL cation‐Li+ complexes are formed assisting lithium‐ion transport. Cross‐linking does not affect considerably the results (decrease of less than 7 % for cl‐20:2:1O7 vs. the linear TSPE). Increasing the IL content to 20:2:2 yields a further increase of diffusion coefficients, in accordance with ionic conductivity. The Li+ transference numbers were estimated by using either the self‐diffusion coefficients or the “Bruce and Vincent” electrochemical technique1,4TFSI with Pyr1,(2O)7TFSI in 20:2:1 TSPE, a doubling of tLi+ can be reached at 40 °C (i.e. from 0.05±0.01 to 0.10±0.01) and even higher for the cross‐linked TSPEs. The increase of tLi+ is likely due to the enhanced solvation by the Pyr1,(2O)7+ cation, affording a similar tLi+ compared to the 20:2:0 “dry” SPE. Increasing the IL content in the 20:2:2 and cl‐20:2:2 membranes keeps tLi+ on a similar level to 20:2:1 but boosts the Li+ ion conductivity even higher because of the higher ionic conductivity.Switching from Pyr+ and pyrrolidinium ions. In particular, we simulated TSPEs with oligo(ethylene oxide)‐based ILs with side chain lengths of one, four, and eight ethylene oxide monomers at a molar ratio of 20:2:1.To unravel the molecular lithium‐ion transport mechanism, MD simulations were performed. We extended our previous studyO1, 20:2:1O4, and 20:2:1O8, respectively  side chain length of the IL cation, that is, the Li+ ion motion along the backbone becomes more efficient as the Li+ ions become progressively coordinated to the IL cations (see discussion of pIL), rendering the ions that remain coordinated to PEO more mobile. Interestingly, the segmental dynamics expressed by the Rouse time τRτ2 (bound monomers) are approximately constant for all side chain lengths. In this context, we demonstrated that ILs act as plasticizers in PEO membranes, which increases the dynamics of the lithium ions attached to the polymer chains as the polymer motion itself is enhanced by the plasticizer.[τ2 values from Table τ2. We observe that τ3 decreases by almost a factor of two when going from 20:2:1O1 to 20:2:1O8, illustrating that, for sufficiently long oligo(ethylene oxide) chains at the IL cation, Li+ is structurally and dynamically decoupled from the PEO chains, resulting in a shorter polymer residence time. This change is accompanied by a concomitant increase of the average time τIL during which Li+ ion is only coordinated by IL ions (either cations or anions) from about 1 to 27 ns when comparing 20:2:1O1 to 20:2:1O8 (Table From Table DLi in the limit of long chains (Table O1) to 20:2:1O4 or 20:2:1O8, DLi increases by factors of 1.7 and 3.7, respectively. Although the absolute DLi values in Table DLi observed in Figure As in our previous work,s Table O to 20:2:σ can then be derived from equilibrium simulations via the expression[So far, we focused on the self‐diffusion of the lithium ions. However, for quantities such as the ionic conductivity or the transference number , the cooperative motion of distinct ions—as expressed by dynamical ion correlations—is important as well.30, 31M is the total number of ion pairs in the simulation box with volume V, e is the elementary charge, Δt the observation or lag time, kB T the thermal energy, iz and jz the (integer) charge numbers and Δri and Δrj the displacement vectors of ions i and j. The terms with i=j correspond to the self‐diffusion contribution, whereas the dynamical ion correlations are expressed by the terms with i≠j. A typical example for the impact of these correlations includes the cooperative motion of cation‐anion pairs, which decreases σ because the product iz jz is negative and the dot product ⟨ΔriΔrj⟩ positive due to the cooperative motion of the ions. In the present case, however, one would expect that the IL cations carry the lithium ions over larger distances, resembling a shuttling mechanism and thus an increase of the ionic conductivity and the Li+ transference number. Unfortunately, extracting absolute values for ionic conductivities or transference numbers from MD data is challenging for statistical reasons, especially for a polymer host with large intrinsic relaxation times .where + ions, we computed ⟨ΔrLi+(Δt)ΔrPyr(Δt)⟩c for all initially coordinated pairs of lithium ions and IL cations  as a function of Δt . We find that ⟨ΔrLi+ΔrPyr⟩c increases substantially when increasing the oligo(ethylene oxide) side chain length and becomes even comparable to the lithium ion MSD for a length of eight monomers. This suggests that the correlated motions between Li+ ions and IL cations significantly contributes to the overall lithium‐ion conductivity, as observed from the experimentally determined transference numbers of 20:2:11,4 and 20:2:1O7  chains is substantially lower. On larger time scales, the correlation diminishes due to the exchange of Li+/IL cation pairs. This decay is slowest for 20:2:1O8, reflected by an average pair lifetime of about 100 ns, followed by 20:2:1O4 (≈10 ns) and 20:2:1O1 .Figure + ions over larger distances (nanometers), significantly enhancing both the single‐ion transport (DLi) and the cooperative motion between Li+ and IL cations in this way.Our simulations confirm our approach to employ functionalized pyrrolidinium cations with sufficiently long oligo(ethylene oxide) side chains, which can indeed carry LiO7 and cl‐20:2:11,4 were explored in terms of ESW on copper for cathodic stability and on stainless steel and LiNi1.5Mn0.5O4 (LNMO) for anodic stability , which is commonly observed.+) close to the ESW of pure Pyr1,(2O)7TFSI  as well, considering a 0.1 mA cm−2 limit in both cases. Nevertheless, common lithium‐metal polymer batteries (LMPB) composite electrodes, such as LFP, exhibit higher surface area and a vastly different surface reactivity and the TSPEs stability was thus probed using a high voltage spinel LNMO composite electrode as well. It resulted in a similar oxidative stability for both TSPEs of ≈4.6 V (vs. Li|Li+).Electrochemical requirements for any electrolyte, either polymer or liquid, are its ability to facilitate sufficient ion transport at the electrode|electrolyte interface while being subjected to as little electrochemical degradation (anodic/cathodic) as possible. The cross‐linked samples, cl‐20:2:1y Figure . An impoO7). The decrease in voltage over time is likely due to some evolution of SEI resistance during cycling. At a current density of 0.1 mA cm−2, the cell voltage is reduced by ≈35 % for cl‐20:2:1O7 compared to cl‐20:2:11,4 (0.15 vs. 0.23 V) at 40 °C and the effect is even more marked at 60 °C . A closer look at the voltage profile in Figure VIR drop) at the beginning of each step is assigned to the initial resistance, dominated by the SEI, the contact and electrolyte resistance. The second part is an asymptotical voltage increase during each step, which is attributed to the establishment of ionic concentration gradients (ΔVpolarization) that become steeper and steeper until a steady‐state is reached . A significantly lower voltage plateau due to a lower ΔVpolarization can be observed for cl‐20:2:1O7 . These improvements result in no short circuit after 1000 h for cl‐20:2:1O7. In fact, cl‐20:2:1O7 also allows maintaining higher steady‐state currents and, as a result, obtaining more homogenous plating onto Cu . To verify that the prevention of a short circuit does not result from increased mechanical stability, dynamic shear rheometry experiments were performed 7TFSI TSPE or a Pyr1,4TFSI analog. The initial Coulombic efficiency (CE) increased from 91.6 % for cl‐20:2:11,4 to 95.7 % for cl‐20:2:1O7 and from 99.93 % to 99.96 % in the following cycles, which would have a highly beneficial effect on cycle life with a smaller Li metal electrode. Beside the excellent CEs, the specific discharge capacities are higher for cl‐20:2:1O7 . Most importantly, the