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.     

Splitting CO2 into CO and O2 by a single catalyst

The metal complex [(tpy)(Mebim-py)Ru^II(S)]²⁺ (tpy = 2,2^′ : 6^′,2^′′-terpyridine; Mebim-py = 3-methyl-1-pyridylbenzimidazol-2-ylidene; S = solvent) is a robust, reactive electrocatalyst toward both water oxidation to oxygen and carbon dioxide reduction to carbon monoxide. Here we describe its use as a single electrocatalyst for CO₂ splitting, CO₂ → CO + 1/2 O₂, in a two-compartment electrochemical cell.     

A quantum-chemical picture of hemoglobin affinity

Understanding the molecular mechanism of hemoglobin cooperativity remains an enduring challenge. Protein forces that control ligand affinity are not directly accessible by experiment. We demonstrate that computational quantum mechanics/molecular mechanics methods can provide reasonable values of ligand binding energies in Hb, and of their dependence on allostery. About 40% of the binding energy differences between the relaxed state and tense state quaternary structures result from strain induced in the heme and its ligands, especially in one of the pyrrole rings. The proximal histidine also contributes significantly, in particular, in the alpha-chains. The remaining energy difference resides in protein contacts, involving residues responsible for locking the quaternary changes. In the alpha-chains, the most important contacts involve the FG corner, at the "hinge" region of the alpha(1)beta(2) quaternary interface. The energy differences are spread more evenly among the beta-chain residues, suggesting greater flexibility for the beta- than for the alpha-chains along the quaternary transition. Despite this chain differentiation, the chains contribute equally to the relaxed substitute state energy difference. Thus, nature has evolved a symmetric response to the quaternary structure change, which is a requirement for maximum cooperativity, via different mechanisms for the two kinds of chains.     

Mechanism of thermal decomposition of carbamoyl phosphate and its stabilization by aspartate and ornithine transcarbamoylases

Carbamoyl phosphate (CP) has a half-life for thermal decomposition of <2 s at 100 degrees C, yet this critical metabolic intermediate is found even in organisms that grow at 95-100 degrees C. We show here that the binding of CP to the enzymes aspartate and ornithine transcarbamoylase reduces the rate of thermal decomposition of CP by a factor of >5,000. Both of these transcarbamoylases use an ordered-binding mechanism in which CP binds first, allowing the formation of an enzyme.CP complex. To understand how the enzyme.CP complex is able to stabilize CP we investigated the mechanism of the thermal decomposition of CP in aqueous solution in the absence and presence of enzyme. By quantum mechanics/molecular mechanics calculations we show that the critical step in the thermal decomposition of CP in aqueous solution, in the absence of enzyme, involves the breaking of the C O bond facilitated by intramolecular proton transfer from the amine to the phosphate. Furthermore, we demonstrate that the binding of CP to the active sites of these enzymes significantly inhibits this process by restricting the accessible conformations of the bound ligand to those disfavoring the reactive geometry. These results not only provide insight into the reaction pathways for the thermal decomposition of free CP in an aqueous solution but also show why these reaction pathways are not accessible when the metabolite is bound to the active sites of these transcarbamoylases.     

Aborted double bicycle-pedal isomerization with hydrogen bond breaking is the primary event of bacteriorhodopsin proton pumping

Quantum mechanics/molecular mechanics calculations based on ab initio multiconfigurational second order perturbation theory are employed to construct a computer model of Bacteriorhodopsin that reproduces the observed static and transient electronic spectra, the dipole moment changes, and the energy stored in the photocycle intermediate K. The computed reaction coordinate indicates that the isomerization of the retinal chromophore occurs via a complex motion accounting for three distinct regimes: (i) production of the excited state intermediate I, (ii) evolution of I toward a conical intersection between the excited state and the ground state, and (iii) formation of K. We show that, during stage ii, a space-saving mechanism dominated by an asynchronous double bicycle-pedal deformation of the C10═C11─C12═C13─C14═N moiety of the chromophore dominates the isomerization. On this same stage a N─H/water hydrogen bond is weakened and initiates a breaking process that is completed during stage iii.     

Experimental demonstration of radicaloid character in a RuV=O intermediate in catalytic water oxidation

Water oxidation is the key half reaction in artificial photosynthesis. An absence of detailed mechanistic insight impedes design of new catalysts that are more reactive and more robust. A proposed paradigm leading to enhanced reactivity is the existence of oxyl radical intermediates capable of rapid water activation, but there is a dearth of experimental validation. Here, we show the radicaloid nature of an intermediate reactive toward formation of the O-O bond by assessing the spin density on the oxyl group by Electron Paramagnetic Resonance (EPR). In the study, an ¹⁷O-labeled form of a highly oxidized, short-lived intermediate in the catalytic cycle of the water oxidation catalyst cis,cis-[(2,2-bipyridine)₂(H₂O)Ru^IIIORu^III(OH₂)(bpy)₂]⁴⁺ was investigated. It contains Ru centers in oxidation states [4,5], has at least one Ru^V = O unit, and shows |A_xx| = 60G ¹⁷O hyperfine splittings (hfs) consistent with the high spin density of a radicaloid. Destabilization of π-bonding in the d³ Ru^V = O fragment is responsible for the high spin density on the oxygen and its high reactivity.     

