Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

Anomalous water diffusion in salt solutions

The dynamics of water exhibits anomalous behavior in the presence of different electrolytes. Recent experiments [Kim JS, Wu Z, Morrow AR, Yethiraj A, Yethiraj A (2012) J Phys Chem B 116(39):12007–12013] have found that the self-diffusion of water can either be enhanced or suppressed around CsI and NaCl, respectively, relative to that of neat water. Here we show that unlike classical empirical potentials, ab initio molecular dynamics simulations successfully reproduce the qualitative trends observed experimentally. These types of phenomena have often been rationalized in terms of the “structure-making” or “structure-breaking” effects of different ions on the solvent, although the microscopic origins of these features have remained elusive. Rather than disrupting the network in a significant manner, the electrolytes studied here cause rather subtle changes in both structural and dynamical properties of water. In particular, we show that water in the ab initio molecular dynamics simulations is characterized by dynamic heterogeneity, which turns out to be critical in reproducing the experimental trends.Despite being one of the most-studied liquids, the properties of water and the nature of its interactions with other physical systems continue to be at the forefront of current research in many fields of science (1–8). One important facet of this vast research is the role of water in the solvation of ions. Thus, understanding the effect that ions have on the structural and dynamical properties of water has been a subject of numerous experimental and theoretical studies (6, 9–23). Besides being a classical textbook problem in physical chemistry, the coupling between solutes such as ions and molecules and the surrounding solvent has deep implications on a plethora of biologically relevant processes (24–26).Over six decades ago, Gurney introduced the notion of “structure makers” and “structure breakers” within the context of how different ions would perturb water’s hydrogen bond (HB) network (27). These ideas have generally been accepted and applied to explain various phenomena observed in electrolyte solutions (6, 28). One such example is the celebrated Hofmeister series, a list of cations and anions empirically discovered by Hofmeister, who found that different ions have varying tendencies to salt-out proteins from solution (6, 26). Although there exist some similarities between this series and various phenomenological measures of structure making and breaking, how exactly the structure of water and the extent of hydrogen bonding should be measured remains an open problem. Experimental studies from the Bakker group (13, 23) probing the rotational mobility of water, for example, have in fact suggested that the presence of ions does not even result in the enhancement or breakdown of the HB network of liquid water.Perhaps more interesting are questions concerning the connection between structural perturbations and the changes that ions induce on the dynamical properties of water. One important measure of this effect that will form the focus of this study is the self-diffusion coefficient of water molecules (D_W) in electrolyte solutions. In particular, NMR experiments have shown that below 3 M salt concentration, D_W for electrolytes like CsI increases as a function of concentration whereas the opposite trend is observed for NaCl (29). Our interest in these experiments is also piqued by the fact that recent molecular dynamics (MD) simulations using both fixed charge and polarizable force fields (FF) of the same systems do not succeed in even qualitatively predicting the experimental trends––D_W decreased with salt concentration for all of the systems studied (29)! We cannot exclude the possibility that an empirical potential can be constructed to reproduce these phenomena. However, our results from this work raise serious concerns about the use of empirical potentials in simulating electrolyte solutions in different applications and hence fail to provide a model that could be used to get a better understanding of the microscopic origins behind the anomalous water diffusion.Herein we revisit this problem using state-of-the-art ab initio molecular dynamics (AIMD) simulations where the electronic degrees of freedom are explicitly treated. Unlike the empirical simulations, we find that the AIMD qualitatively reproduce the trends observed in the experimental D_W. First, our analysis of various dynamical properties, such as residence times, unequivocally shows that there is a characteristic dynamic heterogeneity in the water ensemble that is present in the AIMD but absent in the empirical simulations. Rather than inducing significant perturbations to the dynamical properties, we find that the ions result in subtle but measurable changes in the tails of the dynamical ensemble. Although our analysis of various structural features indicates that there are effects that could be likened to structure making and breaking, the HB network is not disrupted or broken in any significant manner. In similar spirit to some recent work from our group that looked at directional correlations in the HB network relevant for proton and hydroxide diffusion (7), we show that ions such as Na⁺, Cl⁻, Cs⁺, and I⁻ substitute the role of water molecules in the network participating in directed ring structures with similar “network rules” present in neat water. The AIMD and empirical HB network exhibit qualitative differences which provide clues into the origins of the discrepancies previously noted.     

Efficient water oxidation catalyzed by homogeneous cationic cobalt porphyrins with critical roles for the buffer base

A series of cationic cobalt porphyrins was found to catalyze electrochemical water oxidation to O₂ efficiently at room temperature in neutral aqueous solution. Co–5,10,15,20-tetrakis-(1,3-dimethylimidazolium-2-yl)porphyrin, with a highly electron-deficient meso-dimethylimidazolium porphyrin, was the most effective catalyst. The O₂ formation rate was 170 nmol⋅cm⁻²⋅min⁻¹ (k_obs = 1.4 × 10³ s⁻¹) with a Faradaic efficiency near 90%. Mechanistic investigations indicate the generation of a Co^IV-O porphyrin cation radical as the reactive oxidant, which has accumulated two oxidizing equivalents above the Co^III resting state of the catalyst. The buffer base in solution was shown to play several critical roles during the catalysis by facilitating both redox-coupled proton transfer processes leading to the reactive oxidant and subsequent O–O bond formation. More basic buffer anions led to lower catalytic onset potentials, extending below 1 V. This homogeneous cobalt-porphyrin system was shown to be robust under active catalytic conditions, showing negligible decomposition over hours of operation. Added EDTA or ion exchange resin caused no catalyst poisoning, indicating that cobalt ions were not released from the porphyrin macrocycle during catalysis. Likewise, surface analysis by energy dispersive X-ray spectroscopy of the working electrodes showed no deposition of heterogeneous cobalt films. Taken together, the results indicate that Co–5,10,15,20-tetrakis-(1,3-dimethylimidazolium-2-yl)porphyrin is an efficient, homogeneous, single-site water oxidation catalyst.Water oxidation is a key step in photosynthesis that efficiently harvests and stores solar energy (1). The oxidation of H₂O to O₂ is a four-electron, four-proton process in which O–O bond formation is the key chemical step. In photosystem II, these proton-coupled electron transfers (PCETs) occur via a tyrosine at the Mn₄Ca oxygen-evolving complex (2). An important thermodynamic aspect of photosynthesis is the efficient conversion of photonic energy to electrical potential, thus providing 99% of the driving force required to convert CO₂ to carbohydrates (Eqs. 1 and 2):andThe development of synthetic catalysts that can mediate water oxidation under mild conditions with a minimal energy cost has become an appealing challenge for chemists (3–7). Among various approaches, homogeneous molecular catalysts have shown attractive features such as controllable redox properties, ease of investigating reaction mechanisms, and strategies for the characterization of reactive intermediates (8, 9). Recently, such efforts have resulted in the development of a significant number of systems based on single-site and multinuclear transition metal complexes including Mn, Fe, Co, Cu, Ru, and Ir (9–15). Examples of cobalt-based molecular catalysts include a cobalt phthalocyanine (16), a cobalt “hangman” corrole (17), multipyridine cobalt complexes (18, 19), a dinuclear Co-peroxo species (20), and most recently, Co-porphyrins (21). Determining if these molecular complexes retain their homogenous nature during catalysis or merely act as precursors of truly active heterogeneous species such as films and nanoparticles has proven to be problematic (8, 22, 23). This scenario is especially critical for Co-based molecular catalysts because heterogeneous CoO_x species are highly active, so that even small amounts of this surface oxide layer could contribute significantly to the catalytic activity (24–27). Further, very little detailed mechanistic information for these systems is available to date, and the assignment of the key oxidants responsible for oxidizing a water molecule has been challenging (28). Here we describe mechanistic studies of a series of water-soluble, cationic cobalt porphyrins that effectively catalyze water oxidation electrochemically under mild conditions at neutral pH. The homogeneity of these catalysts was confirmed by a variety of techniques including electrochemical, spectroscopic, and surface analysis that exclude the alternative formation of heterogeneous cobalt oxide films. Our results indicate the generation of a reactive, high-valent Co^IV-porphyrin cation radical that has accumulated two oxidizing equivalents above the resting Co^III catalyst. Further, we demonstrate a critical role for the buffer base that accepts a proton during the O–O bond-formation event.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Slow hydrogen-bond switching dynamics at the water surface revealed by theoretical two-dimensional sum-frequency spectroscopy