long‐term capacity retention is considerably improved. After 200 cycles, the cl‐20:2:1O7 cell retains 99.3 % of its initial capacity (referred to the 5th cycle) vs. 67.2 % for cl‐20:2:11,4). The reasons for this can be attributed to the slower Li+ ion transport in cl‐20.2:11,4. Although LFP is known for a very flat voltage plateau at 3.4 V (vs. Li|Li+),1,4 cells is more sloped than that of the cl‐20:2:1O7 cell  for cl‐20:2:11,4 compared to 99.8 % with cl‐20:2:1O7. Even at 1 C, a capacity retention of 96.8 % is reached for cl‐20:2:1O7. At 2 C, the differences in capacity retention are even stronger with 50.0 % for cl‐20:2:11,4 vs. 93.5 % for cl‐20:2:1O7. This clearly illustrates the improved rate performance induced by Pyr1,(2O)7TFSI. In both cells, the initial specific discharge capacity at 0.1 C can be reached after the rate test, showing that at constant charge rate, the cells  were not overly affected by the discharge (i.e. lithium electrodissolution). It shows, however, that the much faster lithium transport results in much higher rate performance capability of the LMPB cells, regardless of the degradation of the lithium‐metal interface.Finally, the effect of the improved Lil Figure , which rs Figure . The cap1,(2O)7TFSI was specifically designed with an oligo(ethylene oxide) side chain on its pyrrolidinium cation to overcome the limitations of N‐alkyl‐N‐alkyl pyrrolidinium‐based ILs in terms of tLi+ when used as plasticizers for PEO‐based TSPEs. This IL is not only highly promising for formulating super‐concentrated binary liquid electrolytes, since it allows reaching a 3:1 7TFSI mol:mol) liquid composition, but it also enables PEO‐based polymer membranes with far higher performance than Pyr1,4TFSI analogues, although having similar physicochemical properties. In particular, the cross‐linked TSPE cl‐20:2:1O7 possesses a similar ionic conductivity as the state‐of‐the‐art cl‐20:2:11,4 electrolyte but exhibits a Li+ ion conductivity three times higher with a tLi+ of 0.10±0.01 at 40 °C . This increase in tLi+ reflects the excellent solvating properties of Pyr1,(2O)7TFSI that enable fast lithium‐ion transport, especially via enabling an additional “vehicular” transport mode since the IL cation is able to solvate one Li+ ion. Our MD simulations confirmed the cooperative motion of Li+ ions and IL cations both via a simplified model for the Li+ ion transport and an explicit analysis of dynamical ion correlations. These insights show that, the use of ionic shuttle molecules opens up new avenues to improve the ion‐transport properties of TSPEs. Practically, thermally stable and elastic membranes allow significantly higher rate performance of LFP∥Li LMPB cells. Long‐term cycling of symmetrical Li∥Li and LFP∥Li cells show that the faster lithium transport results in well‐maintained cell performance with no deterioration over cycling of the lithium transport, contrary to what happens with Pyr1,4TFSI‐based TSPEs in the same conditions. In the latter case, the increasingly slower lithium transport over cycling is attributed to the evolution of the lithium‐metal interface as slow lithium transport favors the formation of HSAL. On the contrary, a 99.3 % capacity retention is reached with cl‐20:2:1O7  after 200 cycles. This demonstrates that faster lithium transport results not only in higher power capability but also in safer and longer living LMPB cells.PyrThe 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.


This anion‐coordination‐induced aggregation effect serves as a powerful sensory mechanism, capable of luminescence chloride sensing at parts per billion concentration. Furthermore, the doubly substituted geometric isomers act as unprecedented photoswitchable XB donor anion receptors, where the composition of the photostationary state can be modulated by the presence of a coordinating halide anion.A series of tetraphenylethene (TPE) derivatives functionalized with highly potent electron‐deficient perfluoroaryl iodo‐triazole halogen bond (XB) donors for anion recognition are reported.  A series of halogen bonding (XB) tetraphenylethene (TPE) based anion receptors are reported. The receptors exhibit a remarkable anion‐induced turn‐on luminescence response, in which halogen bonding anion interactions drive the formation of emissive nanoscale aggregates. Disubstituted XB TPE derivatives function as photoswitchable anion receptors, in which modulation of photostationary states is intimately related to anion affinity. Organic luminescent materials frequently exhibit significant concentration dependent photophysical properties. Traditional chromophores, such as extended planar aromatic systems, are typically strongly fluorescent under dilute conditions, but suffer considerable attenuation in emissive properties with increasing concentration. This phenomenon is known as aggregation‐caused quenching (ACQ),THF, monitoring the binding induced chemical shift perturbations. In the case of 1⋅XB, 2⋅XB and 2⋅XBE, chloride binding induced a downfield shift of the aryl signal directly adjacent to the iodo‐triazole moiety, consistent with XB mediated anion complexation.With the series of mono‐ and di‐functionalised TPE receptors in hand, we examined their chloride anion‐recognition behaviour by 1⋅XB using the Bindfit programme[K1:1(1⋅XB)=2380 M−1, representing the binding affinity of chloride to one iodotriazole XB donor. The integration of additional XB donors to the TPE core enables higher host‐guest complex stoichiometries to be achieved, which in the case of 2⋅XB, 2⋅XBZ and 2⋅XBE corresponds to a 1:2 host‐anion stoichiometry. Depending on the geometric arrangement of the two XB donors, anion complexation occurs with either no cooperativity (binding of the first anion does not affect binding of the second) or negative cooperativity (binding of the first anion inhibits binding of the second). In the former case, the 1:1 and 1:2 host‐guest binding constants, K1:1 and K1:2 respectively, are related by the expression K1:1=4 K1:2, accounting for statistical factors. Analysis of the chloride binding isotherms for the di‐iodotriazole derivatives 2⋅XB, 2⋅XBZ and 2⋅XBE , quantifies the binding of the anion to a single iodotriazole binding site. The 1:1 binding constant for the di‐iodotriazole derivatives 2⋅XB, 2⋅XBE and 2⋅XBZ is given by 2 K1:1(1⋅XB) in the absence of chelate cooperativity and when statistical factors associated with binding of one anion to a ditopic host are considered. Inspection of the values of K1:1 for the di‐iodotriazole derivatives reveals that, in the case of 2⋅XB and 2⋅XBE, the two iodotriazole donors are too far apart to allow for chelation of the anion in a bidentate fashion: K1:1(2⋅XB) and K1:1(2⋅XBE)≈2 K1:1(1⋅XB). In contrast, in Z2⋅XB the two iodotriazole derivatives are closer in space, which allows for effective bidentate cooperative binding of the first chloride anion, such that K1:1(Z2⋅XB)≈10K1:1(1⋅XB). The pronounced negative cooperativity in binding of the second equivalent of anion to 2⋅XBZ, reflected in the interaction parameter α=0.17, presumably therefore arises due to the combination of mutual anion‐anion electrostatic repulsion and the requirement to break the bidentate XB‐chloride interactions upon formation of the ternary complex.Analysis of the 1:1 stoichiometric binding constants for the three di‐iodotriazole geometric isomers 1H NMR anion titration experiments of 2⋅XBZ with the heavier halides revealed that bromide and iodide produced similar binding isotherms with somewhat attenuated association constants, consistent with anion basicity trends .Attention then turned towards the synthesis of the tetra‐substituted receptors 1H NMR chloride titration experiments were conducted on 4⋅XB, Ph4⋅XB and 4⋅HB, to elucidate the role and potency of XB interactions on the anion‐recognition