Synergic mechanism and fabrication target for bipedal nanomotors

Inspired by the discovery of dimeric motor proteins capable of undergoing transportation in living cells, significant efforts have been expended recently to the fabrication of track-walking nanomotors possessing two foot-like components that each can bind or detach from an array of anchorage groups on the track in response to local events of reagent consumption. The central problem in fabricating bipedal nanomotors is how the motor as a whole can gain the synergic capacity of directional track-walking, given the fact that each pedal component alone often is incapable of any directional drift. Implemented bipedal motors to date solve this thermodynamically intricate problem by an intuitive strategy that requires a hetero-pedal motor, multiple anchorage species for the track, and multiple reagent species for motor operation. Here we performed realistic molecular mechanics calculations on molecule-scale models to identify a detailed molecular mechanism by which motor-level directionality arises from a homo-pedal motor along a minimally heterogeneous track. Optimally, the operation may be reduced to a random supply of a single species of reagents to allow the motor's autonomous functioning. The mechanism suggests a distinct class of fabrication targets of drastically reduced system requirements. Intriguingly, a defective form of the mechanism falls into the realm of the well known Brownian motor mechanism, yet distinct features emerge from the normal working of the mechanism.     

Structure, initial excited-state relaxation, and energy storage of rhodopsin resolved at the multiconfigurational perturbation theory level

We demonstrate that a "brute force" quantum chemical calculation based on an ab initio multiconfigurational second order perturbation theory approach implemented in a quantum mechanics/molecular mechanics strategy can be applied to the investigation of the excited state of the visual pigment rhodopsin (Rh) with a computational error <5 kcal.mol(-1). As a consequence, the simulation of the absorption and fluorescence of Rh and its retinal chromophore in solution allows for a nearly quantitative analysis of the factors determining the properties of the protein environment. More specifically, we demonstrate that the Rh environment is more similar to the "gas phase" than to the solution environment and that the so-called "opsin shift" originates from the inability of the solvent to effectively "shield" the chromophore from its counterion. The same strategy is used to investigate three transient structures involved in the photoisomerization of Rh under the assumption that the protein cavity does not change shape during the reaction. Accordingly, the analysis of the initially relaxed excited-state structure, the conical intersection driving the excited-state decay, and the primary isolable bathorhodopsin intermediate supports a mechanism where the photoisomerization coordinate involves a "motion" reminiscent of the so-called bicycle-pedal reaction coordinate. Most importantly, it is shown that the mechanism of the approximately 30 kcal.mol(-1) photon energy storage observed for Rh is not consistent with a model based exclusively on the change of the electrostatic interaction of the chromophore with the protein/counterion environment.     

Highly efficient and robust molecular ruthenium catalysts for water oxidation

Water oxidation catalysts are essential components of light-driven water splitting systems, which could convert water to H₂ driven by solar radiation (H₂O + hν → 1/2O₂ + H₂). The oxidation of water (H₂O → 1/2O₂ + 2H⁺ + 2e^-) provides protons and electrons for the production of dihydrogen (2H⁺ + 2e^- → H₂), a clean-burning and high-capacity energy carrier. One of the obstacles now is the lack of effective and robust water oxidation catalysts. Aiming at developing robust molecular Ru-bda (H₂bda = 2,2^′-bipyridine-6,6′-dicarboxylic acid) water oxidation catalysts, we carried out density functional theory studies, correlated the robustness of catalysts against hydration with the highest occupied molecular orbital levels of a set of ligands, and successfully directed the synthesis of robust Ru-bda water oxidation catalysts. A series of mononuclear ruthenium complexes [Ru(bda)L₂] (L = pyridazine, pyrimidine, and phthalazine) were subsequently synthesized and shown to effectively catalyze Ce^IV-driven [Ce^IV = Ce(NH₄)₂(NO₃)₆] water oxidation with high oxygen production rates up to 286 s^-1 and high turnover numbers up to 55,400.     

Energetic basis of catalytic activity of layered nanophase calcium manganese oxides for water oxidation

Previous measurements show that calcium manganese oxide nanoparticles are better water oxidation catalysts than binary manganese oxides (Mn₃O₄, Mn₂O₃, and MnO₂). The probable reasons for such enhancement involve a combination of factors: The calcium manganese oxide materials have a layered structure with considerable thermodynamic stability and a high surface area, their low surface energy suggests relatively loose binding of H₂O on the internal and external surfaces, and they possess mixed-valent manganese with internal oxidation enthalpy independent of the Mn³⁺/Mn⁴⁺ ratio and much smaller in magnitude than the Mn₂O₃-MnO₂ couple. These factors enhance catalytic ability by providing easy access for solutes and water to active sites and facile electron transfer between manganese in different oxidation states.     

