Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory (DFT) calculations indicate that both active site states are stable. The hydroperoxo formed is potentially an intermediate in the mechanism of hydrogen peroxide formation in the absence of substrate. We establish that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by mining minima free energy calculations we establish His157 conformational flexibility in solution that is abolished by steric hindrance in the crystal. A neutron crystal structure of NcLPMO9D at low pH supports occlusion of the active site which prevents protonation of His157 at acidic conditions.

Modelling of Phase Formation in Solid–Solid and Solid–Liquid Interactions: New Developments

Recent developments (after 2016) in modelling of phase formation during solid–solid and solid–liquid reactions by SKMF (Stochastic Kinetic Mean-Field) method, Monte Carlo simulation and phenomenological modelling are reviewed. Reasonable results of multiphase reactive diffusion modelling demonstrating distinct concentration plateau for each intermediate ordered compound and distinct concentration steps between these phases are obtained by the SKMF and Monte Carlo methods, if one takes into account interatomic interactions within two coordination shells and if the signs of mixing energies are ‘minus’ for the first coordination shell and ‘plus’ for the second one. Second possibility for reasonable modelling results is consideration of interatomic interactions depending on local concentration with maxima around stoichiometric composition. In phenomenological modelling, the generalization of Wagner diffusivity concept and respective superposition rule are introduced. New mechanism of the lateral grain growth in the growing phase layers during reactive diffusion is suggested. Anomalously fast grain growth at the final stages of soldering in sandwich-like Cu–Sn–Cu contacts is reported and explained. Simple model of Zn-additions’ influence on the Cu–Sn reaction is described.

Bulk and Confined Benzene-Cyclohexane Mixtures Studied by an Integrated Total Neutron Scattering and NMR Method

Herein mixtures of cyclohexane and benzene have been investigated in both the bulk liquid phase and when confined in MCM-41 mesopores. The bulk mixtures have been studied using total neutron scattering (TNS), and the confined mixtures have been studied by a new flow-utilising, integrated TNS and NMR system (Flow NeuNMR), all systems have been analysed using empirical potential structure refinement (EPSR). The Flow NeuNMR setup provided precise time-resolved chemical sample composition through NMR, overcoming the difficulties of ensuring compositional consistency for computational simulation of data ordinarily found in TNS experiments of changing chemical composition—such as chemical reactions. Unique to the liquid mixtures, perpendicularly oriented benzene molecules have been found at short distances from the cyclohexane rings in the regions perpendicular to the carbon–carbon bonds. Upon confinement of the hydrocarbon mixtures, a stronger parallel orientational preference of unlike molecular dimers, at short distances, has been found. At longer first coordination shell distances, the like benzene molecular spatial organisation within the mixture has also found to be altered upon confinement.

Structure of lithium tellurite and vanadium lithium tellurite glasses by high-energy X-ray and neutron diffraction

xLi2O–(100 − x)TeO2 (x = 20 and 25 mol%) and xV2O5–(25 − x)Li2O–75TeO2 (x = 1, 2, 3, 4 and 5 mol%) glasses were prepared by melt-quenching and their thermal and structural properties were characterized by differential scanning calorimetry, Raman spectroscopy, high-energy X-ray diffraction and neutron diffraction and reverse Monte Carlo (RMC) simulations. The glass transition temperature increases steadily with an increase in V2O5 mol% in lithium tellurite glasses due to an increase in the average single bond energy of the glass network. The X-ray and neutron diffraction structure factors were modelled by RMC technique and the Te–O distributions show the first peak in the range 1.85–1.90 Å, with V–O = 1.75–1.95 Å, Li–O = 1.85–2.15 Å and O–O = 2.70–2.80 Å. The average Te–O coordination number decreases with an increase in Li2O mol% in lithium tellurite glasses, and the V—O coordination decreases from 5.12 to 3.81 with an increase in V2O5 concentration in vanadium lithium tellurite glasses. The O–Te–O, O–V–O, O–Li–O and O–O–O linkages have maxima in the ranges 86°–89°, 82°–87°, 80°–85° and at 59o, respectively. The structural analysis of tellurite glasses reveal significant short-range and medium-range disorder due to the existence of a wide range of Te–O and Te–Te distances in the first coordination shell.

