How Is Substrate Halogenation Triggered by the Vanadium Haloperoxidase from Curvularia inaequalis?

Vanadium haloperoxidases (VHPOs) are unique enzymes in biology that catalyze a challenging halogen transfer reaction and convert a strong aromatic C-H bond into C-X (X = Cl, Br, I) with the use of a vanadium cofactor and H2O2. The VHPO catalytic cycle starts with the conversion of hydrogen peroxide and halide (X = Cl, Br, I) into hypohalide on the vanadate cofactor, and the hypohalide subsequently reacts with a substrate. However, it is unclear whether the hypohalide is released from the enzyme or otherwise trapped within the enzyme structure for the halogenation of organic substrates. A substrate-binding pocket has never been identified for the VHPO enzyme, which questions the role of the protein in the overall reaction mechanism. Probing its role in the halogenation of small molecules will enable further engineering of the enzyme and expand its substrate scope and selectivity further for use in biotechnological applications as an environmentally benign alternative to current organic chemistry synthesis. Using a combined experimental and computational approach, we elucidate the role of the vanadium haloperoxidase protein in substrate halogenation. Activity studies show that binding of the substrate to the enzyme is essential for the reaction of the hypohalide with substrate. Stopped-flow measurements demonstrate that the rate-determining step is not dependent on substrate binding but partially on hypohalide formation. Using a combination of molecular mechanics (MM) and molecular dynamics (MD) simulations, the substrate binding area in the protein is identified and even though the selected substrates (methylphenylindole and 2-phenylindole) have limited hydrogen-bonding abilities, they are found to bind relatively strongly and remain stable in a binding tunnel. A subsequent analysis of the MD snapshots characterizes two small tunnels leading from the vanadate active site to the surface that could fit small molecules such as hypohalide, halide, and hydrogen peroxide. Density functional theory studies using electric field effects show that a polarized environment in a specific direction can substantially lower barriers for halogen transfer. A further analysis of the protein structure indeed shows a large dipole orientation in the substrate-binding pocket that could enable halogen transfer through an applied local electric field. These findings highlight the importance of the enzyme in catalyzing substrate halogenation by providing an optimal environment to lower the energy barrier for this challenging aromatic halide insertion reaction.

Instructive electroactive electrospun silk fibroin-based biomaterials for peripheral nerve tissue engineering.

Aligned sub-micron fibres are an outstanding surface for orienting and promoting neurite outgrowth; therefore, attractive features to include in peripheral nerve tissue scaffolds. A new generation of peripheral nerve tissue scaffolds is under development incorporating electroactive materials and electrical regimes as instructive cues in order to facilitate fully functional regeneration. Herein, electroactive fibres composed of silk fibroin (SF) and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) were developed as a novel peripheral nerve tissue scaffold. Mats of SF with sub-micron fibre diameters of 190 ± 50 nm were fabricated by double layer electrospinning with thicknesses of ∼100 µm (∼70-80 µm random fibres and ∼20-30 µm aligned fibres). Electrospun SF mats were modified with interpenetrating polymer networks (IPN) of PEDOT:PSS in various ratios of PSS/EDOT (α) and the polymerisation was assessed by hard X-ray photoelectron spectroscopy (HAXPES). The mechanical properties of electrospun SF and IPNs mats were characterised in the wet state tensile and the electrical properties were examined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The cytotoxicity and biocompatibility of the optimal IPNs (α = 2.3 and 3.3) mats were ascertained via the growth and neurite extension of mouse neuroblastoma x rat glioma hybrid cells (NG108-15) for 7 days. The longest neurite outgrowth of 300 µm was observed in the parallel direction of fibre alignment on laminin-coated electrospun SF and IPN (α = 2.3) mats which is the material with the lowest electron transfer resistance (Ret, ca. 330 Ω). These electrically conductive composites with aligned sub-micron fibres exhibit promise for axon guidance and also have the potential to be combined with electrical stimulation treatment as a further step for the effective regeneration of nerves.

A versatile route to edge-specific modifications to pristine graphene by electrophilic aromatic substitution.

Electrophilic aromatic substitution produces edge-specific modifications to CVD graphene and graphene nanoplatelets that are suitable for specific attachment of biomolecules.

Aldehyde-Mediated Protein-to-Surface Tethering via Controlled Diazonium Electrode Functionalization Using Protected Hydroxylamines.