An atomistic picture of the regeneration process in dye sensitized solar cells

A highly efficient mechanism for the regeneration of the cis-bis(isothiocyanato)bis(2,2′-bipyridyl-4,4′-dicarboxylato)-ruthenium(II) sensitizing dye (N3) by I^- in acetonitrile has been identified by using molecular dynamics simulation based on density functional theory. Barrier–free complex formation of the oxidized dye with both I^- and , and facile dissociation of and from the reduced dye are key steps in this process. In situ vibrational spectroscopy confirms the reversible binding of I₂ to the thiocyanate group. Additionally, simulations of the electrolyte near the interface suggest that acetonitrile is able to cover the (101) surface of anatase with a passivating layer that inhibits direct contact of the redox mediator with the oxide, and that the solvent structure specifically enhances the concentration of I^- at a distance which further favors rapid dye regeneration.     

Revealing the catalytic potential of an acyl-ACP desaturase: tandem selective oxidation of saturated fatty acids

It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of “unusual” e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. ¹⁸O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O₂-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.     

Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase beta

Based on a recent ternary complex crystal structure of human DNA polymerase beta with a G:A mismatch in the active site, we carried out a theoretical investigation of the catalytic mechanism of incorrect nucleotide incorporation using molecular dynamics simulation, quantum mechanics, combined quantum mechanics, and molecular mechanics methods. A two-stage mechanism is proposed with a nonreactive active-site structural rearrangement prechemistry step occurring before the nucleotidyl transfer reaction. The free energy required for formation of the prechemistry state is found to be the major factor contributing to the decrease in the rate of incorrect nucleotide incorporation compared with correct insertion and therefore to fidelity enhancement. Hence, the transition state and reaction barrier for phosphodiester bond formation after the prechemistry state are similar to that for correct insertion reaction. Key residues that provide electrostatic stabilization of the transition state are identified.     

Enhanced localized dipole of Pt-Au single-site catalyst for solar water splitting

Solar water splitting is regarded as holding great potential for clean fuels production. However, the efficiency of charge separation/transfer of photocatalysts is still too low for industrial application. This paper describes the synthesis of a Pt-Au binary single-site loaded g-C₃N₄ nanosheet photocatalyst inspired by the concept of the dipole. The existent larger charge imbalance greatly enhanced the localized molecular dipoles over adjacent Pt-Au sites in contrast to the unary counterparts. The superposition of molecular dipoles then further strengthened the internal electric field and thus promoted the charge transportation dynamics. In the modeling photocatalytic hydrogen evolution, the optimal Pt-Au binary site photocatalysts (0.25% loading) showed 4.9- and 2.3-fold enhancement of performance compared with their Pt and Au single-site counterparts, respectively. In addition, the reaction barrier over the Pt-Au binary sites was lowered, promoting the hydrogen evolution process. This work offers a valuable strategy for improving photocatalytic charge transportation dynamics by constructing polynary single sites.

Solar water splitting has attracted worldwide attention due to its immense potential in clean fuel production. It is roughly composed of two processes: photogenerated charge separation/transfer and surface chemical reactions. The photogenerated charge separation and transfer is widely regarded as the rate-limiting step, and its low efficiency is the crucial challenge that needs to be addressed (1–3). Therefore, much effort has been devoted to developing new strategies in surface/interface engineering of photocatalysts to improve the dynamics of charge separation/transportation, and the key is to construct effective built-in electric fields or even tune the diffusion process of charges (1, 3, 4).Recently, single-atom or single-site catalysts (SACs) sparked considerable research interest in photocatalysis and many other heterogeneous catalytic processes (5–9). Generally, due to their unique electronic and geometric structures, single metal sites can improve the photocatalytic performance mainly by optimizing the charge evolved surface chemical reactions (e.g., the adsorption of reactants or charge transfer from catalysts to reactants) (10, 11). However, single metal sites do not obviously change the charge transportation process of the photocatalyst and thus hardly contribute to the enhancement of charge separation and transfer, which greatly limits the promotion of SACs for particulate photocatalysis. Inspired by the synergy between metal species in classical catalytic reactions, very recently, binary metal site catalysts have been developed to achieve the promotional effect of binary atomic sites, and a few successful cases have been investigated (12–14). However, it is still a challenge to construct effective binary sites.It is known that the dipole with one positive center and one negative center is the basic unit of electric fields, which are the main driving force of charge separation and transfer. Enlightened by this concept, we have designed and constructed a Pt-Au binary single-site loaded g-C₃N₄ nanosheet photocatalyst (Pt-Au SAC), which resulted in enhanced localized molecular dipoles in the zone of adjacent Pt-Au sites due to their larger charge imbalance compared with those in unary counterparts. Therefore, via the superposition of these enhanced molecular dipoles, the internal electric field was effectively strengthened for Pt-Au SAC, which is stronger than those of Pt and Au single-site counterparts. As confirmed by multiple spectroscopic methods, including femtosecond transient absorption spectroscopy (TA), the stronger internal electric field of Pt-Au SAC can significantly enhance the charge transportation dynamics. As a result, in solar water splitting, the hydrogen evolution rate of optimum Pt-Au SAC (0.25% metal loading) was boosted to 1.88 mmol g⁻¹ h⁻¹ (apparent quantum efficiency 1.72% at 365 nm), 4.9 and 2.3 times over Pt and Au single-site counterparts (Pt SAC and Au SAC, respectively). Besides, a lower energy barrier was also realized over the Pt-Au binary sites, further stimulating its catalytic performance. Previously, several reports about other binary single sites catalysts have shown their advances as the synergistic reaction centers for optimizing the reaction barriers or rate-determining steps (12–14). In our case, the Pt-Au binary sites not only contribute to the lower reaction barriers but also finely contribute to charge transportation dynamics by enhancing the localized molecular dipoles. This discovery offered a strategy for improving the surface charge separation/transfer of photocatalysis, which is one of the central problems for improving the photocatalytic process.     