process. Chloride binding titration experiments with 4⋅XB revealed a chemical shift perturbation profile with an inflection point similar to that observed with 2⋅XBZ, in which up to two equivalents of TBACl induced an almost linear upfield perturbation of the aryl proton a, indicative of near quantitative formation of a 1:2 receptor:anion complex. Addition of further equivalents caused a progressive downfield shift, indicating the formation of higher receptor:anion stoichiometries, presumably up to 1:4 . Furthermore, the preference for the lighter halides observed for 2⋅XBZ appears to also translate to 4⋅XB, upon visual analysis of the bromide and iodide binding isotherms  persistently yielded the NaCl complex of 4⋅XB  solution of 4⋅XB in the presence of excess NaI. Single‐crystal X‐ray analysis revealed the NaI⋅4⋅XB complex displays an analogous four‐fold anion coordination via C−I⋅⋅⋅I− XB interactions in an approximately square planar geometry around the iodide, however the sodium cation in this case resides in a vacancy between the central aromatic units constituting the tetraphenylethene core . This is presumably due to the reduced electrostatic interaction between the larger iodide anion and the sodium cation. The structural similarity of the obtained sodium halide complexes reveals the crucial role of potent XB‐anion interactions in the assembly of the aggregates. Encouraged by these findings, attention then turned to investigating whether these anion‐induced aggregates persist in solution phase.It is noteworthy that repeated attempts to crystallize B Figure . The pros Figure . Close es Figure . To esta−5 M) revealed that 4⋅XB is weakly emissive (λex=350 nm). A significant increase in emission intensity with increasing water fractions (fw) was observed at fw>60 %, indicative of the anticipated aggregation‐induced emission behaviour of TPE derivative 4⋅XB , with a three‐fold increase observed after the addition of 10 equivalents . Addition of the non‐coordinating anion hexafluorophosphate did not affect the emission spectrum, further demonstrating that XB‐mediated coordination to chloride is required to mediate aggregation . Dynamic light scattering (DLS) measurements of freshly prepared THF solutions (10−5 M) of 4⋅XB indicate that the free receptor is non‐aggregated in the absence of chloride, but the addition of 1 equivalent of TBACl induces the formation of aggregates on the order of 100 nm in diameter  which, consistent with results from DLS, revealed that the addition of 1 equiv. of TBACl to a 1 mM solution of 4⋅XB in THF led to the formation of ≈100 nm sized particles, while no such species were observed for the free receptor in the absence of chloride .Addition of increasing equivalents of chloride to a THF solution of s Figure , top, acr Figure , bottom.4⋅XB and 2⋅XB, and the formation of aggregates in the presence of 1 equivalent of TBACl in both cases was confirmed by DLS measurements. In contrast, 1⋅XB, 2⋅XBZ and 2⋅XBE exhibited minor perturbations in fluorescence intensity upon the addition of 10 equivalents of TBACl, and DLS analysis revealed no evidence of aggregated species. Together, these results confirm that the observed emission enhancement arises from XB‐mediated chloride induced aggregation. The absence of anion‐coordination‐induced aggregation with 2⋅XBZ, despite the receptor possessing enhanced chloride affinity relative to all other mono‐ and di‐substituted TPE derivatives, demonstrates that spatial orientation of the XB donors is crucial in the formation of the chloride induced XB aggregates. Under analogous conditions Ph4⋅XB and the hydrogen bonding analogue 4⋅HB also demonstrated no significant emission intensity change, providing further strong evidence for the requirement for multiple potent XB‐anion interactions to drive aggregate formation.To further explore the role of spatial orientation and potency of the halogen bond donors in the formation of the anion‐mediated aggregates, the relative fluorescence response for all receptors was recorded in the presence of 10 equivalents of TBACl, and the data summarised in Figure 4⋅XB in THF was measured in the presence of 10 equivalents of a range of anions  of 5.14 μΜ (183 ppb), constituting one of the most sensitive supramolecular anion sensors to‐date .[Analogous experiments were also undertaken to investigate how the nature of the anionic guest influences aggregate formation, in which the emission intensity of s Figure . In all 2⋅XBE and 2⋅XBZ are prime candidates for developing photo‐switchable anion receptors due to the contrast in chloride binding affinity of the two geometric isomers , and fortuitously, could be isolated by column chromatography.In addition to the highly desirable luminescent properties of TPE, unsymmetrical doubly substituted TPE derivatives have the capability to function as molecular photo‐switches in which photoisomerisation switches between different spatial arrangements of functional groups. Photo‐switchable anion receptors in particular have attracted interest for applications including ion extraction and transport, enabling control over when and where the hosts bind or release their targets.2⋅XBE with 405 nm light triggered photo‐isomerisation, generating an approximately equal ratio of both isomers in the photo‐stationary state (52:48 2⋅XBZ:2⋅XBE), as determined by integration of the NMR signals of the mixture. The effect of 405 nm irradiation on the UV vis spectrum of a solution of 2⋅XBE is shown in Figure 2⋅XBZ or 2⋅XBE was observed under ambient conditions in THF solution over the course of the experiment when excluded from light. Analogous irradiation experiments starting with 2⋅XBZ generated a similar, but reversed set of perturbations in the UV‐vis profile, namely an increase in absorption at 335 nm and a hypsochromic shift in the local λmax absorption at approximately 265 nm.Irradiation of a solution of a pure sample of Z isomer by coordination to an anion. To this end, analogous photoisomerisation experiments were conducted by irradiating the sample with 405 nm light in the presence of 10 equivalents of TBACl. In the presence of the halide, the PSS is biased in favour of the Z isomer, due to strong XB coordination to the chloride (68:32 2⋅XBZ:2⋅XBE)  Figure . Similar1H NMR anion titration experiments elucidate the varied binding modes and stoichiometries of the family of halogen bonding TPE derivatives. Fluorescence anion titration experiments demonstrate that the tetra‐substituted perfluorinated 4⋅XB receptor, experiences a dramatic increase in emission intensity upon the addition of chloride, whilst the non‐fluorinated Ph4⋅XB or hydrogen bonding 4⋅HB analogues elicit no photophysical response. Detailed analysis of the fluorescence spectra, dynamic light scattering measurements, TEM imaging and X‐ray crystal structure determination demonstrate that the origin of this sensory response is an unprecedented XB‐anion coordination‐induced aggregation mechanism. Furthermore, successful isolation of difunctionalised geometric isomers 2⋅XBZ and 2⋅XBE facilitated the investigation of these TPE derivatives as photoswitchable anion receptors, in which it was demonstrated that the position of the photostationary state could be significantly biased towards 2⋅XBZ on the basis of enhanced halide anion affinity. These results not only provide further evidence for the advantages of XB interactions in the design of potent anion receptors, but also point to new opportunities in the development of XB anion‐responsive materials, switches and sensors.In conclusion, we report a series of halogen bonding TPE‐based anion receptors, in which the multivalency and relative orientation of the strongly potent perfluoroaryl XB donors is systematically investigated. 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.