We report a diazonium electro-grafting method for the covalent modification of conducting surfaces with aldehyde-reactive hydroxylamine functionalities that facilitate the wiring of redox-active (bio)molecules to electrode surfaces. Hydroxylamine near-monolayer formation is achieved via a phthalimide-protection and hydrazine-deprotection strategy that overcomes the multilayer formation that typically complicates diazonium surface modification. This surface modification strategy is characterized using electrochemistry (electrochemical impedance spectroscopy and cyclic voltammetry), X-ray photoelectron spectroscopy, and quartz crystal microbalance with dissipation monitoring. Thus-modified glassy carbon, boron-doped diamond, and gold surfaces are all shown to ligate to small molecule aldehydes, yielding surface coverages of 150-170, 40, and 100 pmol cm-2, respectively. Bioconjugation is demonstrated via the coupling of a dilute (50 µM) solution of periodate-oxidized horseradish peroxidase enzyme to a functionalized gold surface under biocompatible conditions (H2O solvent, pH 4.5, 25 °C).

A Method for Metal/Protein Stoichiometry Determination Using Thin-Film Energy Dispersive X-ray Fluorescence Spectroscopy.

A convenient approach is proposed for the quantitation of elemental cofactors in proteins and the determination of metal/protein stoichiometry, on the basis of energy dispersive X-ray fluorescence spectroscopy (EDXRF). The analysis of proteins containing the metals Cu, Fe, Zn, and Ca and also the nonmetallic element P is shown as a demonstration of the generality of the method. In general, the reported method gives limit of detection (LOD) and limit of quantification (LOQ) values in the low ppm range and requires only a few microliters of protein sample at micromolar concentrations. Moreover, sample preparation does not require any digestion steps before the analysis. The expected metal/protein stoichiometry was observed for each protein analyzed, highlighting the precision and accuracy of the method in all the tested cases. Furthermore, it is shown that the method is compatible with multimeric proteins and those with post-translational modifications such as glycosylation.

Tailoring the Performance of Organic Solvent Nanofiltration Membranes with Biophenol Coatings.

This study reports a systematic investigation of fine-tuning the filtration performance of nanofiltration membranes with biophenol coatings to produce solvent-resistant membranes with 390-1550 g mol-1 molecular weight cutoff (MWCO) and 0.5-40 L m-2 h-1 bar-1 permeance. Six kinds of inexpensive, commercial biophenols (dopamine, tannic acid, vanillyl alcohol, eugenol, morin, and quercetin) were subjected to identical oxidant-promoted polymerization to coat six kinds of loose asymmetric membrane supports: polyimide (PI), polyacrylonitrile (PAN), polysulfone (PSf), polyvinylidene difluoride (PVDF), polybenzimidazole (PBI), and polydimethylsiloxane (PDMS). The coatings were characterized by Fourier-transform infrared spectroscopy (FTIR), and the morphologies were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The long-term stability of 42 membranes were tested in 12 organic solvents, including emerging green solvents MeTHF and Cyrene. The biophenol coatings led to tighter membranes with a decrease in MWCO of 12-79% at a penalty of a 22-92% permeance decrease in acetone.

Robust Covalently Cross-linked Polybenzimidazole/Graphene Oxide Membranes for High-Flux Organic Solvent Nanofiltration.

Robust, readily scalable, high-flux graphene oxide (GO) mixed matrix composite membranes were developed for organic solvent nanofiltration. Hydroxylated polybenzimidazole was synthesized by N-benzylation of polybenzimidazole with 4-(chloromethyl)benzyl alcohol, which was confirmed by FTIR and NMR spectroscopy. Flat-sheet composite membranes comprising of polybenzimidazoles and 1 or 2 wt % GO were fabricated via conventional blade coating and phase inversion. Subsequently, GO was covalently anchored to the hydroxyl groups of the polymer using a diisocyanate cross-linking agent. The even distribution of GO in the membranes was mapped by visible-light microscopy. Hydroxylation and incorporation of GO in the polymer matrix increased the permeance up to 45.2 ± 1.6 L m-2 h-1 bar-1 in acetone, nearly 5 times higher than the unmodified benchmark membrane. The enhancement in permeance from the addition of GO did not compromise the solute rejection. The composite membranes were found to be tight in seven organic solvents, having molecular weight cut-offs (MWCO) as low as 140 g mol-1. Permeance increased with increasing solvent polarity, while rejection of a 420 g mol-1 pharmaceutical remained over 93%. The covalent anchoring resulted in robust composite membranes that maintained constant performance over 14 days in a continuous cross-flow configuration.

Features of reactive cysteines discovered through computation: from kinase inhibition to enrichment around protein degrons.

Large-scale characterisation of cysteine modification is enabling study of the physicochemical determinants of reactivity. We find that location of cysteine at the amino terminus of an α-helix, associated with activity in thioredoxins, is under-represented in human protein structures, perhaps indicative of selection against background reactivity. An amino-terminal helix location underpins the covalent linkage for one class of kinase inhibitors. Cysteine targets for S-palmitoylation, S-glutathionylation, and S-nitrosylation show little correlation with pKa values predicted from structures, although flanking sequences of S-palmitoylated sites are enriched in positively-charged amino acids, which could facilitate palmitoyl group transfer to substrate cysteine. A surprisingly large fraction of modified sites, across the three modifications, would be buried in native protein structure. Furthermore, modified cysteines are (on average) closer to lysine ubiquitinations than are unmodified cysteines, indicating that cysteine redox biology could be associated with protein degradation and degron recognition.