Theoretical Evaluation of Potential Cytotoxicity of Graphene Quantum Dot to Adsorbed DNA

As a zero-dimensional (0D) nanomaterial, graphene quantum dot (GQD) has a unique physical structure and electrochemical properties, which has been widely used in biomedical fields, such as bioimaging, biosensor, drug delivery, etc. Its biological safety and potential cytotoxicity to human and animal cells have become a growing concern in recent years. In particular, the potential DNA structure damage caused by GQD is of great importance but still obscure. In this study, molecular dynamics (MD) simulation was used to investigate the adsorption behavior and the structural changes of single-stranded (ssDNA) and double-stranded DNA (dsDNA) on the surfaces of GQDs with different sizes and oxidation. Our results showed that ssDNA can strongly adsorb and lay flat on the surface of GQDs and graphene oxide quantum dots (GOQDs), whereas dsDNA was preferentially oriented vertically on both surfaces. With the increase of GQDs size, more structural change of adsorbed ssDNA and dsDNA could be found, while the size effect of GOQD on the structure of ssDNA and dsDNA is not significant. These findings may help to improve the understanding of GQD biocompatibility and potential applications of GQD in the biomedical field.     

Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP⁺. The neutron data were collected to 2.0-Å resolution using a 3.6-mm³ crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pK_a value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.Dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP⁺ oxidoreductase) (DHFR) is a housekeeping enzyme that catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8,-tetrahydrofolate (THF). Various redox states of THF are used in several one-carbon transfer reactions to generate thymidine, methionine, glycine, serine, and other molecules (1–3). Given its role in biosynthesis, DHFR is a target for anticancer, antimicrobial, and rheumatoid arthritis drugs, such as methotrexate (MTX) and trimethoprim (4–7).Although the kinetics, structure, and biophysical properties of Escherichia coli DHFR (ecDHFR) have been well characterized, unresolved questions with respect to its catalytic mechanism remain (1, 3, 8–13), as evidenced by the recent controversy over whether millisecond time-scale structural fluctuations can directly affect the chemical step in catalysis (14, 15). Folate is a poor substrate for DHFR, whereas DHF is reduced more efficiently (2). In addition, unlike DHF, folate cannot be further oxidized in solution. Thus, the abortive DHFR-folate-NADP⁺ complex is an excellent mimic of the DHFR-DHF-NADPH Michaelis complex (3, 16), and its stability makes it well suited for structural studies.During catalysis, a proton is donated to the N5 atom of the DHF pterin ring and a hydride equivalent is transferred from NADPH to the C6 atom of the pterin. With folate as a substrate, proton donation occurs at the N8 atom (10). The five intermediates in the catalytic cycle are E-NADPH, E-NADPH-DHF, E-NADP⁺-THF, E-THF, and E-NADPH-THF (3), with product release as the rate-limiting step at neutral pH. THF is released on binding of a new NADPH molecule. The enzyme displays pH dependence with a characteristic pK_a value of 6.5 (8).Previous crystallographic and NMR studies of the DHFR binary and ternary complexes have revealed the locations of the folate and nicotinamide cofactor optimal for hydride transfer and the juxtaposition of the substrate with respect to the catalytic Asp27, which forms hydrogen bonds with the N3 and NA2 atoms of folate (3, 12). The DHFR-folate-NADP⁺ complex structure is considered the closest mimic of the Michaelis complex and has been used as a reference model in studies of the molecular details required for proton donation and hydride transfer (2, 3, 14, 17). Although it is clear from the structure that the nicotinamide ring is optimally positioned for hydride transfer to the C6 atom of the DHF substrate, how a proton can be donated to the N5 atom is unclear, especially considering that the conserved Asp27 is almost 5 Å distant from it. Disagreement abounds as to the protonation state of the Asp27 during catalysis (10, 18, 19). The mutation of the other residues contacting the substrate diminishes but does not abrogate activity, suggesting that the enzyme is flexible and has built-in redundancies (9, 20).Several catalytic mechanisms have been proposed based on X-ray and NMR structures, molecular dynamics, enzyme kinetic measurements, and Raman spectroscopy studies (1, 17, 18, 21). According to Maharaj et al. (21), the pK_a of the N5 atom of DHF is 2.6 in solution. When bound in a binary complex to DHFR, its N5 pK_a remains strongly acidic. However, the pK_a is elevated from <4 in the binary complex to 6.5 in the catalytic mimic complex, where NADP⁺ is bound as well (1, 21). This value matches the pK_a describing the hydride transfer step (8), suggesting that the kinetic pK_a describes the level of N5 protonated substrate available. The accompanying article by Liu et al. (22) further explores the kinetic pH profile for ecDHFR and its relationship to the hydride transfer step, as well as the sequential order of the mechanism. Outstanding questions remain, including, but