The mechanisms of concerted transfer enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules at particular redox states. Here, neutron diffraction of two redox-controlled manganese superoxide dismutase crystals reveal the all-atom structures of Mn3+ and Mn2+ enzyme forms. The structures deliver direct data on protonation changes between oxidation states of the metal. Observations include glutamine deprotonation, the involvement of tyrosine and histidine with altered pKas, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond. We report a concerted proton and electron transfer mechanism for human manganese superoxide dismutase from the direct visualization of active site protons in Mn3+ and Mn2+ redox states.Human manganese superoxide dismutase is a critical oxidoreductase found in the mitochondrial matrix. Concerted proton and electron transfers are used by the enzyme to rid the mitochondria of O 3+SOD and Mn2+SOD, revealing changes in the protonation states of key residues in the enzyme active site during the redox cycle.Human manganese superoxide dismutase (MnSOD) is an oxidoreductase that uses concerted proton and electron transfers to reduce the levels of superoxide radicals in mitochondria, but mechanistic insights into this process are limited. Here, the authors report neutron crystal structures of Mn Concerted proton and electron transfer (CPET) permits a thermodynamically favorable redox reaction, is efficient, and is an integral part of enzymes with the fastest catalytic rates4. Particularly noteworthy is the prominence of CPET enzymes that regulate the concentration of reactive oxygen species (ROS) in the cell. ROS levels are central to programmed cell death and abnormal regulation by these oxidoreductases play significant roles in cancer and cardiovascular diseases5. CPETs are therefore of significant interest to study but a mechanistic understanding of these enzymes is still lacking. Deciphering these fundamental biochemical reactions is not only significant for its role in diseases, but for the biomedical design of CPET-dependent therapeutic interventions, irradiation protectants, and electrochemical biosensors7.Oxidoreductases are a large class of enzymes that use unpaired electrons to facilitate redox reactions with other chemical species and are involved in nearly all aspects of life. Oxidoreductase electron transfers are almost always coupled with a proton transfer2•− with the unpaired electrons of the active site metal. The Mn ion is coordinated to inner-sphere residues His26, His74, His163, Asp159, and a single-oxygen species that could be either H2O or –OH 8 and the resulting divalent Mn reduces another O2•− molecule to H2O2 (k2 = 1.1 nM−1 s−1)8. This is the only means the mitochondrial matrix has to keep O2•− levels low enough to avoid damage to macromolecules and destruction of cellular function9.Human manganese superoxide dismutase (MnSOD) is a CPET-based oxidoreductase found in the mitochondrial matrix that reduces ROS levels by eliminating O2•− is from electrons inadvertently leaking from the electron transport chain. Dysfunctional MnSOD activity, therefore, poses significant consequences on the mitochondria that contributes to several diseases. Genetic aberrations of MnSOD are associated with several cancer types, with mammary and prostate cancers being the most frequently noted in curated databases10. Polymorphisms of MnSOD have also been noted to be a predictor for deficient vascular function11. Therefore, the ability for MnSOD to utilize the high reaction rate and efficiency (kcat/Km > ~109 M−1 s−1) of its CPET mechanism is correlated with the preservation of health8.The major endogenous source of OThe CPET mechanism of MnSOD and the majority of other oxidoreductases has yet to be defined at the atomic level. The limitation in studying CPETs is the difficulty in directly detecting the protonation states of ionizable residues, solvent/ligands at the active site, and correlating them with the electronic state of the active site metal. The second sphere of MnSOD harbors five residues  calculations have been applied to X-ray structures for insight into the possible proton transfers. They have suggested protonation of WAT1 from −OH to H2O during the Mn3+ → Mn2+ redox reaction and deprotonation of WAT1 and Tyr34 for H2O2 formation during the Mn2+ → Mn3+ reaction16. These theoretical methods were the only way to study the simultaneous change of electron state and proton locations in the enzyme.Current mechanistic knowledge has relied on X-ray crystal structures and kinetic studies of point mutants to infer the role of second-sphere residues. Mutations of Gln143, Tyr34, or His30 do not significantly alter the active site structure although they are detrimental to the kinetic rates of the enzyme. The Gln143Asn point mutation leads to near ablation of catalysis, Tyr34Phe depletes activity only for the Mn17. The capability of proton visualization and amenability to enzyme redox control that NPC provides creates an avenue to investigate the CPET mechanism of MnSOD experimentally.Neutron protein crystallography (NPC) is an emerging tool for analyzing hydrogen positions of biological macromolecules and possesses attributes that are useful in deciphering CPET mechanisms. In NPC, scattering of deuterium is on par with carbon, nitrogen, and oxygen, increasing the ability to locate proton positions for the entire enzyme. An additional advantage is that neutrons, unlike X-rays, do not alter the electronic state of metals3+ and Mn2+ states and reveal how the atomic locations of all protons in the enzyme active site change when the active site metal goes through a redox cycle. Direct experimental evidence is provided for deprotonation of a conserved glutamine residue and the feasibility of the proton transfer is explored with quantum calculations. Four other conserved residues are discerned to each have short-strong hydrogen bonds (SSHBs) and unexpected attributes that include an ionized tyrosine and a low-barrier hydrogen bond (LBHB) between a separate tyrosine and histidine across the dimer interface. A proposed CPET mechanism is reported from the culmination of data between Mn3+SOD and Mn2+SOD neutron structures.Here, we present room temperature neutron structures of human MnSOD at physiological pH in Mn3+SOD and Mn2+SOD to resolutions of 2.20 and 2.30 Å, respectively. The two data sets collected were from the same crystal treated with appropriate oxidizing and reducing chemicals and the redox conditions were maintained during each data collection18. Neutron data at these resolutions are excellent and permitted ease in the visualization of deuterium atoms. The proton positions of the resting state, apo active sites were specifically sought to define the effects of metal redox state on the surrounding protons independent of substrate binding. We initially sought a crystallographic check for the success of the redox manipulations. WAT1, the Mn ligand, has historically been thought to obtain a proton (−OH → HOH) upon one-electron reduction of Mn3+ to Mn2+. This was inferred from crystallographic bond distances and QM calculations but otherwise had not yet been directly observed and confirmed25. Indeed, careful inspection of the neutron scattering length density between neutron data sets revealed changes in the protonation of WAT1 when the redox state of the Mn ion was altered  molecule and is supported by the Mn–O(WAT1) bond distance of 1.8 Å  atom is acting as a hydrogen bond acceptor from Dε21(Gln143) with a distance of 1.8 Å  bond distance of 2.2 Å26. The D1(WAT1) atom position is similar to that found in the Mn3+SOD counterpart and hydrogen bonds with Oε2 of Asp159, albeit with a longer distance of 2.5 Å  points toward Gln143 suggesting the WAT1 of Mn2+SOD is acting as a hydrogen bond donor to Gln143. This means Gln143 is a hydrogen bond acceptor in Mn2+SOD and its Dε21 atom is absent. Indeed, there is a lack of neutron scattering length density for Dε21 but not for Dε22. This interpretation was supported when attempts to model Dε21 led to negative |Fo| − |Fc| neutron scattering length density. In the Mn2+SOD structure, the hydrogen bond between D2(WAT1) and Nε2(Gln143) is atypical with a bond distance of only 1.6 Å and O(WAT1)– D2(WAT1)–Nε2(Gln143) angle close to 180°. These are characteristics of an SSHB, a type of hydrogen bond that is thought to stabilize particular enzymatic steps and enhance catalytic rates29. This is not to be confused with an LBHB, a type of SSHB where a proton is equidistant between heteroatoms30. SSHBs are noteworthy in several well-studied enzymes, such as α-chymotrypsin that utilizes an SSHB between the His and Asp of its catalytic triad28. This creates a ~7 kcal mol−1 stronger interaction to substantially increase the kinetic rate. For Mn2+SOD, the SSHB between WAT1 and Gln143 may contribute to the stability of the redox state and the high catalytic efficiency of the enzyme.For Mn8 Å Fig. . The dat5 Å Fig. . SurprisKa of primary amides are 16–18. However, pKa studies of less acidic secondary amides suggest pKa values may be depressed to 7–8 depending on how the amide group is polarized 31. This is supported by the known event of proton exchanges occurring at the amide groups of protein backbones. Moreover, several enzyme studies suggest glutamine or asparagine-mediated proton transfers for