Using our newly developed explicit three-body (E3B) water model, we simulate the surface of liquid water. We find that the timescale for hydrogen-bond switching dynamics at the surface is about three times slower than that in the bulk. In contrast, with this model rotational dynamics are slightly faster at the surface than in the bulk. We consider vibrational two-dimensional (2D) sum-frequency generation (2DSFG) spectroscopy as a technique for observing hydrogen-bond rearrangement dynamics at the water surface. We calculate the nonlinear susceptibility for this spectroscopy for two different polarization conditions, and in each case we see the appearance of cross-peaks on the timescale of a few picoseconds, signaling hydrogen-bond rearrangement on this timescale. We thus conclude that this 2D spectroscopy will be an excellent experimental technique for observing slow hydrogen-bond switching dynamics at the water surface.Interfaces play important roles in many disciplines of science. The water liquid/vapor interface, for example, is of great interest in chemistry, biology, and earth science and is an important model system for water in a heterogeneous environment. Of particular interest is understanding the extent to which the structure and dynamics, and ultimately reactivity, of water at the interface differ from those in the bulk. For example, how does the distribution of hydrogen bonds differ between interfacial and bulk water? How anisotropic is the orientation of the water molecules at the interface? In terms of dynamics, how do the diffusion constant, rotational relaxation time, and hydrogen-bond rearrangement time vary as the interface is approached? One can also consider vibrational dynamics processes such as energy relaxation and transfer.One important technique for addressing these questions is computer simulation. Models used in these calculations for the water surface range from rigid, fixed-point-charge two-body models (1–3), to fluctuating charge or polarizable models (4, 5), to ab initio molecular dynamics calculations (6–10). Regarding static properties, for example, some effort has been expended toward understanding what fraction of H atoms in the surface layer are hydrogen bonded, and what fraction of molecules do not donate any hydrogen bonds (nondonors or “acceptor-only” molecules) (6, 9). In terms of dynamics, it is generally found that diffusion is faster at the interface than in the bulk (1, 4, 10), and rotational relaxation is also faster (3, 6, 7, 10). On the other hand, two studies with fixed-charge two-body models show that hydrogen-bond rearrangement is slower at the interface (2, 3), whereas one study with a fluctuating-charge model shows that hydrogen-bond rearrangement is faster (5). In this latter study the authors conclude that this is generally true for polarizable models.Because of its surface sensitivity, vibrational sum-frequency generation (SFG) spectroscopy (11, 12) has become one of the most powerful experimental techniques for the study of interfaces, including the one separating liquid water and its vapor (13–28). In a vibrational SFG experiment, infrared (IR) and visible laser pulses are incident on the interface, and the signal is detected at the sum of the frequencies of these incoming beams. For the water liquid/vapor interface one can think of the SFG intensity as the vibrational spectrum of the water molecules near the surface (29, 30). Intensity-level SFG spectra of the OH stretching mode of water show two major features for this system. A sharp peak near 3,700 cm⁻¹ indicates the existence of dangling or “free” OH groups at the water surface. The other broad band in the frequency region from 3,000 to 3,600 cm⁻¹ is interpreted as arising from hydrogen-bonded OHs (13, 14).Further interpretation of SFG results was catalyzed by two major advances. First, studying the isotopically dilute HOD in D₂O (or H₂O) system has helped in the interpretation of spectra, because the frequency mismatch of OH and OD stretches largely eliminates the effects of vibrational couplings, which greatly complicate the measured spectra for neat water (31, 32). Second, the invention of phase-sensitive SFG enables the direct measurement of the imaginary part of the second-order complex susceptibility χ₂ (whereas the conventional experiments measure |χ₂|²) (33, 34). Because Im(χ₂) contains only resonant contributions, it is analogous to an absorption spectrum and is easier to interpret and to calculate. Moreover, Im(χ₂) is signed, and the sign is related to the projection of the OH (OD) transition dipole onto the surface normal. Recently, Shen and coworkers measured Im(χ₂) for both the neat and the isotope-labeled water liquid/vapor interfaces (35, 36). These phase-sensitive SFG results for HOD/D₂O show three major features: a sharp positive peak at about 3,700 cm⁻¹ corresponding to the upward-pointing dangling OH bonds and negative and positive bands at about 3,500 and 3,300 cm⁻¹, respectively, attributed to hydrogen-bonded water OHs (36). The latter two peaks were interpreted by the authors as “water-like” molecules with downward-pointing OH bonds and “ice-like” molecules with upward-pointing OH bonds, respectively (36, 37).As mentioned above, phase-sensitive SFG allows for a better comparison between experimental results and theoretical calculations. Although recent papers by Geissler, Shen, and coworkers show that in principle magnetic-dipole and electric-quadrupole terms should be included in a correct SFG calculation (38–40), in practice nearly all calculations have used the electric-dipole approximation. Most of the widely used two-body water models fail to reproduce the positive band in the low-frequency region of the phase-sensitive SFG spectrum (41–46). Morita and coworkers have developed a polarizable and flexible classical water model that successfully qualitatively reproduces Im(χ₂) of the neat H₂O surface, and they assigned the positive signal in the hydrogen-bonding region to induced dipoles perpendicular to the water surface (28, 47, 48). This conclusion was also in agreement with their results from hybrid quantum mechanics/molecular mechanics molecular dynamics (MD) simulations (49). Our group has used the newly developed explicit three-body (E3B) water model (50, 51), which includes three-molecule interactions, to calculate Im(χ₂) using a mixed quantum/classical approach. The calculations also qualitatively reproduce the experimental spectra. The two features in the hydrogen-bonding region were found to result from canceling contributions from water molecules with different hydrogen-bonding configurations (46, 52), with especially large contributions from four-hydrogen–bonded double-donor molecules and two-hydrogen–bonded single-donor molecules.Although conventional SFG spectroscopy can provide structural information about an interface, in the case of water, where the spectrum is dominated by inhomogeneous broadening, it is unable to probe the dynamics. To study dynamics, one-dimensional (1D)SFG needs to be extended to two dimensions (2D), much like IR spectroscopy has recently been extended to 2DIR (53). In a time-domain 2DIR experiment, the sample is subjected to three IR pulses, separated by two time intervals t₁ and t₂, and the signal is heterodyne detected at a time t₃ later. The signal is then Fourier transformed in t₁ and t₃, leading to two frequency dimensions ω₁ and ω₃. A series of 2DIR spectra is collected as a function of t₂, the “waiting time.” Roughly speaking, the 2DIR spectrum can be thought of as the joint probability density that the chromophore has frequency ω₁ at time 0 and ω₃ at time t₂. Thus, the experiment naturally reports on dynamic processes such as spectral diffusion and chemical exchange (53). 2DIR spectroscopy has been widely used to study dynamics in bulk water, including hydrogen-bond dynamics (54–60), rotations (61–63), and vibrational energy transfer (64–69). However, 2DIR, a third-order nonlinear spectroscopy, is not surface sensitive. Therefore, 2DSFG, a fourth-order nonlinear spectroscopy, is needed to study the dynamics at the interfacial region.In 2DSFG, four laser beams are incident on the sample: three time-delayed IR pulses followed by a visible pulse (70). Although some related and beautiful ultrafast pump-probe SFG and homodyne-detected 2DSFG experiments have been performed to measure vibrational relaxation, rotational dynamics, and vibrational energy transfer at the water surface (71–76), to the best of our knowledge the heterodyne-detected 2DSFG experiment for the air/water interface (and especially desirably, for HOD/D₂O or HOD/H₂O) has not yet been performed. Indeed, only two heterodyne-detected 2DSFG experiments on any system have been reported (70, 77). Xiong et al. measured the heterodyne-detected 2DSFG spectrum of CO molecules adsorbed on a platinum surface (70), whereas Singh et al. measured the heterodyne-detected 2DSFG spectrum for the interface of HOD/D₂O with the positively charged surfactant cetyltrimethylammonium bromide (77). This latter system provides a stronger signal than that for the air/water interface, because the electric field from the surfactant produces substantial alignment of the water molecules. A theoretical 2DSFG calculation by Nagata et al. using a classical approach has appeared for water at a lipid monolayer interface (78).In this paper, we simulate the liquid/vapor interface using our E3B water model (51, 52). We calculate the hydrogen-bond rearrangement time-correlation function for the surface molecules, finding that it decays on a timescale of about 4 ps, which is significantly slower than the corresponding timescale for the bulk. We can contrast this to the timescale for rotational motion, which for our model is faster at the surface than in the bulk. We also calculate the 2DSFG signal for different polarizations, as a function of waiting time. Cross-peaks grow in on the timescale of the hydrogen-bond rearrangment time. This demonstrates the promise of the 2DSFG technique for an experimental measurement of spectral diffusion and chemical exchange at the water surface and hence an experimental measurement of structural relaxation in the interfacial region.     

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO₃⁻); it thereby serves as a cellular sensor for HCO₃⁻, carbon dioxide (CO₂), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO₃⁻ binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO₃⁻ activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO₃⁻ sensing and a basis for targeting this system with drugs.The ubiquitous second messenger cAMP regulates diverse physiological processes, from fungal virulence to mammalian brain function (1, 2). In mammals, cAMP can be generated by any of 10 differently expressed and regulated adenylyl cyclases (ACs): nine transmembrane enzymes (tmACs) and one soluble AC (sAC) (3). TmACs reside in the cell membrane, where they mediate cellular responses to hormones acting through G protein-coupled receptors (4). In contrast, sAC functions in various intracellular locations, providing cell-specific spatial and temporal patterns of cAMP (5–7) in response to intracellular signals, including calcium, ATP, and bicarbonate (HCO₃⁻) (3, 8–10). HCO₃⁻ regulation of sAC enzymes is a direct effect on their catalytic domains and is conserved across bacterial, fungal, and animal kingdoms (1, 11–13). Via modulation of sAC, and sAC-like cyclase activities, HCO₃⁻ serves as an evolutionarily conserved signaling molecule mediating cellular responses to HCO₃⁻, CO₂, and pH (3, 14). In mammals, sAC acts as a CO₂/HCO₃⁻/pH sensor in processes such as sperm activation (15), acid-base homeostasis (16), and various metabolic responses (10, 17, 18). sAC has also been implicated in skin and prostate cancer and as a target for male contraceptives (19–21).All mammalian ACs are class III nucleotidyl cyclases sharing homologous catalytic domains. Their catalytic cores are formed through symmetrical or pseudosymmetrical association of two identical or highly similar catalytic domains, C₁ and C₂ (22–24); in mammalian ACs, both domains reside on a single polypeptide chain. Such C₁C₂ pseudoheterodimers form two pseudosymmetrical sites at the dimer interface: the active site and a degenerated, inactive pocket (3, 23). A conserved Lys and an Asp/Thr in the active site recognize the base of the substrate ATP, and two conserved Asp residues bind two divalent cations, normally Mg²⁺ (23). The ions, called ion A and ion B, coordinate the substrate phosphates and support the intramolecular 3′-hydroxyl (3′-OH) attack at the α-phosphorous to form cAMP and pyrophosphate (PP_i) (3). In tmACs, the degenerate site binds forskolin (24), a plant diterpene that activates tmACs but has no effect on sAC (25). The forskolin activation mechanism and the existence of physiological ligands for this site in tmACs or in sAC remain unclear.There are two sAC isoforms known to be generated by alternative splicing (26). Full-length sAC comprises N-terminal catalytic domains along with ∼1,100 residues with a little understood function except for an autoinhibitory motif and a heme-binding domain (3, 27, 28). Exclusion of exon 12 (26) generates a truncated isoform, sAC_t (residues 1–490), which comprises just the two sAC catalytic domains (sAC-cat) (25). sAC_t is widely expressed, and it is the isoform most extensively biochemically characterized (3, 8, 11). It is directly activated by Ca²⁺ and HCO₃⁻; Ca²⁺ supports substrate binding, and HCO₃⁻ increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3, 13, 29). In a homodimeric, HCO₃⁻-regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO₃⁻ appeared to facilitate an active site closure required for catalysis (13), but the HCO₃⁻ binding site and its mechanism of activation remained unknown.Here, we present crystal structures of the human sAC-cat in apo form and in complex with substrate, products, bicarbonate, and a pharmacological inhibitor. The structures reveal insights into binding sites and mechanisms for sAC catalysis and for its regulation by physiological and pharmacological small molecules.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Photogeneration of hydrogen from water using CdSe nanocrystals demonstrating the importance of surface exchange