not limited to, the following: (i) When a catalytically competent complex is present, how does the active site environment so drastically increase the N5 pK_a to promote protonation? (ii) What is the protonation state of Asp27 throughout catalysis? And (iii) what is the source and mechanism of proton donation to N5?An oft-proposed general mechanism based on several crystallography and Raman spectroscopy studies invokes a keto-enol tautomerization of the pterin substrate, initiating at the Asp27 and triggering a proton shuttle that ultimately results in a reduction of N5 (9, 10, 23). Two versions of this mechanism have been proposed, the major differences being the protonation state of Asp27 in the ground state and the ultimate proton source for reduction of N5 (SI Materials and Methods). The caveat regarding this mechanism is that keto-enol tautomerization as a critical step in the DHFR catalytic cycle remains a major point of ambiguity (24). Blakley et al. (25) have challenged the idea that substrate undergoes tautomerization during catalysis based on the NMR finding of a persistent substrate in an N3 imino-C4 keto tautomer across a pH range.Alternative catalytic mechanisms propose the direct involvement of water molecules in the proton transfer step. These mechanisms notably omit the necessity for a substrate tautomerization event and the requirement for protonation of Asp27 at some point in the catalytic cycle (17). Asp27 is mainly responsible for binding the substrate in a catalytically favorable conformation and maintaining a negative electrostatic field in the active site, which would be negated if its carboxylate were protonated even transiently. A recent study revealed that Met20 loop dynamics are critical for solvent access to N5, and proposed a mechanism involving direct solvent protonation of the substrate (22). There has been only one previous structural observation of a solvent molecule within hydrogen-bonding distance of the N5 atom, in a crystal structure of E. coli DHFR bound to folate and NADP⁺ (1RA2). It should be noted that the Met20 loop adopts an open conformation in this structure, likely because of crystal packing effects (3). A solvent molecule is typically modeled in this position in crystal structures with only substrate bound (2).A barrier to experimentally testing most proposed enzyme mechanisms is the inability to directly visualize the positions of important catalytic protons. The initial models used for most theoretical calculations are derived from X-ray structures, and determining the location of hydrogen atoms using X-rays is difficult even at atomic resolution (1.2 Å) (26). Neutron crystallography (NC) has a proven ability to determine the positions of hydrogen atoms or ions (protons) essential for catalysis (27–30). In fact, NC defines unique positions of hydrogen atoms within ordered water molecules (31), and H₃O⁺ molecules crucial for catalysis in xylose isomerase were recently identified (32). By virtue of the need to perform hydrogen/deuterium exchange (HDX) on crystals before data collection, NC can accurately identify hydrogen atom positions even at modest resolution. Deuterium coherently scatters neutrons with lengths similar to carbon and nitrogen, whereas hydrogen coherently scatters neutrons with negative lengths, rendering them invisible in positively contoured nuclear density maps. In the past, the determination of NC structures was hindered by the limited number of data collection facilities, low beam fluxes, and the requirement for extremely large crystals (>1 mm³ in volume). Recently, new spallation sources, enhanced deuterium labeling of samples, and improved detectors have allowed the collection of high-quality data from crystals of smaller volume, leading to a dramatic increase in the number of neutron structures deposited in the Protein Data Bank (PDB).In a previous NC study, we resolved a question pertaining to the protonation state of the classical antifolate inhibitor MTX and Asp27 when MTX binds DHFR (28). The DHFR-MTX neutron structure demonstrates that Asp27 is negatively charged, whereas the N1 atom of MTX is protonated and thus positively charged. After our initial success with the DHFR-MTX complex, we conducted NC studies of a DHFR pseudo-Michaelis complex to identify the protonation state of Asp27 in a catalytic mimic complex, the source of protonation for N5, as well as the presence (or absence) of a substrate keto-enol tautomerization event. Here we report the neutron diffraction structure of the DHFR-folate-NADP⁺ complex and complementary ultrahigh-resolution X-ray structures at three different temperatures. Refinement of the neutron structure allowed determination of the positions of crucial protons on the folate substrate and the ionization state of Asp27. Furthermore, our comprehensive map of backbone HDX sheds light on the dynamically driven changes in solvent accessibility of crystalline DHFR. The ultrahigh-resolution X-ray structures provide molecular details of Met20 loop fluctuations required for the entry of solvent, identifying a water molecule possibly involved in proton donation to N5.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Direct calculation of ice homogeneous nucleation rate for a molecular model of water