catalysis and support the plausibility of a Gln143 → WAT1 proton transfer. For example, an asparagine residue has been suggested to be deprotonated in prenyltransferases due to significant polarization from close proximity to a metal cation32. Nakamura and coworkers33 showed neutron data of cellulase Cel45A from Phanerochaete chrysosporium that revealed asparagine deprotonation that is instrumental for the proton relay of the enzyme. Infrared spectroscopy and computational calculations support the involvement of glutamine-mediated proton transfers in GTP hydrolysis by Ras-GAP and photoexcitation of photoreceptor proteins with the flavin-binding BLUF domain34. For MnSOD, the deprotonation of Gln143 for CPET to the active site ligand has not been observed before although it does explain the high efficiency of the enzyme as a result of this internal proton source.The experimental data, therefore, suggest that Gln143 is undergoing deprotonation to form an amide anion and this is unusual because glutamine residues are not expected to act as weak acids since the p37) was used to evaluate the interactions of Gln143 and WAT1 (ref. 38). Reduction of Mn3+ to Mn2+ increases the electronegative character of the O(WAT1) lone pair facing the proximal amide proton of Gln143  increases its basicity and chemically allows the abstraction of the amide proton from glutamine. In terms of hard and soft Lewis acid and base (HSAB) theory, the transition of the metal acid and solvent base from (Mn3+––OH) to (Mn2+–OH2) corresponds to both the acid and base becoming softer. The bonds of the deprotonated amide rearrange to stabilize its new negatively charged state . The Oε1 atom of Gln143 bears the most electronegative charge, and calculations suggest less covalent electrons for the Oε1–Cε1 bond compared to that of Nε2–Cε1, with bond orders of 1.33 and 1.52, respectively  and is a 1.4 kcal mol−1 stabilizing hyperconjugation interaction  calculations of the active site using the neutron structures support our interpretation of the neutron scattering length density for deprotonation of Gln143. Calculations used the atoms of the residues shown in Fig. ate Fig. . The Oε1ton Fig. . Donor–aε1 of Gln143 and Dε1 of Trp123 in the reduced state  harbors a stronger electronegative character during the Mn2+ redox state. This may be a consequence of Gln143 deprotonation to the amide anion during the Mn3+ → Mn2+ redox reaction and negative charge stabilization of its Oε1 atom through hydrogen bonding with Trp123. Charge stabilization is likely to be important for glutamine deprotonation as amide groups are known to deprotonate at neutral pH when electronegatively polarized at the carbonyl O atom31. Trp123 is especially competent at charge stabilization due to its own ability to polarize. CLPOs suggest that lone pair electrons of Nε1(Trp123) delocalize into the highly conjugated aromatic ring of Trp123 when the glutamine amide is deprotonated and is stabilizing  lone pair electron density into the Cε2–Cδ2 π*-antibonding orbital  and Hε1(Trp123). Quantum calculations indicate an important role for Trp123 in the deprotonation of Gln143.A previously undetected change in the interaction of Gln143 with the neighboring Trp123 was observed. An SSHB of 1.5 Å is seen between the Oate Fig. . The sam9 Å Fig. . The neuing Fig. . Donor–atal Fig.  and decrε21(Gln143)–Nε2(Gln143) interaction is needed for redox cycling of Mn, mutation of Gln143 or a nearby residue that may stabilize amide deprotonation should affect catalysis. In the literature, the Gln143Asn mutant has nearly ablated catalysis in both redox states while Trp123Phe can perform catalysis for Mn3+ → Mn2+ at deficient lower rates (20–50%) but not at all for the Mn2+ → Mn3+ transition39. The effect of these mutations suggests that Gln143 is central to catalytic activity while Trp123 is most significant for the Mn2+ → Mn3+ half of the redox cycle. The detrimental effects for the Mn2+ state due to mutating residue Trp123 may therefore reflect their role in stabilizing the Gln143 amide anion. Indeed, the kinetic behaviors of these mutants were puzzling in past studies but amide proton transfer potentially explains them41. Glutamine at the position of Gln143 is conserved in all isoforms of MnSODs and prokaryotic FeSODs. A closer WAT1-Gln distance correlates with increased redox potentials and catalytic rates42. This is perhaps because of an enhanced ability for proton transfers between O(WAT1) and Nε2(Gln143). Past mutagenesis studies, differences in catalytic rates among isoforms, and the high catalytic rate of MnSOD may be explained by Gln143 serving as an internal proton source for CPET via amide deprotonation.If an O(WAT1)–D12. For Mn3+SOD, Tyr34 does not have a nuclear peak for its hydroxyl proton  and O(WAT2)  atom participates in a strong 1.6 Å hydrogen bond with Dε22(Gln143) in the Mn2+SOD structure and is significantly different than the corresponding hydrogen bond distance of 2.3 Å in Mn3+SOD  Fig. . While tnel Fig. . IntriguSOD Fig. . This SS39. Tyr34 has been speculated to be the proton donor to WAT1 for CPET during the Mn3+ → Mn2+ reaction though this conflicts with the pH independence of the reaction between values of 6 and 10 (refs. 43). This was puzzling because CPET mechanisms are expected to have pH dependence as a result of the proton transfer part of their catalysis and Tyr34 is the closest titratable residue. Instead, the MnSOD neutron data suggest that the proton donor to WAT1 is internally sourced from Gln143 without the direct involvement of solvent and cannot be Tyr34 due to its observed deprotonation in the Mn3+ state.Our experimental data for Tyr34 potentially shines a light on the unexplained observations of previous studies investigating its role in catalysisa of 7.10 ± 0.10 at the active site44. The catalytic role of an ionizable Tyr34 for MnSOD is prominent during the Mn2+ → Mn3+ redox cycle and is supported by the inability of the Tyr34Phe mutant to catalyze this step of the reaction45. Since Tyr34 gains a proton during the Mn3+ → Mn2+ redox cycle and loses a proton during the Mn2+ → Mn3+ cycle, it is conceivable that Tyr34 serves as the source for one of the two protons involved in the protonation of the substrate to H2O2. Indeed, spectroscopic data of the Tyr34Phe mutant suggest a prolonged binding of a species to the metal but could also be a result of the inability of Gln143 to deprotonate without stabilization from the Tyr34 hydroxyl group12. Our crystallographic neutron data have shed light on the perplexing role of the conserved Tyr34 residue.An ionized tyrosine residue at physiological pH is unusual though is potentially explained by polarization from the pronounced positive charge of the metal. Such an effect has been visualized in studies of human carbonic anhydrase II (HCA II), a metalloenzyme with diffusion-limited catalytic efficiencies like MnSOD. For HCA II, joint neutron crystallography and NMR demonstrate a tyrosine residue with a pK–OH, –F, or –N3) to Mn2+SOD coinciding with lengthening or displacement of the Mn–Oδ2(Asp159) bond essentially converting it to a 5-coordinate active site spectrophotometrically46. The chemical purpose of this modification in coordination has been unclear. A sixth-coordinate –OH-bound complex has been difficult to visually confirm due to poor scattering of hydrogen by X-rays47. Serendipitously, one of the active sites of the Mn2+SOD neutron structure has density for an unexpected sixth-coordinate –OD ligand  bond is stretched from 1.95 to 2.44 Å. Of note, the buffer system did not include –OD, and only appropriate ratios of K2DPO4 and KD2PO4 were used to achieve a pD of 7.8. Yet the observed nuclear scattering length density for deuterium and the 1.84 Å Mn–O distance of OL supports the identification of OL as –OD20. OL is well ordered with a B-factor that is comparable to other atoms of the active site (Supplementary Table 2+SOD with sixth-coordinate –OH(OL), and suggests stability of the six-coordinate complex while replacing OL with H2O causes disassociation into five-coordinate Mn2+SOD. The observed differences between the Mn2+SOD active site  as proposed by the associative displacement ligand binding model46.Previous spectroscopic studies have suggested the sixth-coordinate binding of a ligand -O(WAT1) 1.4 Å SSHB  SSHB in apo Mn2+SOD  as observed at the other subunit  to initiate covalent bonding with the proximal Dε21(Gln143) proton.Maliekal and coworkerston Fig. . Since TSHB Fig. . This diSOD Fig.  and suggSOD Fig. . Trp123 nit Fig.  and is tnit Fig. . These sδ1 atom is solvent accessible and is hydrogen bonding to WAT2 while the Nε2 atom is not accessible to solvent and hydrogen bonds with the buried Tyr166 from the adjacent subunit  is solvent inaccessible along with Tyr166, the LBHB may be supported by residing within an enclosed environment.His30 is positioned at the active site channel Fig.  and alsonit Fig. . For Mn2Nδ1 Fig. . Interes3+SOD, the D atom between His30 and Tyr166 refines with the average position on Tyr166, and its hydrogen bond distance with Nε2(His30) is of typical length  and may reflect the movement of the proton , a stark contrast with Chain A  rather than a hydrogen bond acceptor, which would agree with the notion that Nδ1(His30) is deprotonated. With Nδ1 and Nε2 of His30 both deprotonated, the imidazole group puzzlingly appears