Unique tripodal S-donor capping agents with an attached carboxylate are found to bind tightly to the surface of CdSe nanocrystals (NCs), making the latter water soluble. Unlike that in similarly solubilized CdSe NCs with one-sulfur or two-sulfur capping agents, dissociation from the NC surface is greatly reduced. The impact of this behavior is seen in the photochemical generation of H₂ in which the CdSe NCs function as the light absorber with metal complexes in aqueous solution as the H₂-forming catalyst and ascorbic acid as the electron donor source. This precious-metal–free system for H₂ generation from water using [Co(bdt)₂]⁻ (bdt, benzene-1,2-dithiolate) as the catalyst exhibits excellent activity with a quantum yield for H₂ formation of 24% at 520 nm light and durability with >300,000 turnovers relative to catalyst in 60 h.Artificial photosynthesis (AP) represents an important strategy for energy conversion from sunlight to storage in chemical bonds (1–4). Unlike natural photosynthesis in which CO₂ + H₂O are converted into carbohydrates and O₂, the key energy-storing reaction in AP is the splitting of water into its constituent elements of hydrogen and oxygen (5–16). As a redox reaction, water splitting can be divided into two half-reactions, of which the light-driven generation of H₂ is the reductive component. Many systems for the photogeneration of H₂ have been described over the years and they typically consist of a light absorber, a catalyst for H₂ formation, and sources of protons and electrons. For systems that function in aqueous media, the protons are provided by water, whereas for nonaqueous systems, the protons are provided by weak, generally organic acids. The source of electrons in these photochemical systems is generally a sacrificial electron donor—that is, a compound that decomposes following one electron oxidation.Reports of the light-driven generation of hydrogen date back more than 30 y, beginning with a multicomponent system containing [Ru(bpy)₃]²⁺ (where bpy is 2,2′-bipyridine) as the chromophore or photosensitizer (PS) and colloidal Pt as the catalyst for making H₂ from protons and electrons (17). In these and many subsequent systems, electron mediators were used to accept an electron from the excited chromophore, PS*— thereby serving as an oxidative quencher—and transfer it to the catalyst. Whereas two of the initial mediators were bpy complexes of rhodium and cobalt (17, 18), the overwhelming majority of electron mediators in these systems were dialkylated 2,2′- and 4,4′-bipyridines and their derivatives (19–22). The most extensively used of these mediators was methyl viologen (MV²⁺, dimethyl-4,4′-bipyridinium, usually as its chloride salt). These mediators were subsequently found to undergo deactivation in their role by hydrogenation (23, 24). The sacrificial electron donors used in these studies depended on system pH and were generally based on compounds having tertiary amine functionality for decomposition following oxidation, such as triethylamine (TEA), triethanolamine (TEOA), and ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) (17, 19–22). A different electron mediator during the early studies on light-driven generation of hydrogen was found to be TiO₂, which when platinized served as both the mediator and the catalyst (25–28).During the more than three decades that have passed since the initial reports (17, 19–22, 25–27), every aspect and component of photochemical proton reduction systems have been investigated with the goal of increasing activity and durability. These include new molecular catalysts and different photosensitizers ranging from other metal complexes with long-lived charge-transfer excited states to strongly absorbing organic dyes. With a view toward the possible long-term utilization of hydrogen from solar-driven water splitting, efforts have expanded over the past decade to use components that contain only earth-abundant elements and thus to remove Pt, Pd, Ru, Ir, and Rh from such systems. In this regard, photochemical proton reduction systems have been reported in which complexes of cobalt, nickel, and iron are found to function as catalysts for hydrogen generation (18, 29–41). A number of these complexes were inspired by the active sites of hydrogenase enzymes in which Fe is, and Ni may be, present, and a pendant organic base is thought to help as a proton shuttle to a postulated metal-hydride intermediate for H₂ formation (30, 31, 35, 37, 42, 43).Another set of complexes investigated as catalysts for proton reduction are complexes of Co having diglyoxime-type ligands that form a pseudomacrocyclic structure (that is, two diglyoxime ligands linked together by either H bonds or BF₂ bridges) (44–54). Although many of these studies with regard to catalyst development were, and are, based on electrocatalytic generation of H₂ (44–50), more recent efforts have used the cobaloxime catalysts in light-driven systems (51–54). Photosensitizers in these investigations have been either charge transfer metal complexes of Ru(II), Ir(III), Re(I), and Pt(II) or organic dyes. Although some of these systems exhibited significant activity for making H₂, all of them suffered from instability that led to cessation of activity after periods ranging from 6 h to 30 h.The use of the cobaloxime catalyst CoCl(pyr)(dmg)₂ (where dmg is dimethylglyoximate anion) in conjunction with organic dyes as PS provided the first molecular systems for visible light-driven proton reduction to H₂ that were free of precious metals (55–57). The most effective of these used a Se-derivatized rhodamine dye as the chromophore with TEOA as the sacrificial electron donor, yielding good activity with an initial turnover frequency (TOF) > 5,000/h (vs. PS) and total turnover number (TON) of 9,000 after 8 h (57). Analysis of this system revealed that it functioned via reductive quenching of PS* by TEOA and subsequent electron transfer from PS^– to the catalyst. Another organic dye-catalyst system that also exhibited good activity used fluorescein (Fl) as PS and a nickel pyridinethiolate (pyS) catalyst in pH 11 media with TEA as the sacrificial donor. This system also was found to function via reductive quenching of PS* by the electron donor, rather than by direct electron transfer from PS* to the catalyst or an electron mediator (37). A significant number of reviews provide detailed accounts of the various systems studied and their effectiveness with regard to H₂ generation (1, 58–64). However, all of them, which contain molecular light absorbers [charge transfer (CT) metal complexes and organic dyes], suffer from photoinstability during prolonged irradiation. Additionally, the molecular catalysts for H₂ generation may undergo deactivation, as has been established for the Co glyoximate complexes.In an analysis for genuinely viable systems for proton reduction and water oxidation in solar-driven water splitting, Bard and Fox addressed the question of component stability and indicated a need to focus on the use of semiconductors (SCs) as light absorbers based on the wide energy range of SC bandgaps, the electron transfer properties of excited semiconductors, and their potential stability under prolonged irradiation (65). Although the use of semiconductors for photochemical water splitting dates back to a report by Fujishima and Honda in 1972 with TiO₂ and UV light, the challenge was to use SCs with absorption maxima that better matched the solar spectrum (66). There have been numerous reports describing efforts in this direction and several recent reviews offer a summary of systems used and results obtained (67–75). Semiconductor nanoparticles that exhibit size-constrained electronic properties represent a large and important class of possible light absorbers for the two half-reactions of water splitting. These nanoparticles, which are referred to as quantum dots (QDs) and nanocrystals (NCs), represent a fertile area of study in the context of energy conversion because their bandgaps can be adjusted via their preparation and their solubility can be controlled by their surface stabilizers or capping agents (76). In this way, NCs can offer unique size-dependent optical properties and stronger light absorption over a wider spectral range than do molecular PSs (68, 76). In fact, NCs as light absorbers in combination with precious metal proton reduction catalysts or with Fe-Fe hydrogenase have been studied, yielding interesting photocatalytic systems (77–81).We recently communicated such a system for carrying out H₂ formation from aqueous protons that possessed great durability and impressive activity. The light absorber in this system was water-solubilized CdSe NCs, the catalyst was an in situ-formed complex of Ni²⁺ with the water-solubilizing agent dihydrolipoic acid (DHLA), the electron source was ascorbic acid (AA), and the system medium was water at pH 4.5. TONs of more than 600,000 were reported for one set of conditions, using 520 nm light, whereas for a different set of conditions durability over 15 d was found (82). Water solubilization of CdSe NCs using agents such as 3-thiopropionic acid and DHLA has been known for some time, with DHLA more strongly binding via chelation (83–85). In the system that we previously reported for H₂ production, however, dissociation of DHLA from the CdSe NCs was an essential aspect of its operation to form the Ni-DHLA catalyst (82). On the other hand, the dissociation of DHLA from the CdSe NCs was also found to negatively affect the examination of preformed catalysts because of competing exchange reactions involving DHLA and the catalyst ligands.In our current study, we report a unique hydrogen-generating system using CdSe NCs with much less labile water-solubilizing capping agents. This unique system, which is more durable, allows assessment of the activity of successful H₂-generating catalysts that had been established electrochemically or in a different photochemical system. The reduced lability of the water-solubilizing agent is based on having three S donors in close proximity to each other for the formation of a more stable bridging structure to the CdSe nanoparticle.     

