Local structure and vibrational dynamics in indium-doped barium zirconate

Barium zirconate (BaZrO3), when substituted with trivalent acceptor ions to replace Zr4+, is a proton conducting material of interest for several electrochemical applications. The local coordination environments, and vibrational dynamics, of the protons are known to critically influence the material\u27s proton conducting properties, however, the nature of the static and dynamic structure around the protons and, especially, how it is affected by the dopant atoms for high doping concentrations, remains to be elucidated. Here we report results from X-ray powder diffraction, infrared (IR) spectroscopy, inelastic neutron scattering (INS) and ab initio molecular dynamics (AIMD) simulations on a hydrated sample of BaZrO3 substituted with 50% In3+. The investigation of the momentum-transfer (Q) dependence of the INS spectrum is used to aid the analysis of the spectra and the assignment of the spectral components to fundamental O-H bend and O-H stretch modes and higher-order transitions. The AIMD simulations show that the INS spectrum is constituted of the overlapping spectra of protons in several different local structural environments, whereas the local proton environments for specific protons are found to vary with time as a result of thermally activated vibrations of the perovskite lattice. It is argued that, converse to more weakly doped systems, such as 20% Y-doped BaZrO3, the dopant-proton association effect does not hinder the diffusion of protons due to the presence of percolation paths of dopant atoms throughout the perovskite lattice

Local structure and vibrational dynamics in indium-doped barium zirconate

Barium zirconate (BaZrO3), when substituted with trivalent acceptor ions to replace Zr4+, is a proton conducting material of interest for several electrochemical applications. The local coordination environments, and vibrational dynamics, of the protons are known to critically influence the material\u27s proton conducting properties, however, the nature of the static and dynamic structure around the protons and, especially, how it is affected by the dopant atoms for high doping concentrations, remains to be elucidated. Here we report results from X-ray powder diffraction, infrared (IR) spectroscopy, inelastic neutron scattering (INS) and ab initio molecular dynamics (AIMD) simulations on a hydrated sample of BaZrO3 substituted with 50% In3+. The investigation of the momentum-transfer (Q) dependence of the INS spectrum is used to aid the analysis of the spectra and the assignment of the spectral components to fundamental O-H bend and O-H stretch modes and higher-order transitions. The AIMD simulations show that the INS spectrum is constituted of the overlapping spectra of protons in several different local structural environments, whereas the local proton environments for specific protons are found to vary with time as a result of thermally activated vibrations of the perovskite lattice. It is argued that, converse to more weakly doped systems, such as 20% Y-doped BaZrO3, the dopant-proton association effect does not hinder the diffusion of protons due to the presence of percolation paths of dopant atoms throughout the perovskite lattice

The role of oxygen vacancies on the vibrational motions of hydride ions in the oxyhydride of barium titanate

Perovskite-type oxyhydrides, BaTiO3-xHx, represent a novel class of hydride ion conducting materials of interest for several electrochemical applications, but fundamental questions surrounding the defect chemistry and hydride ion transport mechanism remain unclear. Here we report results from powder X-ray diffraction, thermal gravimetric analysis, nuclear magnetic resonance spectroscopy, inelastic neutron scattering (INS), and density functional theory (DFT) simulations on three metal hydride reduced BaTiO3 samples characterized by the simultaneous presence of hydride ions and oxygen vacancies. The INS spectra are characterized by two predominating bands at around 114 (ω⊥) and 128 (ω∥) meV, assigned as fundamental Ti-H vibrational modes perpendicular and parallel to the Ti-H-Ti bond direction, respectively, and four additional, weaker, bands at around 99 (ω1), 110 (ω2), 137 (ω3) and 145 (ω4) meV that originate from a range of different local structures associated with different configurations of the hydride ions and oxygen vacancies in the materials. Crucially, the combined analyses of INS and DFT data confirm the presence of both nearest and next-nearest neighbouring oxygen vacancies to the hydride ions. This supports previous findings from quasielastic neutron scattering experiments, that the hydride ion transport is governed by jump diffusion dynamics between neighbouring and next-nearest neighbouring hydride ion-oxygen vacancy local structures. Furthermore, the investigation of the momentum transfer dependence of the INS spectrum is used to derive the mean square displacement of the hydride ions, which is shown to be in excellent agreement with the calculations. Analysis of the mean square displacement confirms that the hydrogen vibrational motions are localized in nature and only very weakly affected by the dynamics of the surrounding perovskite structure. This insight motivates efforts to identify alternative host lattices that allow for a less localization of the hydride ions as a route to higher hydride ion conductivities

Protein Engineering on Azurin. Expression Mutagenesis and Characterisation of Copper Site Mutants