Ice formation is ubiquitous in nature, with important consequences in a variety of environments, including biological cells, soil, aircraft, transportation infrastructure, and atmospheric clouds. However, its intrinsic kinetics and microscopic mechanism are difficult to discern with current experiments. Molecular simulations of ice nucleation are also challenging, and direct rate calculations have only been performed for coarse-grained models of water. For molecular models, only indirect estimates have been obtained, e.g., by assuming the validity of classical nucleation theory. We use a path sampling approach to perform, to our knowledge, the first direct rate calculation of homogeneous nucleation of ice in a molecular model of water. We use TIP4P/Ice, the most accurate among existing molecular models for studying ice polymorphs. By using a novel topological approach to distinguish different polymorphs, we are able to identify a freezing mechanism that involves a competition between cubic and hexagonal ice in the early stages of nucleation. In this competition, the cubic polymorph takes over because the addition of new topological structural motifs consistent with cubic ice leads to the formation of more compact crystallites. This is not true for topological hexagonal motifs, which give rise to elongated crystallites that are not able to grow. This leads to transition states that are rich in cubic ice, and not the thermodynamically stable hexagonal polymorph. This mechanism provides a molecular explanation for the earlier experimental and computational observations of the preference for cubic ice in the literature.Ice nucleation affects the behavior of many systems (1–6). For example, the formation of ice crystals inside the cytoplasm can damage living cells (1). The amount of ice in a cloud determines both its light-absorbing properties (5) and its precipitation propensity (6), and is therefore an important input parameter in many meteorological models (7, 8). However, current experiments are incapable of uncovering the kinetics and the molecular mechanism of freezing due to their limited spatiotemporal resolution. The ice that nucleates homogeneously in the atmosphere and vapor chamber experiments is predominantly comprised of the cubic-rich stacking-disordered polymorph, not the thermodynamically stable hexagonal polymorph (9, 10). This observation has been rationalized invoking the Ostwald step rule (11). However, the molecular origin of this preference is unknown, due to the limited spatiotemporal resolution of existing experimental techniques. Furthermore, experimental measurements of nucleation rates are only practical over narrow ranges of temperatures (12), with any extrapolation being prone to large uncertainties.Computer simulations are attractive alternatives in this quest, as they make it possible to obtain, at any given thermodynamic condition, a statistically representative sample of nucleation events that can then be used to estimate the rates and identify the mechanism of nucleation. This, however, has only been achieved (13–15) for coarse-grained representations of water, such as the monoatomic water (mW) model (16). For the more realistic molecular force fields, all of the existing studies have relied either on launching a few-microseconds-long molecular dynamics (MD) trajectories (17, 18), or on applying external fields (19), or biasing potentials along prechosen reaction coordinates (20) to drive nucleation, and the generation of statistically representative nucleation trajectories that can allow direct and accurate rate predictions has so far been beyond reach.In this work, we achieve this goal in a system of 4,096 water molecules at 230 K and 1 bar by introducing a novel coarse-graining modification to the path sampling method known as forward-flux sampling (FFS) (21). In the FFS approach, the nucleation process is sampled in stages defined by an order parameter, λ. In crystallization studies, λ is typically chosen as the size of the largest crystalline nucleus in the system (13–15). Individual molecules are labeled as solid- or liquid-like based on the Steinhardt order parameters (22), and the neighboring solid-like molecules are connected to form a cluster (for further details, refer to SI Text, Fig. S1). The cumulative probability of growing a crystallite with λ molecules is then computed from the success probabilities at individual stages (e.g., Fig. 1). If a sufficiently large number of trajectories are sampled at each stage, the nucleation mechanism can be accurately determined by inspecting the ensemble of pseudotrajectories that connect the liquid and crystalline basins. We use the term “pseudotrajectory” as, during FFS, all velocities are randomized at any given milestone.Open in a separate windowFig. 1.Cumulative transition probability vs. size of the largest crystalline nucleus in the TIP4P/Ice system at 230 K and 1 bar. The inflection region is shown in shaded purple. Several representative crystallites are also depicted. The cumulative probability curve for the LJ system simulated at k_BT/? = 0.82 and pσ³/? = 5.68 is shown in the Inset with ε and σ the LJ energy and size parameters. No inflection region is observed in the LJ system.

Table S1.