absent of protons which is chemically infeasible. Differences in B values between the subunits of solvent-exposed residues could degrade the quality of the density but this does not explain the absence of proton density at Nδ1(His30) for Chain B. In fact, these residues in Chain B have lower refined B values than in Chain A (Supplementary Table ε2(His30) and this is the most plausible explanation. The chemical function may be to modulate protonation at the solvent accessible Nδ1 of His30. Protonation of Nε2(His30) would coincide with deprotonation of Nδ1(His30) leading to shifting between singly Nδ1- or Nε2-protonated tautomers  that utilizes CPETs55. The tyrosine–histidine pair of (PSII) appears to have an SSHB that needs to be maintained for catalysis with measured pKa values ranging between 7.3 and 8.0. For MnSOD, the observed atypical hydrogen bonding observed across the dimer interface between Tyr166 and His30 is probably important in the enzymatic mechanism.Similarly, unusual hydrogen bonding between residues has been observed in other enzymes. The most notable are histidines found in catalytic triads of proteases, where changes in protonation of one nitrogen could be tied to the protonation state of the other with the help of an SSHB interactionε2(His30) and Oη(Tyr166) that may coincide with changes in protonation at Nδ1(His30). His30Gln is the only His30 mutant that has been studied that maintains the hydrogen bonding at the active site and does not significantly affect the positions of other residues at the active site50. Kinetically, the His30Gln rate for k1 (Mn3+ → Mn2+) is 38% of the wild type while k2 (Mn2+ → Mn3+) is 72%56. The rates indicate an important role for His30 k1 that may correspond with our observations of its changes in protonation in Mn3+SOD. It should be noted that previous studies refrain from attributing differential protonations to His30 due to the similar redox potentials between wild type (393 ± 29 mV) and His30Gln (380 ± 30 mV)57. However, the investigations do not consider whether compensatory protonations or deprotonations occur at nearby residues as a result of the mutation giving the appearance of an inconsequential effect. Indeed, the Tyr34Phe mutant also has an insignificant change of redox potential (435 ± 30 mV), but has a nearly identical rate for k1 (37% of wild type) as His30Gln56. Drawing an inference from the Tyr166Phe mutant is difficult because hydrogen bonding and side-chain conformations are significantly changed at the active site, but this mutant has nearly identical measurements of redox potential (436 ± 10 mV) compared to Tyr34Phe, indicating a synonymous effect to the charge of the active site56. The neutron data for His30 and Tyr166 help explain past observations of MnSOD mutants that were perplexing and ties together changes of protonation state with kinetic and redox potential measurements.Previous mutagenesis investigations suggest that the Tyr166 and His30 interaction is needed for catalysis and support the interpretation of proton transfers occurring between N3+SOD and Mn2+SOD have revealed active site states that have unique configurations of protonations and hydrogen bonds. Two of the configurations are the enzymatic resting states for Mn3+ and Mn2+ characterized by a five-coordinate Mn46. For the oxidized resting state that is described by both chains of the Mn3+SOD neutron structure . Nδ1(His30) is also observed to be both deprotonated or protonated  with a LBHB, His30 is protonated on Nδ1, and Tyr34 is no longer ionized. Gln143 and WAT1 have undergone a proton transfer where WAT1 is now H2O and Gln143 is an amide anion. Gln143 forms SSHBs with WAT1 and Trp123 presumably to stabilize its ionized form. The third active site state is a six-coordinate Mn2+ with –OH-bound opposite Asp159  and also Trp123. In total, five sites undergo changes in protonation state, Tyr166, His30, Tyr34, Gln143, and WAT1 that occur with four instances of SSHBs between pairs Tyr166-His30 (a LBHB), WAT2-Tyr34, Gln143-WAT1, and Gln143-Trp123.The neutron structures of Mnure Fig. , the proted Fig. . For simted Fig. . An SSHBSOD Fig. , Tyr166 159 Fig.  describeKas that are likely a consequence of a polarized environment provided by the metal and limited solvent accessibility. As a result of obtaining neutron structures for both Mn3+ and Mn2+ states, we built a suggested mechanism that details the changes of protonation states as a result of the Mn ion gaining or losing an electron. For the mechanism, the superoxide substrate is not present but is represented strictly as its donation or abstraction of electrons due to the lack of experimental evidence for its precise binding site.In total, the present work provides details for some of the critical aspects of the CPET mechanism of human MnSOD. Through neutron diffraction, direct evidence is observed for (1) an internal protonation mechanism via glutamine deprotonation to the Mn-bound solvent molecule (WAT1) supported by quantum calculations; (2) an SSHB of Trp123 with the anionic form of Gln143 that stabilizes the anion; (3) changes in protonation of Tyr34 that interacts intimately with a solvent molecule (WAT2) when ionized, and (4) alternate protonation states and a LBHB for His30 and Tyr166 across the dimer interface. It is evident that the residues of the active site have unusual p3+ resting state  that coincides with Nδ1(His30) acquiring a proton from the nearest solvent molecule  and Tyr166 gaining partial covalent character from the proton of Nε2(His30) to form a LBHB  to form a six-coordinate Mn2+ complex and may be the same solvent molecule that donated a proton to His30  ceasing its proton sharing with Tyr166 to regenerate five-coordinate Mn3+.Starting from the five-coordinate Mnate Fig. , Mn3+ acBHB Fig. . The Mn2s30 Fig. . The depate Fig. . When thuct Fig. . The chaAltogether, the suggested mechanism utilizes two internal proton transfers where the protons move back-and-forth within the active site and two external proton transfers where the protons originate from solvent molecules to ultimately be consumed to form the product. The proton transfer between WAT1 and Gln143 is central to the mechanism as it permits the cyclic nature of catalysis. From this study, we have revealed, to our knowledge, the first direct coupling of electronic states to protonation states for an oxidoreductase. It is evident that the CPET mechanism of MnSOD is not straightforward as is exemplified by the previous elusiveness of the proton source for WAT1. Tyr34 was assumed to be the donor, but our data instead indicate an unusual and unexpected proton transfer from the Gln143 amide. Likewise, Trp123, His30, and Tyr166 were not assumed to be involved in catalysis. As this is just one biologically relevant oxidoreductase in a sea of many, finding the protonation states at the active sites of other oxidoreductases may reveal further unusual mechanisms for CPET.MnSOD was transformed into Escherichia coli BL21(DE3) cells. Transformed cells were grown in D2O minimal media within a bioreactor vessel using D8-glycerol as the carbon source58. Induction was performed with 1 mM isopropyl β-d-thiogalactopyranoside, 8 mM MnCl2, and fed D8-glycerol until an OD600 of 15.0. Expression was performed at 37 °C for optimal Mn ion metal incorporation59. Harvested cell pastes were stored at −80 °C until purification. For protein purification, cells were resuspended in a solution of 5 mM MnCl2 and 5 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.8. Clarified lysate was incubated at 55 °C to precipitate contaminant proteins that were subsequently removed by centrifugation. Next, soluble protein was diluted with an equal volume of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5 yielding a final concentration of 25 mM. Measurement of pH verified a value of 5.5 after dilution. Protein was applied onto a carboxymethyl sepharose fast flow column  and eluted with a 0-500 mM sodium chloride gradient that contained 50 mM MES pH 6.5 before concentration to 23 mg mL−1. Crystallization was performed using a nine-well glass plate and sandwich box setup (Hampton Research) and the reservoir solution consisted of 1.9 M potassium phosphate adjusted to pH 7.8 by varying ratios of KH2PO4 and K2HPO4. The crystallization drop was a mixture of 60 µL concentrated protein solution (within a buffer of 50 mM MES pH 6.5) and 40 µL of the reservoir solution. Crystals grew up to 0.5 mm3 after 6 weeks at 23 °C. Purification and crystallization were performed with hydrogenated reagents.For deuterated protein expression of MnSOD, the pCOLADuet-1 expression vector harboring full-length cDNA of 2PO4 and K2HPO4 at pH 7.4 (adjusted by varying ratios of dibasic and monobasic forms) that is the equivalent pD value of 7.8. The pD value was calculated from pD = pHa (apparent reading from pH meter) + 0.4. A crystal in a quartz capillary was soaked in deuterated solutions of 2.3 M KD2PO4/K2HPO4 at pD 7.8 (measured pH 7.4) containing either 6.4 mM potassium permanganate (KMnO4) to achieve the Mn3+ state or 300 mM sodium dithionite (Na2S2O4) to achieve the Mn2+ state. After drying the crystal from soaking solutions, the crystal was flanked in the capillary by slugs of the deuterated reservoir soaking solutions. Fortuitously, the decomposition products of the redox agents are unable to enter the active site of MnSOD18. Methods