Prediction of Reduction Potentials of Copper Proteins with Continuum Electrostatics and Density Functional Theory.

Blue copper proteins, such as azurin, show dramatic changes in Cu2+ /Cu+ reduction potential upon mutation over the full physiological range. Hence, they have important functions in electron transfer and oxidation chemistry and have applications in industrial biotechnology. The details of what determines these reduction potential changes upon mutation are still unclear. Moreover, it has been difficult to model and predict the reduction potential of azurin mutants and currently no unique procedure or workflow pattern exists. Furthermore, high-level computational methods can be accurate but are too time consuming for practical use. In this work, a novel approach for calculating reduction potentials of azurin mutants is shown, based on a combination of continuum electrostatics, density functional theory and empirical hydrophobicity factors. Our method accurately reproduces experimental reduction potential changes of 30 mutants with respect to wildtype within experimental error and highlights the factors contributing to the reduction potential change. Finally, reduction potentials are predicted for a series of 124 new mutants that have not yet been investigated experimentally. Several mutants are identified that are located well over 10 Šfrom the copper center that change the reduction potential by more than 85 mV. The work shows that secondary coordination sphere mutations mostly lead to long-range electrostatic changes and hence can be modeled accurately with continuum electrostatics.

Quartz Crystal Microbalance Assay of Clinical Calcinosis Samples and Their Synthetic Models Differentiates the Efficacy of Chelation-Based Treatments.

This paper sets out in vitro protocols for studying the relative effectiveness of chelators used in the dissolution-based treatment of hard calcinosis. Pulverized hard calcinosis samples from human donors or synthetic hydroxyapatite nanoparticles were deposited by electrophoretic deposition on the surface of a quartz crystal microbalance sensor. Over 150 deposits of <20 µg were dissolved over the course of 1 h by aliquots of buffered, aqueous solutions of two calcium chelators, EDTA and citrate, with the surface-limited dissolution kinetics monitored with <1 s time resolution. There was no statistically significant difference in dissolution rate between the four synthetic hydroxyapatite materials in EDTA, but the dissolution rates in citrate were lower for hydroxyapatite produced by acetate or nitrate metathesis. Hard calcinosis and synthetic hydroxyapatites showed statistically identical dissolution behavior, meaning that readily available synthetic mimics can replace the rarer samples of biological origin in the development of calcinosis treatments. EDTA dissolved the hydroxyapatite deposits more than twice as fast as citrate at pH 7.4 and 37 °C, based on a first-order kinetic analysis of the initial frequency response. EDTA chelated 6.5 times more calcium than an equivalent number of moles of citrate. Negative controls using nonchelating N,N,N',N'-tetraethylethylenediamine (TEEDA) showed no dissolution effect. Pharmaceutical dissolution testing of synthetic hydroxyapatite tablets over 6 h showed that EDTA dissolved the tablets four to nine times more quickly than citrate.

A sensitivity metric and software to guide the analysis of soft films measured by a quartz crystal microbalance.

This article introduces a set of mathematical and computational tools for use with a quartz crystal microbalance (QCM) to aid in experimental planning and data interpretation. The optimisation tools are based on a metric we term the total parameter matrix sensitivity (TPM-sensitivity). TPM-sensitivity is defined mathematically as the Jacobian determinant of a QCM's responses (e.g., frequency change or dissipation/bandwidth change for a given harmonic) with respect to changes in the physical properties of a soft film and surrounding solution (e.g., density or viscosity). Large TPM-sensitivity values denote conditions where the sensor responses are not only large but also allow the selected unknown physical properties to be mathematically decoupled. In some cases, the viscoelastic properties of an adlayer can be determined using only frequency responses. We validated this method using experimentally obtained data of an ageing adlayer of the enzyme bilirubin oxidase from Myrothecium verrucaria. Fits to these measurements produced more realistic film parameters when responses, including frequency-only combinations, were selected to maximise TPM sensitivity. We provide documented MATLAB code with a graphical user interface to enable other QCM users to employ this analysis. The current software can be applied to any single, homogeneous adlayer that obeys a Kelvin-Voigt viscoelastic model and sits under a semi-infinite Newtonian fluid. Only initial estimates of the film values are required, with the analysis providing guidance and predictions, allowing users to create testable hypothesis and determine the physical changes on the surface rather than have pre-existing values for them.

Optimizing the Mass-Specific Activity of Bilirubin Oxidase Adlayers through Combined Electrochemical Quartz Crystal Microbalance and Dual Polarization Interferometry Analyses.