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10⁻¹⁴ cm³/s or 3.5(±1.0) × 10⁸ water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10⁸ water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10⁸ water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10⁷ water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10⁵ pores per μm²) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10³ pores per μm²). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.The discovery of the high water and gas permeability of aquaporins (AQPs) and the development of artificial analogs, carbon nanotubes (CNTs), have led to an explosion in studies aimed at incorporating such channels into materials and devices for applications that use their unique transport properties (1–9). Areas of application include liquid and gas separations (10–13), drug delivery and screening (14), DNA recognition (15), and sensors (16). CNTs are promising channels because they conduct water and gas three to four orders of magnitude faster than predicted by conventional Hagen–Poiseuille flow theory (11). However, their use in large-scale applications has been hampered by difficulties in producing CNTs with subnanometer pore diameters and fabricating membranes in which the CNTs are vertically aligned (4). AQPs also efficiently conduct water across membranes (∼3 billion molecules per second) (17) and are therefore being studied intensively for their use in biomimetic membranes for water purification and other applications (1, 2, 18). The large-scale applications of AQPs is complicated by the high cost of membrane protein production, their low stability, and challenges in membrane fabrication (1).Artificial water channels, bioinspired analogs of AQPs created using synthetic chemistry (19), ideally have a structure that forms a water-permeable channel in the center and an outer surface that is compatible with a lipid membrane environment (1). Interest in artificial water channels has grown in recent years, following decades of research and focus on synthetic ion channels (19). However, two fundamental questions remain: (i) Can artificial channels approach the permeability and selectivity of AQP water channels? (ii) How can such artificial channels be packaged into materials with morphologies suitable for engineering applications?Because of the challenges in accurately replicating the functional elements of channel proteins, the water permeability and selectivity of first-generation artificial water channels were far below those of AQPs (SI Appendix, Table S1) (20–25). In some cases, the conduction rate for water was much lower than that of AQPs as a result of excess hydrogen bonds being formed between the water molecules and oxygen atoms lining the channel (20). The low water permeability that was measured for first-generation water channels also highlights the experimental challenge of accurately characterizing water flow through low-permeability water channels. Traditionally, a liposome-based technique has been used to measure water conduction, in which the response to an osmotic gradient is followed by measuring changes in light scattering (26, 27) or fluorescence (28). The measured rates are then converted to permeability values. These measurements suffer from a high background signal due to water diffusion through the lipid bilayer, which, in some cases, can be higher than water conduction through the inserted channels, making it challenging to resolve the permeability contributed by the channels (29). Thus, there is a critical need for a method to accurately measure single-channel permeability of artificial water channels to allow for accurate comparison with those of biological water channels. Furthermore, first-generation artificial water channels were designed with a focus on demonstrating water conduction and one-dimensional assembly into tubular structures (20–24), but how the channels could be assembled into materials suitable for use in engineering applications was not explored. To derive the most advantage from their fast and selective transport properties, artificial water channels are ideally vertically aligned and densely packed in a flat membrane. These features have been long desired but remain a challenge for CNT-based systems (4).Here we introduce peptide-appended pillar[5]arene (PAP; Fig. 1) (30) as an excellent architecture for artificial water channels, and we present data for their single-channel permeability and self-assembly properties. Nonpeptide pillar[5]arene derivatives were among first-generation artificial water channels (1, 23). Pillar[5]arene derivatives, including the one used in this study, have a pore of ∼5 Å in diameter and are excellent templates for functionalization into tubular structures (31–34). However, the permeability of hydrazide-appended pillar[5]arene channels was low (∼6 orders of magnitude lower than that of AQPs; SI Appendix, Table S1). We addressed the challenges of accurately measuring single-channel water permeability and improving the water conduction rate over first-generation artificial water channels by using both experimental and simulation approaches. The presented PAP channel contains more hydrophobic regions (30) compared with its predecessor channel (23), which improves both its water permeability and its ability to insert into membranes. To determine single-channel permeability of PAPs, we combined stopped-flow light-scattering measurements of lipid vesicles containing PAPs with fluorescence correlation spectroscopy (FCS) (35, 36). Stopped-flow experiments allow the kinetics of vesicle swelling or shrinking to be followed with millisecond resolution and water permeability to be calculated, whereas FCS makes it possible to count the number of channels per vesicle (36, 37). The combination of the two techniques allows molecular characterization of channel properties with high resolution and demonstrates that PAP channels have a water permeability close to those of AQPs and CNTs. The experimental results were corroborated by molecular dynamics (MD) simulations, which also provided additional insights into orientation and aggregation of the channels in lipid membranes. Finally, as a first step toward engineering applications such as liquid and gas separations, we were able to assemble PAP channels into highly packed planar membranes, and we experimentally confirmed that the channels form 2D arrays in these membranes.Open in a separate windowFig. 1.Structure of the peptide-appended pillar[5]arene (PAP) channel. (A) The PAP channel (C₃₂₅H₃₂₀N₃₀O₆₀) forms a pentameric tubular structure through intramolecular hydrogen bonding between adjacent alternating d-l-d phenylalanine chains (d-Phe-l-Phe-d-Phe-COOH). (B) Molecular modeling (Gaussian09, semiempirical, PM6) of the PAP channel shows that the benzyl rings of the phenylalanine side chains extend outward from the channel walls (C, purple; H, white; O, red; N, blue). (C and D) MD simulation of the PAP channel in a POPC bilayer revealed its interactions with the surrounding lipids. The five chain-like units of the channel are colored purple, blue, ochre, green, and violet, with hydrogen atoms omitted. In C, the POPC lipids are represented by thin tan lines; in D, water is shown as red (oxygen) and white (hydrogen) van der Waals spheres.     

Crossing the divide between homogeneous and heterogeneous catalysis in water oxidation

Enhancing the surface binding stability of chromophores, catalysts, and chromophore–catalyst assemblies attached to metal oxide surfaces is an important element in furthering the development of dye sensitized solar cells, photoelectrosynthesis cells, and interfacial molecular catalysis. Phosphonate-derivatized catalysts and molecular assemblies provide a basis for sustained water oxidation on these surfaces in acidic solution but are unstable toward hydrolysis and loss from surfaces as the pH is increased. Here, we report enhanced surface binding stability of a phosphonate-derivatized water oxidation catalyst over a wide pH range (1–12) by atomic layer deposition of an overlayer of TiO₂. Increased stability of surface binding, and the reactivity of the bound catalyst, provides a hybrid approach to heterogeneous catalysis combining the advantages of systematic modifications possible by chemical synthesis with heterogeneous reactivity. For the surface-stabilized catalyst, greatly enhanced rates of water oxidation are observed upon addition of buffer bases and with a pathway identified in which O-atom transfer to OH⁻ occurs with a rate constant increase of 10⁶ compared to water oxidation in acid.Heterogeneous catalysis plays an important role in industrial chemical processing, fuel reforming, and energy-producing reactions. Examples include the Haber–Bosch process, steam reforming, Ziegler–Natta polymerization, and hydrocarbon cracking (1–8). Research in heterogeneous catalysis continues to flourish (9–15) but iterative design and modification are restricted by limitations in materials preparation and experimental access to surface mechanisms. By contrast, synthetic modification of molecular catalysts is possible by readily available routes; a variety of experimental techniques is available for monitoring rates and mechanism in solution for the investigation of homogeneous catalysis (16–23). Transferring this knowledge and the reactivity of homogeneous molecular catalysts to a surface could open the door to heterogeneous applications in fuel cells, dye sensitized photoelectrochemical cells, and multiphase industrial reactions.Procedures are available for immobilization of organometallic and coordination complexes on the surfaces of solid supports. Common strategies include surface derivatization of metal oxides by carboxylate, phosphonate, and siloxane bindings (24–27), carbon-grafted electrodes (28–30), and electropolymerization (31–33). These approaches provide a useful bridge to the interface and a way to translate mechanistic understanding and ease of synthetic modification of solution catalysts to heterogeneous applications with a promise of higher reactivity under milder conditions.A significant barrier to this approach arises from the limited stability of surface binding. Surface-bound carboxylates are typically unstable to hydrolysis in water, whereas phosphonates are unstable in neutral or basic solutions (27, 34). For water oxidation catalysis this is particularly detrimental given the accelerated rates that are accessible for catalytic water oxidation as the pH is increased due to the intervention of base-catalyzed pathways with concerted atom–proton transfer accompanying O—O bond formation (35).We report here the results of a designed strategy for the systematic surface stabilization of molecular catalysts on solid oxide surfaces. In the strategy we use indium tin oxide (ITO) electrodes and first bind a phosphonate-derivatized molecular water oxidation catalyst to the surface of the electrode. The derivatized electrode is then coated with a conformal nanoscale TiO₂ overlayer applied by layer-by-layer atomic layer deposition (ALD). The overlayer of TiO₂ acts to block hydrolysis of the phosphonate groups from the surface, Fig. 1. ALD stabilization has been used previously to demonstrate significantly enhanced photostability of surface-bound chromophores in acidic and neutral solutions (36–38). In this article we apply the ALD stabilization procedure to surface stabilization of a known water oxidation catalyst and show remarkably enhanced surface binding stability even in basic solutions. Retention of electrocatalytic reactivity on the surface is demonstrated and water oxidation catalysis investigated over a wide pH range. Clear evidence is found in these studies that added proton acceptor bases enhance the kinetic pathways in the key, rate-limiting step (O—O bond formation) via an atom–proton transfer (APT) mechanism (22, 35). In addition, a facile pathway has been identified with direct attack by OH⁻ on an activated oxo form of the catalyst with rate enhancements of up to 10⁶ for water oxidation.Open in a separate windowFig. 1.Schematic representation of the ALD overlayer protection strategy for a catalyst surface-attached to nanoITO protected by TiO₂. (A) Illustrating the electrochemical device architecture showing the surface derivatized electrode and water oxidation. (B) Underivatized electrodes exposed to basic aqueous conditions showing detachment of the catalysts from the electrode surface. (C) ALD protection of surface attachment even basic aqueous conditions. This figure was adapted from ref. 38.     

Phase transition-induced band edge engineering of BiVO4 to split pure water under visible light