for manipulating the Mn metal of MnSOD to either Mn3+ or Mn2+ are described in more detail in previous work18.Before redox manipulation, initial deuterium exchange of crystals was performed by vapor diffusion in capillaries using deuterated solutions of 2.3 M KD3 perdeuterated crystal using the MaNDi instrument61 at the ORNL SNS using all neutrons with wavelengths between 2-4 Å. Data collection of each diffraction pattern was from the crystal held in a stationary position, with successive diffraction patterns being collected after 20° rotations along the Φ axis. A KMnO4-treated perdeuterated crystal of 0.46 mm3 in volume at 296 K was recorded to 2.20 Å resolution for the Mn3+SOD form and subsequently treated with Na2S2O4 to achieve the Mn2+SOD state where 2.30 Å data were collected  were present. Afterwards, each individual exchangeable position outside the active site was inspected for residual |Fo|−|Fc| neutron scattering length density and modeled with D atoms appropriately before more iterations of refinement. Next, the O atoms of solvent molecules were first modeled manually outside the active site and refined to determining whether to model solvent as O, OD, or DOD using residual |Fo| − |Fc| neutron scattering length density. The omit density peaks were also used to discern the appropriate orientation of the solvent molecules. After refinement of the solvent structure outside the active site, non-D atoms of the active site were modeled, including Mn and the O of solvent. Last, D atoms of the active site were modeled and refined manually.Neutron data were integrated with MANTID68. The COSMO solvation model was implemented into the geometry optimizations to model the solution phase and utilized an energy convergence value of 1E−6 atomic units69. The def2-TZVPD basis set was used for the Mn ion whereas the 6–31+G Pople basis set was specifically used for all other atoms due to its use in predicting pKas under the B3LYP functional71. The QM models utilized for DFT calculations encompassed the active site residues that had the O and N atoms of the peptide backbone truncated and the Cα atoms fixed. Additional fixed restraints were placed on aromatic residues found on the periphery of the active site  to mimic the packing found in the native enzyme. The Mn ion used the high-spin quintet and sextet states for trivalent and divalent systems, respectively, per experimental observations72.Computational methods are discussed with further depth in the Supplementary Methods. All QM DFT calculations were performed with the NWChem 6.8 software74. These are bonding and antibonding orbitals with maximum electron density computed through a series of localized basis set transformations. CLPOs are calculated with the same target quantity as Natural Bond Orbital (NBO) methods and yield comparable results75. The electron delocalization stabilization/destabilization energies utilized second-order perturbation theory analysis of the Fock matrix defined by the NBO methods76 but were done in the CLPO basis of JANPA. The energy associated with electron delocalization from lone pair or bonding orbital i to antibonding orbital j is defined in Eq. (1) where qi is the donor orbital occupancy and 76 Values The JANPA software package was used to calculate CLPOs from open-shell DFT geometry optimizationsSupplementary InformationPeer Review File


By adjusting reaction time and cooling rate, a morphology of as‐prepared Li2Se is controlled, resulting in nanoparticles, nanorods, or nanowalls with a dominant (220) plane parallel to the (110) plane of the Li metal substrate. Uniaxial pressing the layers under a pressure of 50 MPa for a cell preparation transforms more compact and denser. Dual compatibility of the Li2Se layers with strong chemical bonds to Li metal and uniform physical contact to a Li6PS5Csulfide electrolyte prevents undesirable side reactions and enables a homogeneous charge transfer at the interface upon cycling. As a result, a full cell coupled with a LiCoO2‐based cathode shows significantly enhanced electrochemical performance and demonstrates the practical use of Li anodes with Li2Se layers for all solid‐state battery applications.Lithium is considered to be the ultimate anode material for high energy‐density rechargeable batteries. Recent emerging technologies of all solid‐state batteries based on sulfide‐based electrolytes raise hope for the practical use of lithium, as it is likely to suppress lithium dendrite growth. However, such devices suffer from undesirable side reactions and a degradation of electrochemical performance. In this work, nanostructured Li 2Se including nanoparticles, nanorods, and nanowalls are epitaxially grown directly on Li metal via chemical vapor deposition for all solid‐state lithium metal batteries. These unique materials have dual compatibility with Li metal and sulfide‐based solid electrolytes and an electrochemical stability, suppressing undesirable side reactions and enabling homogeneous charge distribution as well as long‐term cyclability.Nanostructured Li The replacement of flammable liquid organic electrolytes with solid‐state electrolytes (SSEs) can prevent explosions and fires, which makes ASSBs the most promising candidate for application in electric vehicles. Breakthroughs have been made with the development of sulfide‐based electrolytes such as Li2S–P2S5, Li4‐xGe1‐xPxS4, and Li10GeP2S12 with high Li+ ionic conductivity of ≈10−3 S cm−2 at room temperature. However, sulfide‐based solid electrolytes are not stable when in contact with lithium metal, resulting in the decomposition and the formation of various lithium compounds at voltages below 0 V versus Li/Li+. Lithium alloys have been considered as an alternative to Li metal for ASSB applications. Alloying Li with Al, Ga, In, Sn, or Sb forms a stable interface with sulfide‐based electrolytes and allows for long‐term cycling. Among them, lithium indium (Li/In) is commonly used due to the long plateau at 0.622 V versus Li/Li+. However, the utilization of Li/In alloys decreases both the cell voltage and energy density, and the high cost of indium is not suitable for large‐scale applications.All solid‐state batteries (ASSBs) have recently attracted significant attention due to their enhanced safety compared to conventional LIBs.1 The  First, discontinuous interface contact causes the growth of lithium dendrite. Compared to liquid electrolytes, solid‐state electrolytes normally consist of particles. The physical contact between the SSEs and the Li metal is likely to be a point‐to‐point case. The voids and holes at the interface allow for the dendritic growth of lithium. Second, grain boundary induces lithium dendrite penetration. For most SSEs, polycrystalline are more common. The grain boundaries act as pathways for lithium deposition and propagation in the SSEs. Third, interphase effects on lithium dendrite growth. Most SSEs are thermodynamically unstable against Li metal. The reaction between the SSEs and Li leads to the formation of interphase layers with different properties, leading to an increase in a local current density and a growth of lithium dendrite. In this regard, the introduction of an artificial protective layer could be an effective and straightforward strategy.Another main issue is the growth of lithium dendrite. According to previous reports, there are main scenarios for the growth of lithium dendrite in solid‐state batteries.5 Firs2Se) as an artificial solid electrolyte interface for ASSBs. The direct deposition of selenium on Li metal and the subsequent formation of Li2Se is a facile and effective method to build a low‐resistance interface between the Li and SSE. The Li2Se as a protective layer is thoroughly characterized by various methods, including scanning electron microscopy (SEM), energy dispersive X‐Ray (EDX) spectroscopy, X‐ray diffraction (XRD), and X‐ray photoelectron spectroscopy (XPS) analyses. The advantages of the Li2Se protective layer for a stable ASSB operation are demonstrated by direct microscopic observations and electrochemical evaluations.Here, we first report on lithium selenide (Li 2) selenium is less reactive toward lithium and more controllable, 3) depending on vapor pressure, a small number of atoms and molecules of liquid‐state selenium easily evaporates from the surface in the range of 100–500 ℃ above the melting temperature and below the boiling temperature, and gas‐phase selenium can react with metals, resulting in various metal compounds, and 4) one of metal selenides, Li2Se, has low electrical conductivity (bandgap: ≈2.997 eV) and could have higher ionic conductivity than Li2S (≈10−5 S cm−1), which is suitable for a protective layer.Figure2Se materials with three different structures epitaxially grown on Li metal. First, a Li metal attached onto the copper foil and a selenium power are placed into a quartz tube in a CVD system. During heating at 300 °C under a flow of Ar gas, liquid Li metal reacts with vaporized Se, resulting in the formation of a lithium selenium compound with a stoichiometry of Li2Se. After holding for 1min and quenching, Li2Se nanoparticles are formed on the surface of the Li metal (Li/Li2Se‐NP). In contrast, slow cooling of the specimens leads to the Li2Se nanorods and nanowalls  depending on the holing time for 1min and 10 min, respectively. Due to a poor wettability of a copper