Technical specifications of the MD simulations and the order parameter

Pathway for Mn-cluster oxidation by tyrosine-Z in the S2 state of photosystem II

Water oxidation in photosynthetic organisms occurs through the five intermediate steps S₀–S₄ of the Kok cycle in the oxygen evolving complex of photosystem II (PSII). Along the catalytic cycle, four electrons are subsequently removed from the Mn₄CaO₅ core by the nearby tyrosine Tyr-Z, which is in turn oxidized by the chlorophyll special pair P680, the photo-induced primary donor in PSII. Recently, two Mn₄CaO₅ conformations, consistent with the S₂ state (namely, S2A and S2B models) were suggested to exist, perhaps playing a different role within the S₂-to-S₃ transition. Here we report multiscale ab initio density functional theory plus U simulations revealing that upon such oxidation the relative thermodynamic stability of the two previously proposed geometries is reversed, the S2B state becoming the leading conformation. In this latter state a proton coupled electron transfer is spontaneously observed at ∼100 fs at room temperature dynamics. Upon oxidation, the Mn cluster, which is tightly electronically coupled along dynamics to the Tyr-Z tyrosyl group, releases a proton from the nearby W1 water molecule to the close Asp-61 on the femtosecond timescale, thus undergoing a conformational transition increasing the available space for the subsequent coordination of an additional water molecule. The results can help to rationalize previous spectroscopic experiments and confirm, for the first time to our knowledge, that the water-splitting reaction has to proceed through the S2B conformation, providing the basis for a structural model of the S₃ state.For 2.5 Gy photosynthetic organisms have used the photosystem II complex (PSII) to capture light energy from the sun and convert it into chemical energy stored within energy-rich carbohydrates (1). The water oxidation reaction, occurring in the reaction center of PSII, represents the central step of the natural photosynthetic process, leading to the formation of molecular oxygen and hydrogen equivalents. A deep understanding of the photosynthetic water-splitting mechanism may serve as a valuable source of inspiration for the development of artificial devices able to store solar energy in environmentally friendly fuels, such as molecular hydrogen (2–6). The active site of the PSII enzyme, where the water-splitting reaction takes place, consists of a core of four Mn ions and one Ca ion connected together through μ-oxo bridges in a cubane-like aggregate (7). Water oxidation proceeds through five sequential S₀–S₄ steps known as the Kok cycle (8). At each step of the catalytic cycle the absorption of photons turns out the oxidation of the tyrosyl group of a nearby tyrosine (i.e., Tyr161, also known as Tyr-Z in the D1 subunit of PSII), which acts as an intermediate in the electron transfer between the Mn₄CaO₅ cluster and the primary donor P680 (9, 10).The molecular structure for the different states of the Kok cycle was largely investigated in the past decades by extended X-ray absorption fine structure experiments (11–14) and X-ray crystallography (15–17), thus revealing atomic details at an increasing level of accuracy (7). In parallel the electronic and magnetic properties, characterizing steps of the catalytic cycle, were investigated by EPR experiments (18–24), with particular attention to the well-characterized S₂ state (25). The combination of this large number of experimental results within a computational modeling framework gave the opportunity to understand some of the atomic details underlying the water-splitting reaction in the oxygen-evolving complex (OEC) (26–35). Recently, Pantazis et al. (36) have suggested the presence of two possible interconvertible structures representative of the S₂ state. The two models, namely, the S2A state, characterized by a S = 1/2 spin ground state, and the S2B state, characterized by a S = 5/2 spin ground state (Fig. 1), can explain the presence of the two distinct EPR signals revealed at cryogenic temperatures (i.e., a multiline signal indicative of a ground state characterized by a spin S = 1/2 and a second signal at g = 4.1 consistent with a spin S = 5/2). In a recent work (37) we characterized by extensive quantum mechanics/molecular mechanics (QM/MM) ab initio simulations the free-energy profiles for the interconversion between the two above-mentioned conformations, suggesting that the transition from the S₂ to S₃ state should proceed passing first by the S2A and subsequently through the S2B state. Still, clear evidence confirming such a hypothesis is missing.Open in a separate windowFig. 1.Ab initio QM/MM model of photosystem II. (Right) The QM region, consisting of 224 atoms, is shown in balls and sticks representation. (Upper Left) A selection of the the most important residues and distances involved in the oxidation of the Mn₄CaO₅ cluster by the radical Tyr-Z are sketched. (Lower Left) Representation of the two investigated conformations S2A and S2B.Here, using the same approach previously adopted, we characterized different (spin) energy surfaces along the interconversion path between the S2A and S2B states after the removal of one electron from the QM region (Fig. 1). QM/MM molecular dynamic simulations were additionally carried out for the two models in their respective spin ground state: the low spin (LS) state for the S2A model and the high spin (HS) state for the S2B model. The present results show for the first time, to our knowledge, the occurrence of a proton coupled electron transfer (PCET) in the S2B state resulting in the oxidation of the Mn4 ion by the tyrosyl group of Tyr-Z.     