Through phase transition-induced band edge engineering by dual doping with In and Mo, a new greenish BiVO₄ (Bi_1-XIn_XV_1-XMo_XO₄) is developed that has a larger band gap energy than the usual yellow scheelite monoclinic BiVO₄ as well as a higher (more negative) conduction band than H⁺/H₂ potential [0 V_RHE (reversible hydrogen electrode) at pH 7]. Hence, it can extract H₂ from pure water by visible light-driven overall water splitting without using any sacrificial reagents. The density functional theory calculation indicates that In³⁺/Mo⁶⁺ dual doping triggers partial phase transformation from pure monoclinic BiVO₄ to a mixture of monoclinic BiVO₄ and tetragonal BiVO₄, which sequentially leads to unit cell volume growth, compressive lattice strain increase, conduction band edge uplift, and band gap widening.Photocatalytic water splitting with a particulate semiconductor powered by sunlight is an ideal route to cost-effective, large-scale, and sustainable hydrogen production because of its extreme simplicity. However, it is challenging, because it requires a rare photocatalyst that carries a combination of suitable band gap energy, appropriate band positions, and photochemical stability (1–5). Thus, reproducible photocatalytic systems for visible light-driven overall water splitting (OWS) by one-step photoexcitation are also rare, although there were several reports of such systems (4–6). In the best-known successful case, Domen and coworkers (5) reported in 2005 that a solid solution of GaN and ZnO [(Ga_1−xZn_x)(N_1−xO_x)] was a stable photocatalyst that could split water into H₂ and O₂ under visible light when modified with a cocatalyst. This system remains the most active and reproducible one-step OWS photocatalyst responsive to visible light so far (4).Scheelite monoclinic (m-) BiVO₄ is a well-documented photocatalyst having suitable band gap energy (E_g ∼ 2.4 eV) for absorbing visible light (7–10). Also, it is chemically stable in aqueous solution under light irradiation. Thus, it functions as an excellent photocatalyst for O₂ evolution under visible light in the presence of an appropriate electron acceptor (e.g., AgNO₃). However, because the bottom of its conduction band is located at a more positive potential than the potential of water reduction [0 V_RHE (reversible hydrogen electrode) at pH 7], it is incapable of evolving H₂. In addition, it shows poor charge transport characteristics (11) and weak surface adsorption properties (12), causing low photocatalytic activity. To overcome these weaknesses, a variety of strategies, such as heterojunction structure formation (11, 13, 14), loading cocatalysts (8, 15–17), and impurity doping (1, 7, 12, 18–23), has been attempted. These strategies were successful in improving BiVO₄’s oxidation capability for photoelectrochemical water oxidation (1, 11, 13, 15, 19, 24–28) as well as the Z scheme (two-photon excitation) water splitting system (29). Also, cocatalyzed Bi_xY_1-xVO₄ (x ∼ 0.5) (7, 8) and cocatalyzed Bi_0.5La_0.5VO₄ (23) promoted OWS by raising the conduction band edge (CBE) position, but OWS under visible light irradiation over BiVO₄-based photocatalysts has not been fully shown.To meet this challenge, we developed greenish BiVO₄ (GBVO_x; x = atom ratio of In and Mo), Bi_1-xIn_xV_1-xMo_xO₄, by simultaneously substituting In³⁺ for Bi³⁺ and Mo⁶⁺ for V⁵⁺ in the host lattice of m-BiVO₄. The new GBVO_x photocatalyst has a slightly larger band gap energy than the usual yellow scheelite m-BiVO₄ as supported by the unique color change to green and a higher (more negative) conduction band than H⁺/H₂ potential (0 V_RHE at pH 7). Consequently, as depicted in Fig. 1, GBVO_x is able to split water into H₂ and O₂ under visible light irradiation without using any sacrificial reagents (e.g., CH₃OH or AgNO₃). Herein, we report the dual-metal doping effects on the optical absorption behavior, crystal structure, and electronic band structure of BiVO₄, which led to one-photon OWS under visible light irradiation. We elucidate the physical origin of the augmented photoresponse behaviors of GBVO_x through density functional theory (DFT) calculation of electronic structure as well as a variety of physical and electrochemical characterizations.Open in a separate windowFig. 1.OWS reaction mechanism by GBVO_x.     

Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation

Cyanobacteria, algae, and plants oxidize water to the O₂ we breathe, and consume CO₂ during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO₂/ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast’s lumen, resulting in a light-driven production of O₂ and CO₂. Depletion of from the media leads, in the absence of added buffers, to a reversible down-regulation of O₂ production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis and conclude the more than 50-y-long quest for the function of CO₂/ in photosynthetic water oxidation.Oxygenic photosynthesis in cyanobacteria, algae, and higher plants leads to the reduction of atmospheric CO₂ to energy-rich carbohydrates. The electrons needed for this process are extracted in a cyclic, light-driven process from water that is split into dioxygen (O₂) and protons. This reaction is catalyzed by a penta-µ-oxo bridged tetra-manganese calcium cluster (Mn₄CaO₅) within the oxygen-evolving complex (OEC) of photosystem II (PSII) (1–4). The possible roles of inorganic carbon, , in this process have been a controversial issue ever since Otto Warburg and Günter Krippahl (5) reported in 1958 that oxygen evolution by PSII strictly depends on CO₂ and therefore has to be based on the photolysis of H₂CO₃ (“Kohlensäure”) and not of water. These first experiments were indirect and, as became apparent later, were wrongly interpreted (6–8). Several research groups followed up on these initial results and identified two possible sites of C_i interaction within PSII (reviewed in refs. 9–12). Functional and spectroscopic studies showed that facilitates the reduction of the secondary plastoquinone electron acceptor (Q_B) of PSII by participating in the protonation of . Binding of (or ) to the nonheme Fe between the quinones Q_A and Q_B was recently confirmed by X-ray crystallography (3, 13, 14). Despite this functional role at the acceptor side, the very tight binding of to this site makes it impossible for the activity of PSII to be affected by changing the C_i level of the medium; instead inhibitors such as formate need to be added to induce the acceptor-side effect (15). Consequently, the water-splitting electron-donor side of PSII has also been studied intensively (for recent reviews, see refs. 11 and 12). Although a tight binding of C_i near the Mn₄CaO₅ cluster is excluded on the basis of X-ray crystallography (3, 14), FTIR spectroscopy (16), and mass spectrometry (17, 18), the possibility that a weakly bound affects the activity of PSII at the donor side remains a viable option (reviewed in refs. 10 and 19).In the present study using higher plant PSII membranes, we specifically evaluate a recently suggested role of weakly bound , namely, that it acts as an acceptor for, and transporter of, protons produced by water splitting in the OEC (20–22).     

Detection of an intermediary,protonated water cluster in photosynthetic oxygen evolution

In photosynthesis, photosystem II evolves oxygen from water by the accumulation of photooxidizing equivalents at the oxygen-evolving complex (OEC). The OEC is a Mn₄CaO₅ cluster, and its sequentially oxidized states are termed the S_n states. The dark-stable state is S₁, and oxygen is released during the transition from S₃ to S₀. In this study, a laser flash induces the S₁ to S₂ transition, which corresponds to the oxidation of Mn(III) to Mn(IV). A broad infrared band, at 2,880 cm⁻¹, is produced during this transition. Experiments using ammonia and ²H₂O assign this band to a cationic cluster of internal water molecules, termed “W₅⁺.” Observation of the W₅⁺ band is dependent on the presence of calcium, and flash dependence is observed. These data provide evidence that manganese oxidation during the S₁ to S₂ transition results in a coupled proton transfer to a substrate-containing, internal water cluster in the OEC hydrogen-bonded network.Internal proton transfer reactions play important catalytic roles in many integral membrane proteins. In these enzymes, including bacteriorhodopsin, light-driven or redox-coupled proton-transfer reactions lead to the production of a transmembrane, electrochemical gradient. Amino acid side chains often participate in the acid/base chemistry that occurs in proton-transfer pathways. However, internal bound water clusters can also play essential roles as proton donors or acceptors (reviewed in ref. 1).In photosystem II (PSII), proton transfer contributes to the generation of a transmembrane potential, and chemical protons are released from the substrate, water, during the light-driven reactions that produce molecular oxygen (2). PSII is a complex membrane protein consisting of both integral, membrane-spanning subunits and extrinsic subunits (3). A monomeric unit of PSII consists of at least 20 distinct protein subunits, which are composed of 17 integral subunits and 3 extrinsic polypeptides (4, 5). The primary subunits that make up the reaction center and bind most of the redox-active cofactors are D1, D2, CP43, and CP47. The light-induced electron transfer pathway in the reaction center involves the dimeric chlorophyll (chl) donor, P₆₈₀, and accessory chl molecules. One light-induced charge separation oxidizes the primary donor, P₆₈₀, and reduces a bound plastoquinone acceptor, Q_A. P₆₈₀⁺ oxidizes a tyrosine residue, YZ, Y161 of the D1 polypeptide, which is a powerful oxidant. YZ• oxidizes the oxygen-evolving complex (OEC) on each photoinduced charge separation (reviewed in ref. 6).The OEC is a Mn₄CaO₅ cluster (Fig. 1 A, Inset) (5). Oxygen release from the OEC fluctuates with period four (7). The OEC cycles through five sequentially oxidized states, called the S_n states. A single flash given to a dark-adapted sample (S₁ state) generates the S₂ state (Fig. 1A), which corresponds to the oxidation of Mn(III) to Mn(IV) (8). Subsequent flashes advance the remaining manganese ions to higher oxidation states, with an accompanying deprotonation of two bound water molecules. The O–O bond is formed, and oxygen is evolved during the transition from S₃ to S₀ (Fig. 1A). Despite decades of study, many aspects of the water-oxidation mechanism remain to be elucidated. In this work, we obtain previously unknown information concerning proton-coupled electron transfer reactions during the S₁-to-S₂ and other S-state transitions.Open in a separate windowFig. 1.Photosynthetic water oxidation and protonation of a water cluster during the S₁-to-S₂ transition. (A) S-state cycle of photosynthetic water oxidation (7). (A, Inset) Predicted hydrogen-bond network of water molecules in the OEC of PSII (5). Amino acids are shown as sticks. Oxygen atoms of water molecules are shown in blue, with hydrogen bonds to peptide carbonyl groups shown as dashed lines. (B) Mechanism proposed for deprotonation of a terminal water ligand during the S₁-to-S₂ transition in PSII (14). Hydrogen bonds are shown as dashed lines. Putative substrate water molecules are shown in blue. (C) Schematic diagram showing the formation of a cationic water cluster, W₅⁺, on the S₁ to S₂ transition in PSII. (D) Diagram showing method generating the reaction-induced FTIR spectrum, corresponding to the S₂-minus-S₁ spectrum. Difference FTIR spectra for the other S-state transitions are produced with two (S₃-minus-S₂), three (S₀-minus-S₃), or four (S₁–S₀) flashes (see cycle in A).Many mechanisms have been proposed for photosynthetic oxygen evolution (reviewed in refs. 9–13). Fig. 1B shows a possible mechanism for the S₁-to-S₂ transition based on quantum mechanics (QM)/molecular mechanics (MM) calculations (ref. 14; but also see ref. 13). The calcium ion in the metal cluster has been proposed to bind a substrate water molecule and to activate the substrate (15–18). Deprotonation of terminal water ligands is important in decreasing the midpoint potential necessary for oxygen evolution, which is mediated by YZOH/YZ• (midpoint potential; 1V vs. normal hydrogen electrode) and which, therefore, occurs with a low driving force (19).A hydrogen-bonding network containing bound water molecules (Fig. 1 A, Inset) has been assigned in a recent X-ray structure (5). This network has been hypothesized to play a role in the water-oxidizing cycle (18, 20). Recent reaction-induced FTIR studies of the S₁-to-S₂ transition showed that the frequencies of hydrogen-bonded amide C=O groups were markers of hydrogen-bonding changes in the network. In another approach, the recombination kinetics of YZ• were used as a probe of electrostatic changes in the network in the S₂ and S₀ states (21, 22). In both studies, ammonia, a substrate-based inhibitor (23–25), was used to perturb hydrogen bonding in the OEC and was shown to have significant effects on the spectroscopic signals (18, 20, 22).Proton-coupled electron-transfer reactions occur during the S-state cycle (11, 12). Although the S₁-to-S₂ transition is not accompanied by a net proton release to sucrose-containing buffers, proton release accompanies the other S-state transitions (26). Proton-transfer pathways have been proposed based on site-directed mutagenesis and the 3D arrangement of amino acid side chains (5, 27, 28). However, the idea that the water network itself may act as a proton acceptor has not yet been critically evaluated. Spectroscopic signals from protonated water clusters (Fig. 1C) have been identified in model compounds (29) and in proteins (30–33). The OH frequencies of these clusters are red-shifted from bulk water, with a frequency related to the size of the cluster (34, 35). For example, in bacteriorhodopsin, a cluster of internal water molecules acts as a proton donor during the L-to-M transition (36, 37).Here, spectroscopic evidence for the formation of a cationic water cluster, termed W₅⁺, during the S₁-to-S₂ transition is presented (Fig. 1C). The results suggest that deprotonation of a terminal water ligand or a μ–OH bridge occurs on this transition, that the W₅ water cluster acts as a proton acceptor, and that this proton is not released to bulk solvent until later S states. The formation of the protonated cluster is shown to be dependent on temperature, S state, and calcium, consistent with a role for an internal water cluster in photosynthetic oxygen evolution.     