Azurin belongs to a family of small blue copper proteins or cupredoxins which participate in electron transfer processes in plants and bacteria. The type 1 copper site in these proteins is characterised by an intense blue colour, a narrow hyperfine coupling in the EPR signal and, generally, a high reduction potential. A common feature in the cupredoxin structure is the eight stranded b-barrel and a copper binding site situated in a loop between two b-strands. Three of the four copper ligands, two histidines and one cysteine, are conserved in all cupredoxins, whereas the fourth ligand, a methionine, is exchanged in stellacyanin. The gene encoding azurin from Pseudomonas aeruginosa has been expressed in large amounts in Escherichia coli. Cassette mutagenesis was used to exchange the methionine ligand for all other amino acids, including a stop codon. The properties of the mutant proteins were investigated by optical and EPR spectroscopy. The effect of the amino acid exchange on the reduction potential was studied, XAFS was used to analyse the structure of the copper site and the X-ray structure was determined for the Met121Glu mutant protein. It was concluded that the ligand methionine is not needed to create a type 1 copper site. The exchange of the methionine results in mutant proteins with large changes in spectroscopic and redox properties. The introduction of polar residues gives rise to spectroscopic properties similar to properties found in stellacyanin or pseudoazurin. A large increase in reduction potential is observed when hydrophobic residues are introduced, whereas a decrease is observed when the copper ion is solvent exposed. The introduction of a negative group directly coordinating the copper is accompanied by a change in the copper site geometry, a decrease in reduction potential and a drastic change in the spectroscopic properties

Protein Engineering on Azurin. Expression Mutagenesis and Characterisation of Copper Site Mutants

Azurin belongs to a family of small blue copper proteins or cupredoxins which participate in electron transfer processes in plants and bacteria. The type 1 copper site in these proteins is characterised by an intense blue colour, a narrow hyperfine coupling in the EPR signal and, generally, a high reduction potential. A common feature in the cupredoxin structure is the eight stranded b-barrel and a copper binding site situated in a loop between two b-strands. Three of the four copper ligands, two histidines and one cysteine, are conserved in all cupredoxins, whereas the fourth ligand, a methionine, is exchanged in stellacyanin. The gene encoding azurin from Pseudomonas aeruginosa has been expressed in large amounts in Escherichia coli. Cassette mutagenesis was used to exchange the methionine ligand for all other amino acids, including a stop codon. The properties of the mutant proteins were investigated by optical and EPR spectroscopy. The effect of the amino acid exchange on the reduction potential was studied, XAFS was used to analyse the structure of the copper site and the X-ray structure was determined for the Met121Glu mutant protein. It was concluded that the ligand methionine is not needed to create a type 1 copper site. The exchange of the methionine results in mutant proteins with large changes in spectroscopic and redox properties. The introduction of polar residues gives rise to spectroscopic properties similar to properties found in stellacyanin or pseudoazurin. A large increase in reduction potential is observed when hydrophobic residues are introduced, whereas a decrease is observed when the copper ion is solvent exposed. The introduction of a negative group directly coordinating the copper is accompanied by a change in the copper site geometry, a decrease in reduction potential and a drastic change in the spectroscopic properties

Apo-azurin folds via an intermediate that resembles the molten-globule.

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism

Apo-azurin folds via an intermediate that resembles the molten-globule.

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism

Probing the influence on folding behavior of structurally conserved core residues in P. aeruginosa apo-azurin.

The effects on folding kinetics and equilibrium stability of core mutations in the apo-mutant C112S of azurin from Pseudomonas aeruginosa were studied. A number of conserved residues within the cupredoxin family were recognized by sequential alignment as constituting a common hydrophobic core: I7, F15, L33, W48, F110, L50, V95, and V31. Of these, I7, V31, L33, and L50 were mutated for the purpose of obtaining information on the transition state and a potential folding nucleus. In addition, residue V5 in the immediate vicinity of the common core, as well as T52, separate from the core, were mutated as controls. All mutants exhibited a nonlinear dependence of activation free energy of folding on denaturant concentration, although the refolding kinetics of the V31A/C112S mutant indicated that the V31A mutation destabilizes the transition state enough to allow folding via a parallel transition state ensemble. Phi-values could be calculated for three of the six mutants, V31A/C112S, L33A/C112S, and L50A/C112S, and the fractional values of 0.63, 0.33, and 0.50 (respectively) obtained at 0.5 M GdmCl suggest that these residues are important for stabilizing the transition state. Furthermore, a linear dependence of ln k(obs)(H2O) on DeltaG(U-N)(H2O) of the core mutations and the putative involvement of ground-state effects suggest the presence of native-like residual interactions in the denatured state that bias this ensemble toward a folding-competent state

Probing the influence on folding behavior of structurally conserved core residues in P. aeruginosa apo-azurin.

The effects on folding kinetics and equilibrium stability of core mutations in the apo-mutant C112S of azurin from Pseudomonas aeruginosa were studied. A number of conserved residues within the cupredoxin family were recognized by sequential alignment as constituting a common hydrophobic core: I7, F15, L33, W48, F110, L50, V95, and V31. Of these, I7, V31, L33, and L50 were mutated for the purpose of obtaining information on the transition state and a potential folding nucleus. In addition, residue V5 in the immediate vicinity of the common core, as well as T52, separate from the core, were mutated as controls. All mutants exhibited a nonlinear dependence of activation free energy of folding on denaturant concentration, although the refolding kinetics of the V31A/C112S mutant indicated that the V31A mutation destabilizes the transition state enough to allow folding via a parallel transition state ensemble. Phi-values could be calculated for three of the six mutants, V31A/C112S, L33A/C112S, and L50A/C112S, and the fractional values of 0.63, 0.33, and 0.50 (respectively) obtained at 0.5 M GdmCl suggest that these residues are important for stabilizing the transition state. Furthermore, a linear dependence of ln k(obs)(H2O) on DeltaG(U-N)(H2O) of the core mutations and the putative involvement of ground-state effects suggest the presence of native-like residual interactions in the denatured state that bias this ensemble toward a folding-competent state