Reversible catalytic dehydrogenation of alcohols for energy storage

Reversibility of a dehydrogenation/hydrogenation catalytic reaction has been an elusive target for homogeneous catalysis. In this report, reversible acceptorless dehydrogenation of secondary alcohols and diols on iron pincer complexes and reversible oxidative dehydrogenation of primary alcohols/reduction of aldehydes with separate transfer of protons and electrons on iridium complexes are shown. This reactivity suggests a strategy for the development of reversible fuel cell electrocatalysts for partial oxidation (dehydrogenation) of hydroxyl-containing fuels.Hydrogenation and dehydrogenation reactions are fundamental in synthetic organic chemistry and used in a variety of large- and small-scale processes for manufacturing chemicals, pharmaceuticals, foods, and fuels. An increased need for energy storage technologies, in large part because of the recent deployment of intermittent renewable energy sources, has generated a renewed interest in hydrogen as a form of chemical energy storage. Hydrogen, which may be used in fuel cells or internal combustion engines, is best-suited for longer-term energy storage and can be stored in the form of a compressed gas or a cryogenic liquid or chemically bonded in hydrides (1). The most attractive hydrogen storage media are liquid organic hydrogen carriers (LOHCs), because they have relatively high hydrogen content and can be transported and distributed using the existing liquid fuel infrastructure (2–5).Two strategies for coupling the chemical energy stored in these LOHCs with an energy storage device include thermal dehydrogenation to provide H₂(g) for a polymer electrolyte membrane (PEM) fuel cell (expression 1) (6) and electrochemical dehydrogenation to yield protons and electrons in a direct alcohol fuel cell (expressions 2 and 3) (2, 7). In the former (acceptorless strategy), hydrogen release from LOHCs frequently requires high reaction temperatures and expensive platinum group metal (PGM) catalysts. Regeneration of the hydrogen-depleted compounds can be achieved with both PGM and less expensive non-PGM catalysts (e.g., nickel-based); however, elevated hydrogen pressures are needed (8, 9). The latter was the basis for an Energy Frontier Research Center around Electrocatalysis, Transport Phenomena, and Materials for Innovative Energy Storage funded by the Department of Energy (Acknowledgments), which assumed the use of a single electrocatalyst for dehydrogenation and hydrogenation of LOHCs that would also simplify the conventional hydrogen storage process (10). The envisioned partial electrochemical dehydrogenation of LOHCs typically involves expensive PGM catalysts (11–18), with weak bases to serve as proton scavengers in lieu of a proton exchange membrane. Reversing the applied potential in the presence of these same components provides a mechanism for regenerating the LOHCs without the need for elevated hydrogen pressure:LH_n ? L + n/2?H₂, [1]LH_n ? L + n?H⁺ + n?e^?, [2]andn/2?O₂ + n?H⁺ + n?e^? ? n/2?H₂O.[3]A thermodynamic analysis of a variety of potential LOHCs showed that cyclic hydrocarbons exhibit high hydrogen contents but that nitrogen heterocycles exhibit lower reaction enthalpies (9, 10). Nitrogen heterocycles are also more practical, because they display energy densities that are comparable with those of liquid hydrogen, and the theoretical open cell potentials of these materials are calculated to be close to or exceed the potential of the hydrogen–oxygen fuel cell (11). In addition, the overpotential of their electrooxidation is also smaller compared with cyclic hydrocarbons (12). However, basic nitrogen heterocycles are not compatible with commonly used acidic proton exchange membranes because of the formation of a nonconductive salt. One alternative class of LOHCs that does not suffer from this problem is hydroxyl-containing compounds (e.g., alcohols and diols) that feature reasonable hydrogen content and low oxidation potentials. Both mono- and polysubstituted alcohols have been proposed as hydrogen storage materials (13) and used as fuel for direct alcohol fuel cells, usually in the form of an aqueous alkaline solution (14).Homogeneous catalysts for the acceptorless dehydrogenation of primary and secondary alcohols for the most part contain precious metals, such as Ru (19), Rh (20), and Ir (21). By comparison, the same reaction with nonprecious, earth-abundant metal catalysts is, so far, a relatively unexplored area in the literature. The first cobalt catalyst bearing a noninnocent bis(dicyclohexylphosphino)amine (PNP^Cy) ligand for acceptorless dehydrogenation of alcohols was reported by Hanson and coworkers (22, 23). Recently, Beller and coworkers (24) have shown hydrogen production from methanol in the presence of KOH with octahedral iron complexes (PNP^iPr)Fe(H)(CO)X [X = BH₄ (1) and X = Br (2)]. Remarkably, very low catalyst loadings (parts per million level) were used, and catalysis was performed at 91 °C, which suggests high thermal stability of these iron complexes and related intermediate species. However, Guan and coworkers (25) have accomplished ester hydrogenation using catalyst 1. In addition to these studies, it was recently shown that the same iron complexes can also efficiently catalyze the reversible dehydrogenation–hydrogenation of N-heterocycles (26). Yamaguchi et al. (27) have also reported Cp*Ir complexes with substituted pyridonate ligands that catalyze the reversible acceptorless dehydrogenation of 1,2,3,4-tetrahydroquinoline to quinoline in boiling xylene. Dehydrogenation is harder and routinely produced lower yields than hydrogenation, but with the 5-trifluoromethylpyridonate ligand, a quantitative yield was achieved in both directions (27). A computational analysis of the proposed catalytic cycle showed that two major pathways are possible: through a bifunctional species with coordinated pyridonate ligand or through a monomeric Cp*Ir(H)Cl complex (28).As part of our ongoing effort to develop another strategy, specifically electrocatalysts for reversible partial oxidation (dehydrogenation) of alcohol-based fuels, we concentrated on the possibility of converting a known dehydrogenation catalyst to an electrocatalyst through separation of protons and electrons. We regard separately extracting protons and electrons in a catalytic dehydrogenation reaction together with the microscopic reverse (29) (i.e., separately injecting protons and electrons to effect substrate hydrogenation) as fundamental in the development of a reversible alcohol dehydrogenation–hydrogenation electrocatalyst. It was clear at the outset that the possibility of oxidation of intermediate species containing metals in low-oxidation states during dehydrogenation and the competing proton reduction to H₂ in the reverse reaction substantially limited our selection of possible candidates. We recently reported electrocatalytic properties of an iridium amino-olefin complex [Ir(trop₂DACH)][OTf], which is capable of catalyzing alcohol dehydrogenation with chemical oxidants as well as electrocatalytic dehydrogenation of primary alcohols with excellent faradaic efficiency (30). Two catalytic systems capable of oxidizing alcohols with a chemical oxidant (ferrocenium cation) in the presence of a base as a proton acceptor have been very recently described in literature (30, 31).In this report, we describe catalytic systems that address both strategies outlined above as part of a unified effort to develop catalysts for reversible dehydrogenation of organic fuels in energy generation and storage reactions. These catalysts show reversible acceptorless (expression 1) and partial oxidative (expression 2) dehydrogenation of alcohols using non-PGM (iron-based) and PGM (iridium-based) catalysts, respectively.