From the Cover: Water access points and hydration pathways in CLC H+/Cl? transporters

CLC transporters catalyze transmembrane exchange of chloride for protons. Although a putative pathway for Cl⁻ has been established, the pathway of H⁺ translocation remains obscure. Through a highly concerted computational and experimental approach, we characterize microscopic details essential to understanding H⁺-translocation. An extended (0.4 µs) equilibrium molecular dynamics simulation of membrane-embedded, dimeric ClC-ec1, a CLC from Escherichia coli, reveals transient but frequent hydration of the central hydrophobic region by water molecules from the intracellular bulk phase via the interface between the two subunits. We characterize a portal region lined by E202, E203, and A404 as the main gateway for hydration. Supporting this mechanism, site-specific mutagenesis experiments show that ClC-ec1 ion transport rates decrease as the size of the portal residue at position 404 is increased. Beyond the portal, water wires form spontaneously and repeatedly to span the 15-Å hydrophobic region between the two known H⁺ transport sites [E148 (Glu_ex) and E203 (Glu_in)]. Our finding that the formation of these water wires requires the presence of Cl⁻ explains the previously mystifying fact that Cl⁻ occupancy correlates with the ability to transport protons. To further validate the idea that these water wires are central to the H⁺ transport mechanism, we identified I109 as the residue that exhibits the greatest conformational coupling to water wire formation and experimentally tested the effects of mutating this residue. The results, by providing a detailed microscopic view of the dynamics of water wire formation and confirming the involvement of specific protein residues, offer a mechanism for the coupled transport of H⁺ and Cl⁻ ions in CLC transporters.The chloride channel (CLC) family (1, 2) includes both passive Cl⁻ channels and secondary active H⁺-coupled Cl⁻ transporters (3–8). The latter, also known as H⁺/Cl⁻ exchangers, drive uphill movement of H⁺ by coupling the process to downhill movement of Cl⁻ or vice versa, thereby exchanging the two types of ions across the membrane at fixed stoichiometry (9). ClC-ec1, a CLC from Escherichia coli, has served as the prototype CLC for biophysical studies because of its known crystal structures (10, 11), its tractable biochemical behavior, and its structural and mechanistic similarities to mammalian CLC transporters (3–8, 12–17). Detailed structural and functional studies of ClC-ec1 (9, 11, 18–27) have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl⁻ permeation pathway and its lining residues (10, 18, 25) and established the role of E148, also known as Glu_ex, as the extracellular gate for the Cl⁻ pathway (9, 11).Although much less is known about the H⁺ translocation pathway (and mechanism), experimental studies have provided key information on the involvement of specific residues in H⁺ transport (9, 13, 14, 20, 22, 27, 28). Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H⁺ transport (Fig. 1A): E148 (Glu_ex), which acts as the main extracellular H⁺ binding site (9, 11, 27), and E203 (Glu_in), which plays a similar role on the cytoplasmic side (20, 22, 28). Neutralization of either glutamate eliminates H⁺ translocation by ClC-ec1 (9, 28). However, the discovery of these H⁺ binding sites also raised a mechanistic puzzle (3, 23): How do protons translocate between the two sites, which are separated by a ∼15-Å-long, largely hydrophobic region within the lumen of the protein?Open in a separate windowFig. 1.Cl⁻ and H⁺ permeation pathways in ClC-ec1. (A) View of the ClC-ec1 structure in a lipid bilayer (the simulation system used here), with the identical subunits shown in yellow and orange. The presumed Cl⁻/H⁺ permeation pathways are indicated by green and red lines, respectively. The dashed segment of the red line denotes the pathway investigated in this study. (B) Close-up of the central hydrophobic region, with the residues forming this region shown as orange sticks and labeled. Also shown are key glutamate residues (E202, E203, and E148) as well as the Cl⁻ at the central anion binding site. (C) Hydration of the central hydrophobic region during the 0.4-µs equilibrium simulation, measured as the number of water molecules in this region for each subunit.Since the report of its first crystal structure, a large number of computational studies have aimed at investigating various molecular details related to the CLC H⁺ transport mechanism (27, 29–34). One model emerging from these studies proposes that water molecules may connect the two H⁺ sites (Glu_ex and Glu_in) and, thereby, facilitate H⁺ transport (29, 30, 34). This idea was initially proposed by Kuang and coworkers (29) on the basis of a hole-searching algorithm applied to static crystal structures of ClC-ec1. In their proposed pathway, water molecules are suggested to form two half-wires that are then connected by the hydroxyl group of Y445 to form a complete path for H⁺ transfer. However, it is known from experiments on the Y445F mutant that this hydroxyl is not required for H⁺ transport (20). Wang and Voth (30) proposed another pathway by combining an improved search algorithm for buried water with short molecular dynamics (MD) simulations, thereby taking into account the dynamic nature of the protein. Their pathway did not rely on Y445 but required reorientation of the side chain of E203 to connect the two H⁺ sites. In another study, these investigators further carried out semiempirical free energy calculations to investigate the Cl⁻/H⁺ coupling mechanism (33).Although the idea of water-mediated H⁺ transport is intriguing and could be key to understanding H⁺ transport in ClC-ec1, several questions relevant to a water wire mechanism remain unanswered: Can the hydrophobic region between the two H⁺ sites actually be hydrated under equilibrium conditions? What is the access/entry point or points for water from the bulk into the hydrophobic region, which is buried inside the protein, approximately at the midpoint of the membrane? Is it possible to observe the spontaneous formation of water wires through MD simulations? If so, how much do the simulated wire structures differ from the ones proposed by the prior studies based on search algorithms? How could the protein affect the dynamics and/or the thermodynamics of water wires?In the current study, we have addressed these questions through a combined computational and experimental approach. An extended 0.4-µs MD simulation of a membrane-embedded model of wild-type (WT) ClC-ec1 reveals that the central hydrophobic region can indeed be hydrated by water molecules mainly from the cytoplasmic bulk phase through pathways near the dimer interface via a portal lined by residues E202, E203, and A404. Water wires connecting the two H⁺ sites form spontaneously and repeatedly during the equilibrium simulation. Formation of wires requires a side-chain conformational change of I109 and the occupancy of the central Cl⁻ binding site, S_cen. These simulation results make two strong and testable predictions: that mutations at A404 and I109 will reduce ClC-ec1 activity and that the reduction in activity occurs via effects on the H⁺ branch of the transport mechanism. Our experimental tests and additional simulations performed on one mutant form of the protein fully support these predictions.     

Base-enhanced catalytic water oxidation by a carboxylate–bipyridine Ru(II) complex