substrate toward lithium metal prevents a flow of molten lithium and a morphological change during the CVD process . Top‐view SEM images are displayed in Figure 2Se‐NP sample consists of nanoparticles with a diameter of ≈100 nm without microcracks  appeared at ≈42° and 50°, while Li metal was recrystallized along the (110) plane. In heteroepitaxy, a crystalline layer or film grow on a crystalline substrate of a different material, and newly formed material has one or more well‐defined orientation with respect to the substrate. In this regard, the dominant (220) plane of the Li2Se layer is affected by the (110) plane of the Li metal substrate, which indicates the epitaxial growth of the Li2Se on the Li metal. This is highly possible because Li and Li2Se possess the same cubic crystal structure , and Se atoms are located at the bridge site between two adjacent Li atoms, as shown in a unit cell of Li2Se through <110> projection  plane via strong chemical bonds, and the formation of a Li2S film is thermodynamically favorable. In this respect, the formation of Li2Se on Li metal could also favor in terms of thermodynamic because the Li2Se has the same space group and similar lattice parameters with those of the Li2S . To investigate the chemical composition, XPS spectra were obtained after selenium deposition. Bare Li metal (dashed line) has one main peak at ≈56.1 eV that is originated from Li 1s , Li2Se nanoparticles are formed due to a lack of time for grain growth. In contrast, slow cooling leads to the recrystallization of Li metal along the (110) plane (the close‐packed plane of the bcc structure with minimum energy), and grain growth of Li2Se subsequently occurs along the (220) plane on the (110) surface of Li metal, resulting in the formation of Li2Se nanorods or nanowalls depending on the reaction time.The strategy of introducing lithium selenide is based on several factors; 1) selenium, the immediate neighbor of sulfur, has similar chemical properties and compatibility with sulfide‐based materials,6 2) ss Figure . For thee Figure . For then Figure . Thickney Figure –g. Figurn Figure . Accordis Figure . After sy Figure . An SEM‐6PS5Cl electrolyte prepared by high‐energy ball milling were carried out. SEM images showed a secondary particle size in the range of 5–10 µm . XRD analysis revealed the Li6PS5Cl particles have high crystallinity with no Li‐bearing secondary phases such as Li2S and Li4P2S6. The patterns are well matched with those of previous reports . From EIS data , the ionic conductivity was calculated to be 2 × 10−3 S cm−1 at 25 ℃, which is in the range of 10−4–10−3 S cm−1 from previous reports. For electrochemical test, uniaxial pressure was applied on the electrodes to reduce contact resistance between the components in the cell . SEM observation after pressing shows the Li/Li2Se‐NP sample has smooth and dense surface layer with no discernable pores , and the Li/Li2Se‐NR specimen exhibits a dense surface layer consisting of compressed nanorods . In contrast, the Li/Li2Se‐NW sample became denser due to the pressing and folding of nanowalls, but pores remained between the grains . XRD pattern demonstrates that mechanical pressing does not have an appreciable impact on the crystallinity of Li2Se despite the morphological change .Prior to evaluating electrochemical performance, characterizations of the Lirmation).13 FroFigure−2, a bare Li cell displayed an average overpotential of ≈25 mV, larger than the ≈8, 9, and ≈10 mV values obtained from the Li/Li2Se‐NP, Li/Li2Se‐NR, and Li/Li2Se‐NW cells, respectively . Three cells with the Li2Se protective layers exhibited a gradual augmentation of overpotential as the current densities increased from 0.1 to 0.7 mAh cm−2 while the bare Li cell showed the short circuit under 0.3 mAh cm−2. As a result, the Li metals with the protective layers possess the enhanced cycle and rate performances than those of the pristine Li metal. On the other hand, the larger overpotential of the Li/Li2Se‐NW cell than those of the Li/Li2Se‐NP and Li/Li2Se‐NR is attributed to the thicker layer of the Li2Se protective layer, leading to a longer Li ion pathway. Although there was no a significant difference, the Li/Li2Se‐NR exhibited better Li striping/plating property than that of the Li/Li2Se‐NP. This might be due to the epitaxial growth of the Li2Se nanorods with the dominant (220) plane, which is in accordance with the density functional theory and simulations. Hence, the Li/Li2Se‐NR sample with the best performance was selected for further characterizations and full cell tests.y Figure ,d. In thV Figure , respectV Figure . Under t2 Figure ,f, all t6PS5Cl/Li and Li/Li2Se‐NR/Li6PS5Cl/Li2Se‐NR/Li were measured. Nyquist plots after 1 and 50 cycles were shown in FigureRΩ) from the Li6PS5Cl electrolyte, interfacial resistance (R1) from solid electrolyte interface and/or protective layer, charge transfer resistance (R2) at the Li metal surface, and Warburg impedance (Zw) as the resistance of mass transfer. Calculated values are summarized in Table S2 (Supporting Information). The R1 and R2 of the bare Li cell, as determined from the size of semicircles, were 62 and 91 Ω after 1 cycle and 93 and 124 Ω after 50 cycles, respectively. The overall resistance increased upon subsequent cycling, which may be due to the decomposition of the argyrodite‐type Li6PS5Cl, the formation of lithium compounds with low ionic conductivity such as Li2S, Li3P, LiCl, etc. In contrast, for the cell with Li2Se‐NR, the semicircle corresponding to R1 (81 Ω) was larger than that of R2 (65 Ω), which can be attributed to the dense protective Li2Se layer. However, the Li2Se‐NR shows a much smaller increase in both R1 (90 Ω) and R2 (68 Ω) after 50 cycles, resulting in 127 Ω of total resistance. This is lower than the 163 Ω of total resistance for the bare Li cell. Ex situ SEM on cross‐sections shows the Li cell consists of two layers of the Li metal and the Li6PS5Cl electrolyte , was calculated to be in the range of 0.2–0.3 eV, which indicates they can have lithium ionic conductivity. In addition, the Li2S have been studied as a protective layer for Li‐ion batteries, and they successfully prevent the growth of lithium dendrites during cycling, resulting in enhanced electrochemical performances. Therefore, our experimental data and previous reports demonstrate that lithium chalcogenides such as Li2Se can be a potential candidate for the artificial protective layer.To investigate the internal resistance in the cell, EIS spectra of two symmetric cells, Li/Lie Figure . In contd Figure . The this Figure . In parts Figure , enablins Figure . Accordi2Se as a protective layer, full cells employing three different anodes were tested. Specific charge capacities (delithiation) of 135, 148, and 144 mAh g−1 with coulombic efficiencies of 87, 86 and 84% were obtained for the cells employing bare Li, Li/In alloy, and Li/Li2Se‐NR, respectively, as shown in Figure2Se‐NR anode retained much higher capacity through all rates compared to the full cell employing bare Li anode. The long‐term cyclability at 0.1 C‐rate over 100 cycles is displayed in Figure 2Se‐NR anode achieved such an efficiency within only 5 cycles. In addition, average efficiencies of three samples were 99.5%, 99.9%, and 99.9% in the range of 20–100 cycles. The capacity retentions of the full cells with the Li, the Li/In, and the Li/Li2Se‐NR anodes were 47%, 70%, and 76% after 100 cycles, respectively. In contrast to the continuous capacity fading observed for the Li metal‐based full cell, the full cell employing Li/Li2Se‐NR anode exhibited stabilization at ≈100 mAh g−1 after subsequent cycles, which is almost equivalent to the cyclability of the full cell with Li/In anode. The capacity decrease in the full cell with Li/Li2Se‐NR anode is mainly attributed to degradation of the LiCoO2 cathode, while the capacity drop for the full cell with bare Li metal may be due to a synergetic effect on the cathode and anode side. For a comparison, electrochemical performances of all solid‐state batteries based on the artificial interface layer were summarized in Table S3 (Supporting Information). It should be noted that a capacity retention is in the range of 75–90% over 100 cycles. A reason for the lower value of this work might be a higher voltage range up to 4.3 V versus Li/Li+, which can cause a more electrochemical degradation of the LiCoO2.To explore the practical use of Li2Se was successfully introduced as the protective layer for all solid‐state lithium metal batteries. The Li2Se was formed directly on the Li metal at a low temperature of 300 °C via CVD method. By controlling the reaction time and cooling conditions, as‐prepared Li2Se samples with morphologies consisting of nanoparticles, nanorods, or nanowalls were produced. In symmetric cell tests, the Li/Li2Se‐NR electrode showed the smallest overpotential and more stable cycle performance over 140 cycles than those of the Li and Li/Li2Se‐NW cells. Consequently, the full cell with Li/Li2Se‐NR anode showed enhanced electrochemical performance. The devised strategy of employing an artificial protective layer will be beneficial to the practical use of lithium metal in all solid‐state batteries with sulfide‐based electrolytes.In summary, LiAll experimental details are included in the Supporting Information.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.