Investigation of the Structural Heterogeneity and Corrosion Performance of the Annealed Fe-Based Metallic Glasses

This study investigated the structural heterogeneity, mechanical property, electrochemical behavior, and passive film characteristics of Fe–Cr–Mo–W–C–B–Y metallic glasses (MGs), which were modified through annealing at different temperatures. Results showed that annealing MGs below the glass transition temperature enhanced corrosion resistance in HCl solution owing to a highly protective passive film formed, originating from the decreased free volume and the shrinkage of the first coordination shell, which was found by pair distribution function analysis. In contrast, the enlarged first coordination shell and nanoscale crystal-like clusters were identified for MGs annealed in the supercooled liquid region, which led to a destabilized passive film and thereby deteriorated corrosion resistance. This finding reveals the crucial role of structural heterogeneity in tuning the corrosion performance of MGs.

The BU72-μ opioid receptor crystal structure is a covalent adduct

<div>In the crystal structure of BU72 bound to the μ opioid receptor (μOR), the opioid clashes with an adjacent residue in the N-terminus; strong and unexplained electron density connects the two, centered on a point ~1.6 Å from each. This is too short for non-covalent interactions, implying covalent bonds to an unmodeled non-hydrogen atom. A magnesium ion has recently been proposed as a candidate. However, this would require unrealistically short bonds and an incomplete coordination shell. Moreover, the crystals were prepared without magnesium salts, but with components that can generate reactive oxygen species (ROS): HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fits the unexplained density well, giving a type of covalent adduct known to form in the presence of ROS, with reasonable geometry and no clashes. While the precise structure is tentative, the observed density firmly establishes covalent bonds linking ligand and residue. Severe strain is evident in the ligand, the tethered N-terminus, and the connecting bonds. This strain, along with interactions between the N-terminus and surrounding residues, is likely to distort the receptor conformation. The subsequent μOR-Gi structure, which differs in several features associated with activation, is therefore likely to be a more accurate model of the active receptor. The possibility of reactions like this should be considered in the choice of protein truncation sites and purification conditions.</div>

The BU72-μ Opioid Receptor Crystal Structure Is a Covalent Adduct

<div>In the crystal structure of BU72 bound to the μ opioid receptor, the opioid clashes with an adjacent residue, and unexplained electron density connects the two. It has been reported that this density can be filled by a magnesium ion. However, this proposal requires unrealistically short bonds and an incomplete coordination shell. Moreover, the crystals were prepared without magnesium salts, but with components that can generate reactive oxygen species: HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fills the unexplained density, giving a known type of covalent adduct with reasonable geometry and no clashes. Strain is evident, but is consistent with tension from the tethered N-terminus.</div>

The BU72-μ Opioid Receptor Crystal Structure Is a Covalent Adduct

<div>In the crystal structure of BU72 bound to the μ opioid receptor, the opioid clashes with an adjacent residue, and unexplained electron density connects the two. It has been reported that this density can be filled by a magnesium ion. However, this proposal requires unrealistically short bonds and an incomplete coordination shell. Moreover, the crystals were prepared without magnesium salts, but with components that can generate reactive oxygen species: HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fills the unexplained density, giving a known type of covalent adduct with reasonable geometry and no clashes. Strain is evident, but is consistent with tension from the tethered N-terminus.</div>

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2′/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2′/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

Cellular pathways of calcium transport and concentration towards mineral formation in sea urchin larvae

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy (cryo-SXM) we imaged intra-cellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near edge spectra (XANES) of these Ca-particles. Using the pre-peak/main peak (L2’/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-particles with order parameters, L2’/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1-15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all water, is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.SignificanceOrganisms form mineralized skeletons, many of which are composed of calcium salts. Marine organisms extract calcium ions from sea water. One of the main unresolved issues is how organisms concentrate calcium by more than 3 orders of magnitude, to achieve mineral deposition in their skeleton. Here we determine the calcium state in each of the calcium-containing vesicles inside the spicule-building cells of sea urchin larvae. We show that within one cell there is a wide range of concentrations and states from solution to solid. We hypothesize that calcium concentration increases gradually in each vesicle, starting from sea water levels and until mineral is deposited. This model might well be relevant to other phyla, thus advancing the understanding of biomineralization processes.