In aqueous solution above pH 2.4 with 4% (vol/vol) CH₃CN, the complex [Ru^II(bda)(isoq)₂] (bda is 2,2′-bipyridine-6,6′-dicarboxylate; isoq is isoquinoline) exists as the open-arm chelate, [Ru^II(CO₂-bpy-CO₂⁻)(isoq)₂(NCCH₃)], as shown by ¹H and ¹³C-NMR, X-ray crystallography, and pH titrations. Rates of water oxidation with the open-arm chelate are remarkably enhanced by added proton acceptor bases, as measured by cyclic voltammetry (CV). In 1.0 M PO₄^3–, the calculated half-time for water oxidation is ∼7 μs. The key to the rate accelerations with added bases is direct involvement of the buffer base in either atom–proton transfer (APT) or concerted electron–proton transfer (EPT) pathways.Metal-complex catalyzed water oxidation continues to evolve with new catalysts and new mechanistic insights (1–9). Studies on single-site Ru catalysts such as [Ru^II(Mebimpy)(bpy)(OH₂)]²⁺ [Mebimpy is 2,6-bis(1-methylbenzimidazol-2-yl)pyridine; bpy is 2,2′-bipyridine; Fig. 1], both in solution and on surfaces, reveal mechanisms in which stepwise oxidative activation of aqua precursors to Ru^V=O is followed by rate-limiting O–O bond formation (10–15). The results of kinetic and mechanistic studies have revealed the importance of concerted atom–proton transfer (APT) in the O–O bond-forming step. In APT, the O–O bond forms in concert with H⁺ transfer to water or to an added base (11, 12, 16–19). APT can promote dramatic rate enhancements. In a recent study on surface-bound [Ru(Mebimpy)(4,4′-((HO)₂OPCH₂)₂bpy)(OH₂)]²⁺ [4,4′-((HO)₂OPCH₂)₂bpy is 4,4′-bis-methlylenephosphonato-2,2′-bipyridine] stabilized by atomic layer deposition, a rate enhancement of ∼10⁶ was observed with 0.012 M added PO₄³⁻ at pH 12 compared with oxidation at pH 1 (20).Open in a separate windowFig. 1.Structures of [Ru^II(Mebimpy)(bpy)(OH₂)]²⁺ (Left) and [Ru^II(CO₂-bpy-CO₂)(isoq)₂] [1] (Right).Sun and coworkers (21, 22) have described the Ru single-site water oxidation catalysts, [Ru^II(bda)(L)₂] (H₂bda is 2,2′-bipyridine-6,6′-dicarboxylic acid, HCO₂-bpy-CO₂H; L is isoquinoline, 4-picoline, or phthalazine). They undergo rapid and sustained water oxidation catalysis with added Ce^IV. A mechanism has been proposed in which initial oxidation to seven coordinate Ru^IV is followed by further oxidation to Ru^V(O) with O–O coupling to give a peroxo-bridged intermediate, Ru^IVO–ORu^IV, which undergoes further oxidation and release of O₂ (21, 22). We report here the results of a rate and mechanistic study on electrochemical water oxidation by complex [1], [Ru^II(CO₂-bpy-CO₂)(isoq)₂] (isoq is isoquinoline) (Fig. 1). Evidence is presented for water oxidation by a chelate open form in acidic solutions. The chelate open form displays dramatic rate enhancements with added buffer bases, and the results of a detailed mechanistic study are reported here.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O₂^•−) dismutase-like properties yet were inert to nitric oxide (NO^•) as well as peroxynitrite (ONOO⁻). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO₂ and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O₂^•− to O₂ by PEG-HCCs at >20,000 s⁻¹. The major products of this catalytic turnover are O₂ and H₂O₂, making the PEG-HCCs a biomimetic superoxide dismutase.Reactive oxygen species (ROS), such as superoxide (O₂^•−), hydrogen peroxide (H₂O₂), organic peroxides, and hydroxyl radical (^•OH), are a consequence of aerobic metabolism (1, 2). These ROS are necessary for the signaling pathways in biological processes (3, 4) such as cell migration, circadian rhythm, stem cell proliferation, and neurogenesis (5). In healthy systems, ROS are efficiently regulated by the defensive enzymes superoxide dismutase (SOD) and catalase, and by antioxidants such as glutathione, vitamin A, ascorbic acid, uric acid, hydroquinones, and vitamin E (6). When the production of ROS overwhelms the scavenging ability of the defense system, oxidative stress occurs, causing dysfunctions in cell metabolism (7–16).In addition to ROS, reactive nitrogen species (RNS) such as nitric oxide (NO^•), nitrogen dioxide, and dinitrogen trioxide can be found in all organisms. NO^• can act as an oxidizing or reducing agent depending on the environment (17), is more stable than other radicals (half-life 4–15 s) (18), and is synthesized in small amounts in vivo (17–22). NO^• is a potent vasodilator and has an important role in neurotransmission and cytoprotection (17, 18, 22, 23). Owing to its biological importance and the low concentration found normally in vivo, it is often important to avoid alteration of NO^• levels in biological systems to prevent aggravation of acute pathologies including ischemia and reperfusion.One way to treat these detrimental pathologies is to supply antioxidant molecules or particles that renormalize the disturbed oxidative condition. We recently developed a biocompatible carbon nanoparticle, the poly(ethylene glycolated) hydrophilic carbon cluster (PEG-HCC), which has shown ability to scavenge oxyradicals and protect against oxyradical damage in rodent models and thus far has demonstrated no in vivo toxicity in laboratory rodents (24–27). The carbon cores of PEG-HCCs are ∼3 nm wide and range from 30 to 40 nm long. Based on these data, we estimate that there are 2,000–5,000 sp² carbon atoms on each PEG-HCC core. We have demonstrated the efficacy of PEG-HCCs for normalizing in vivo O₂^•− in models of traumatic brain injury with concomitant hypotension. Simultaneously, we observed normalization in NO^• levels in these experiments (26, 27). A better understanding of these materials is necessary to potentially translate these therapeutic findings to the clinic.In the present work, we evaluated antioxidant properties of PEG-HCCs. Using spin-trap EPR spectroscopy, we demonstrate that PEG-HCCs scavenge O₂^•− with high efficiency. X-ray photoelectron spectroscopy (XPS) indicates that covalent addition of ROS to the PEG-HCCs is not responsible for the observed activity. Direct measurement of O₂^•− concentration using freeze-trap EPR demonstrates that PEG-HCCs behave as catalysts, and measurements made with a Clark oxygen electrode during the reaction reveal that the rate of production of O₂ is above that expected due to self-dismutation of O₂^•− in water. An equivalent amount of H₂O₂ is also simultaneously produced. Finally, selectivity for ROS is confirmed using a hemoglobin and a pyrogallol red assay; PEG-HCCs are unreactive to both NO^• and ONOO⁻. These results clarify the fundamental processes involved in the previously observed in vivo protection against oxygen damage (26, 27).     

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2^−/− mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.Due to its unique chemistry, the redox-active metal ion copper (Cu) is an essential element for human growth and development (1–3). Defects in Cu metabolism are associated with pathologies that include Alzheimer’s disease, peripheral neuropathy, anemia, neutropenia, cardiomyopathy, Menkes disease, and Wilson’s disease (4–9). Although many of the components responsible for Cu uptake, intracellular distribution, detoxification, and efflux have been identified, the mechanisms by which these proteins are regulated are not well understood.The copper transporter 1 (Ctr1) protein is a high-affinity Cu⁺ transporter that functions in copper accumulation in organisms ranging from yeast to mammals (10–16). In mammals Ctr1 localizes to both the plasma membrane and to intracellular vesicles (17–19). Mice bearing a systemic Ctr1 deletion fail to survive gestation, whereas tissue-specific ablation of Ctr1 in the intestinal epithelium, liver, or heart cause a range of phenotypes that include peripheral Cu deficiency, hepatic iron accumulation, and lethal cardiac hypertrophy, respectively (20–24). Moreover, both yeast and mammalian Ctr1 function in acquisition of the chemotherapeutic agent cisplatin (25–29) and Ctr1 expression levels have been correlated to the efficacy of chemotherapy and patient survival (30). The regulation of Ctr1 function and abundance is of great significance to both normal growth and development as well as to the efficacy of platinum-based chemotherapy.The general structure and function of Ctr1 is conserved from yeast to humans, with three membrane-spanning domains and a Met-X₃-Met motif in the second transmembrane domain that is essential for Cu⁺ import (16). The human and mouse protein contains a short ecto-domain with clusters of Met and His. Mutagenic and truncation studies in the context of intact yeast or human Ctr1 indicate that the ecto-domain in general, and the Met residues in particular, play an important role in high-affinity cellular Cu⁺ import, yet all but one key Met near the first transmembrane domain appear to be dispensable for function in cellular Cu⁺ import (31). Studies using model peptides suggest that the Ctr1 Met residues are direct ligands for both Cu⁺ and cisplatin (32–34). In contrast to Cu⁺ uptake, the Met-rich ecto-domain of yeast Ctr1 is required for cisplatin import (27). Moreover, as Ctr1 M-X₃-M mutants are competent for cisplatin uptake, but not Cu⁺ (35), studies suggest that Ctr1-mediated cisplatin uptake may occur via an ecto-domain–dependent receptor-mediated endocytosis mechanism, rather than as an ion channel as for Cu⁺ (27). Ctr1 has been observed in both cell lines and mouse tissues as a full-length glycosylated form and a lower-molecular-weight form, which has been reported to lack a portion of the Cu⁺ and cisplatin-binding ecto-domain (17, 36). However, neither the physiological significance of this truncated form of Ctr1, nor its mode of biogenesis, have been elucidated.The Ctr2 protein is structurally related to Ctr1 and is encoded by a linked gene in both the mouse and the human genome. Recent studies suggest that Ctr2 functions as a low-affinity Cu⁺ importer, a lysosomal Cu⁺ exporter, or as a regulator of cellular macropinocytosis (37–39). However, these studies have been performed in cultured cells, and the physiological role of Ctr2 in animals has not been reported. Here we demonstrate that Ctr2 interacts with Ctr1 in vivo and that Ctr2 knockout mice show increased levels of total copper in several tissues. Mice and mouse embryonic fibroblasts lacking Ctr2 accumulate copper in endosomal compartments and have lower levels of the truncated form of Ctr1 lacking the metal-binding ecto-domain. Whereas truncation of the Ctr1 ecto-domain reduces Cu⁺ import at the plasma membrane, truncated Ctr1 stimulates the mobilization of Cu⁺ from endosomal compartments. These studies demonstrate a critical role for Ctr2 in modulating the accumulation of Ctr1 lacking the Cu⁺ and cisplatin-binding ecto-domain of Ctr1 and, as a consequence, in the regulation of cellular copper uptake and intracellular mobilization. Given the fundamental role for Ctr1 in Cu⁺ import and cisplatin acquisition, the action of Ctr2 represents an important mechanism for the regulation of Ctr1 function.     

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.Since the invention of the Haber–Bosch process, the amount of fixed nitrogen in soils and waters has been increasing, and this trend has significant impact on the global environment (1, 2). Fixed nitrogen is oxidized to nitrite (NO₂⁻) or nitrate (NO₃⁻) by nitrification and then converted to gaseous dinitrogen (N₂) by microbial denitrification, which closes the nitrogen cycle. Microorganisms involved in denitrification couple their respiratory systems to stepwise reduction of nitrogen oxides to N₂ (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (3, 4). The reduction of NO₂⁻ to toxic nitric oxide (NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O) is referred to as the key step in denitrification and catalyzed by either cd₁-heme nitrite reductase (cd₁NiR) or copper nitrite reductase (CuNiR) (3, 4). Although the catalytic mechanism of cd₁NiR is well understood (5, 6), that of CuNiR is controversial (7). CuNiR is a homotrimeric protein containing two distinct Cu sites per monomer (SI Appendix, Fig. S1). Type 1 Cu (T1Cu) with a Cys–Met–His₂ ligand set is an electron acceptor incorporated near the molecular surface, whereas type 2 Cu (T2Cu) with a His₃ ligand set is a catalytic center, which is ∼12 Å distant from the molecular surface and located between two adjacent monomers (7, 8). Spaced ∼12.5 Å apart, the two Cu sites are linked by a Cys–His bridge and a sensor loop. Whereas the Cys–His bridge is an electron pathway, the sensor loop is thought to control electron distribution between T1Cu and T2Cu (9).Two conserved residues, Asp98 and His255 (Alcaligenes faecalis numbering), are located above the T2Cu site and bridged by a water molecule called bridging water (SI Appendix, Fig. S1). They are essential to the CuNiR activity because they assist proton transfer (PT) to the substrate (10–12). Although intramolecular electron transfer (ET) from T1Cu to T2Cu can occur in the resting state (RS) (13, 14), the differences in the redox potentials of T2Cu minus T1Cu are small and sometimes negative in the absence of NO₂⁻, meaning that intramolecular ET before NO₂⁻ binding is not energetically favorable (15, 16). By contrast, intramolecular ET is dramatically accelerated in the presence of NO₂⁻ (15, 17). An explanation for this gating-like phenomenon is that substrate binding raises the redox potential of T2Cu and shifts the equilibrium of the ET reaction (16). However, pH dependence of intramolecular ET in the presence of NO₂⁻ cannot be explained by such a change of redox potentials (15). Instead, Kobayashi et al. (15) proposed that reduction-induced structural change of His255 is responsible for the gating-like mechanism. Because it has been recently proven that intramolecular ET in CuNiR is accompanied by PT and hence proton-coupled ET (PCET) (17, 18), one can readily speculate that intramolecular ET contributes PT to NO₂⁻ and that the structural change of His255 is involved in PCET. Crystal structures of CuNiR from Rhodobacter sphaeroides (RhsNiR) implies this possibility because His287 in RhsNiR, which corresponds to His255, seems to show pH- and redox-dependent conformational changes (19, 20). However, presumably because of X-ray radiation damages implied by rerefinement of RhsNiR structures (21), electron density around His287 was so unusual that interpretation of it is difficult (SI Appendix, Fig. S2).Crystal structures determined by synchrotron radiation crystallography (SRX) have provided insights into the enzymatic mechanism of CuNiR (22–25), and these studies are summarized elsewhere (7). High-resolution nitrite complex (NC) structures revealed an O-coordination of NO₂⁻ showing a near face-on binding mode (22, 23), whereas Cu(II)-NO₂⁻ model complexes show a vertical binding mode (7, 26–29). The near face-on coordination manner is thought to facilitate its conversion to side-on NO, which was observed in the crystal structures of CuNiR exposed to NO (22, 23, 25). Skeptical eyes have, however, been cast on these CuNiR structures because SRX data might be affected by some problems connected to the high radiation dose delivered on the crystals. First, strong synchrotron X-rays cause not only radiation damages to amino acid residues but also photoreduction of metalloproteins (30, 31). Although a comparison between oxidized and reduced states is necessary to closely investigate redox reactions, completely oxidized structures are almost impossible to determine by SRX. Indeed, the Cu centers in CuNiR are rapidly reduced by exposure to synchrotron X-rays (21, 32). Second, following the photoreduction of T2Cu, NO₂⁻ is easily reduced and produces NO and water in SRX (21). Consequently, electron density at the catalytic site of an NC structure is derived from the mixture of both substrate and product, making interpretation of data complicated and unreliable. Third, cryogenic manipulations for reducing radiation damages in SRX have also been focused as a factor that changes the population of amino acid residues (33, 34) and enzyme–substrate complexes (35). Crystallographic (36), computational (37), and spectroscopic (38–40) studies actually show that binding modes of NO₂⁻ and NO in CuNiR crystal structures can differ from those in physiological environments.We here ventured to use photoreduction in SRX to initiate a chemical reaction and to trap an enzymatically produced intermediary state (30, 31). Furthermore, to visualize intact CuNiR structures in the resting and NC states, we applied serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) (41), which enables damage-free structural determination of metalloproteins (42, 43) and evaluation of the native conformational population at room temperature (RT) (44). By comparing SRX and SFX data, we discuss PCET and nitrite reduction in CuNiR.     

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

The inositol 1,4,5-trisphosphate receptor (IP₃R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP₃Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP₃ at a distance. Defective allostery in IP₃R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP₃R type 1 (IP₃R1) and negatively regulates IP₃R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP₃R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.Ligand-gated ion channels function by allostery that is the regulation at a distance; the allosteric coupling of ligand binding with channel gating requires reversible changes in subunit configurations and conformations (1). Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ligand-gated ion channels that release calcium ions (Ca²⁺) from the endoplasmic reticulum (ER) (2, 3). IP₃Rs are allosteric proteins comprising four subunits that assemble a calcium channel with fourfold symmetry about an axis perpendicular to the ER membrane. The subunits of three IP₃R isoforms (IP₃R1, IP₃R2, and IP₃R3) are structurally divided into three domains: the IP₃-binding domain (IBD), the regulatory domain, and the channel domain (3–6). Fitting of the IBD X-ray structures (7, 8) to a cryo-EM map (9) indicates that the IBD activates a remote Ca²⁺ channel by allostery (8); however, the current X-ray structure only spans 5% of each tetramer, such that the mechanism underlying allosteric coupling of the IBD to channel gating remains unknown.The IP₃R in the ER mediates intracellular calcium signaling that impinges on homeostatic control in various subsequent intracellular processes. Deletion of the genes encoding the type 1 IP₃R (IP₃R1) leads to perturbations in long-term potentiation/depression (3, 10, 11) and spinogenesis (12), and the human genetic disease spinocerebellar ataxia 15 is caused by haploinsufficiency of the IP₃R1 gene (13–15). Dysregulation of IP₃R1 is also implicated in neurodegenerative diseases including Huntington disease (HD) (16–18) and Alzheimer’s disease (AD) (19–22). IP₃Rs also control fundamental cellular processes—for example, mitochondrial energy production (23, 24), autophagy regulation (24–27), ER stress (28), hepatic gluconeogenesis (29), pancreatic exocytosis (30), and macrophage inflammasomes (31). On the other hand, excessive IP₃R function promotes cell death processes including apoptosis by activating mitochondrial or calpain pathways (2, 17). Considering these versatile roles of IP₃Rs, appropriate IP₃R structure and function are essential for living systems, and aberrant regulation of IP₃R closely relates to various diseases.Several factors such as cytosolic molecules, interacting proteins, and posttranslational modifications control the IP₃-induced Ca²⁺ release (IICR) through allosteric sites in IP₃Rs. Cytosolic Ca²⁺ concentrations strictly control IICR in a biphasic manner with activation at low concentrations and inhibition at higher concentrations. The critical Ca²⁺ sensor for activation is conserved among the three isoforms of IP₃ and ryanodine receptors, and this sensor is located in the regulatory domain outside the IBD and the channel domain (32). A putative ATP regulatory region is deleted in opisthotonos mice, and IICR is also regulated by this mutation in the regulatory domain (33). Various interacting proteins, such as cytochrome c, Bcl-2-family proteins, ataxin-3, huntingtin (Htt) protein, Htt-associated protein 1A (HAP1A), and G-protein–coupled receptor kinase-interacting protein 1 (GIT1), target allosteric sites in the carboxyl-terminal tail (3–5). The regulatory domain and the carboxyl-terminal tail also undergo phosphorylation by the protein kinases A/G and B/Akt and contain the apoptotic cleavage sites for the protease caspase-3 (4, 5). These factors allosterically regulate IP₃R structure and function to control cellular fates; therefore, understanding the allosteric coupling of the IBD to channel gating will elucidate the regulatory mechanism of these factors.Transglutaminase (TG) catalyses protein cross-linking between a glutamine (Gln) residue and a lysine (Lys) residue via an N^ε-(γ-glutamyl)lysine isopeptide bond (34, 35). TG type 2 (TG2) is a Ca²⁺-dependent enzyme with widespread distribution and is highly inducible by various stimulations such as oxidative stress, cytokines, growth factors, and retinoic acid (RA) (34, 35). TG2 is considered a significant disease-modifying factor in neurodegenerative diseases including HD, AD, and Parkinson’s diseases (PD) (34, 36–45) because TG2 might enzymatically stabilize aberrant aggregates of proteins implicated in these diseases—that is, mutant Htt, β-amyloid, and α-synuclein; however, the causal role of TG2 in Ca²⁺ signaling in brain pathogenesis has been unclear. Ablation of TG2 in HD mouse models is associated with increased lifespan and improved motor function (46, 47). However, TG2 knockout mice do not show impaired Htt aggregation, suggesting that TG2 may play a causal role in these disorders rather than TG2-dependent cross-links in aberrant protein aggregates (47, 48).In this study, we discovered a new mode of chronic and irreversible allosteric regulation in IP₃R1 in which covalent modification of the receptor at Gln2746 is catalyzed by TG2. We demonstrate that up-regulation of TG2 modifies IP₃R1 structure and function in HD models and propose an etiologic role of this modification in the reduction of neuronal signaling and subsequent processes during the prodromal state of the neurodegenerative disease.     

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Proteins containing C2 domains are the sensors for Ca²⁺ and PI(4,5)P₂ in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca²⁺ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P₂ and IP₃ at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3–β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P₂/PI(3,4,5)P₃, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P₂. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca²⁺-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (1–4). Many of these C2 domains have been demonstrated to function in a Ca²⁺-dependent membrane-binding manner and hence act as cellular Ca²⁺ sensors. Calcium ions bind in a cup-shaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca²⁺ ions are incomplete (5–7). This incomplete coordination sphere can be occupied by neutral and anionic (7–9) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P₂ simultaneously (10). This revealed an additional lipid-binding site located in the polybasic region formed by β3–β4 strands that preferentially binds to PI(4,5)P₂ (11–15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10, 16–19). Given the importance of PI(4,5)P₂ for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20–23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca²⁺ and PI(4,5)P₂ combine to orchestrate the vesicle fusion and repriming processes by acting through the two C2 domains existing in each of these proteins (16, 20, 22, 24–28). A myriad of works have explored the 3D structure of the individual C2 domains of both synaptotagmins and rabphilin 3A (5, 26, 27, 29, 30). However, the impossibility of obtaining crystal structures of these domains in complex with Ca²⁺ and phosphoinositides has hindered the understanding of the molecular mechanism driving the PI(4,5)P₂–C2 domain interaction. Here, we sought to unravel the molecular mechanism of Ca²⁺ and PI(4,5)P₂ binding to the C2A domain of rabphilin 3A by X-ray crystallography. A combination of site-directed mutagenesis together with isothermal titration calorimetry (ITC), fluorescence resonance of energy transfer (FRET), and aggregation experiments has enabled us to propose a molecular mechanism of Ca²⁺/PI(4,5)P₂-dependent membrane interaction through two different motifs that could bend the membrane and accelerate the vesicle fusion process. A comparative analysis revealed the structural basis for the different phosphoinositide affinities of C2A and -B domains. Furthermore, the C2A domain of synaptotagmin 1 lacks one of the key residues responsible for the PI(4,5)P₂ interaction, confirming it is a non-PI(4,5)P₂ responder.