Two surface analysis techniques, dual polarization interferometry (DPI) and analysis by an electrochemical quartz crystal microbalance with dissipation capability (E-QCM-D), were paired to find the deposition conditions that give the highest and most stable electrocatalytic activity per adsorbed mass of enzyme. Layers were formed by adsorption from buffered solutions of bilirubin oxidase from Myrothecium verrucaria at pH 6.0 to planar surfaces, under high enzyme loading (≥1 mg mL(-1)) for contact periods of up to 2 min. Both unmodified and carboxylate-functionalized gold-coated sensors showed that a deposition solution concentration of 10-25 mg mL(-1) gave the highest activity per mass of adsorbed enzyme with an effective catalytic rate constant (k(cat)) of about 60 s(-1). The densification of adsorbed layers observed by DPI correlated with reduced bioactivity observed by parallel E-QCM-D measurements. Postadsorption changes in thickness and density observed by DPI were incorporated into Kelvin-Voigt models of the QCM-D response. The modeled response matched experimental observations when the adlayer viscosity tripled after adsorption.

Surface Characterization and in situ Protein Adsorption Studies on Carbene-Modified Polymers.

Polystyrene thin films were functionalized using a facile two-step chemical protocol involving carbene insertion followed by azo-coupling, permitting the introduction of a range of chemical functional groups, including aniline, hexyl, amine, carboxyl, phenyl, phosphonate diester, and ethylene glycol. X-ray photoelectron spectroscopy (XPS) confirmed the success of the two-step chemical modification with a grafting density of at least 1/10th of the typical loading density (10(14)-10(15)) of a self-assembled monolayer (SAM). In situ, real-time quartz crystal microbalance with dissipation (QCM-D) studies show that the dynamics of binding of bovine serum albumin (BSA) are different at each modified surface. Mass, viscoelastic, and kinetic data were analyzed, and compared to cheminformatic descriptors (i.e., c log P, polar surface area) typically used for drug discovery. Results show that functionalities may either resist or adsorb BSA, and uniquely influence its adsorption dynamics. It is concluded that carbene-based surface modification can usefully influence BSA binding dynamics in a manner consistent with, and more robust than, traditional systems based on SAM chemistry.

The birth of protein electrochemistry.

The results from a final-year undergraduate project led to an $876M sale of a spin-out company 19 years later: the 1977 communication from Mark Eddowes and Allen Hill seeded the rich field of protein electrochemistry, the technology that underpins commercial glucose biosensors.

Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase.

X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O(2). In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV-Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O(2) reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account.

Bilirubin oxidase from Myrothecium verrucaria: X-ray determination of the complete crystal structure and a rational surface modification for enhanced electrocatalytic O2 reduction.

The blue multi-copper oxidase bilirubin oxidase (BOx) from the ascomycete plant pathogen Myrothecium verrucaria (Mv) efficiently catalyses the oxidation of bilirubin to biliverdin, with the concomitant reduction of O(2) to water, a reaction of considerable interest for low-temperature bio-fuel cell applications. We have solved the complete X-ray determined structure of Mv BOx at 2.4 Å resolution, using molecular replacement with the Spore Coat Protein A (CotA) enzyme from Bacillus subtilis (PDB code 1GSK) as a template. The structure reveals an unusual environment around the blue type 1 copper (T1 Cu) that includes two non-coordinating hydrophilic amino acids, asparagine and threonine. The presence of a long, narrow and hydrophilic pocket near the T1 Cu suggests that structure of the substrate-binding site is dynamically determined in vivo. We show that the interaction between the binding pocket of Mv BOx and its highly conjugated natural organic substrate, bilirubin, can be used to stabilise the enzyme on a pyrolytic graphite electrode, more than doubling its electrocatalytic activity relative to the current obtained by simple adsorption of the protein to the carbon surface.

Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3).

The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single-site proline variants of CYP102A1 (P450(BM3)) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C-H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non-natural substrates, displays a number of structural similarities to substrate-bound forms of the wild-type enzyme, notably an off-axial water ligand, a drop in the proximal loop, and the positioning of two I-helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second-generation I401P variants gave high in vitro oxidation rates with non-natural substrates as varied as fluorene and propane, towards which the wild-type enzyme is essentially inactive. The substrate-free I401P haem domain had a reduction potential slightly more oxidising than the palmitate-bound wild-type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate-free wild-type enzyme.

Mechanistic studies of the 'blue' Cu enzyme, bilirubin oxidase, as a highly efficient electrocatalyst for the oxygen reduction reaction.

The 'blue copper' enzyme bilirubin oxidase from Myrothecium verrucaria shows significantly enhanced adsorption on a pyrolytic graphite 'edge' (PGE) electrode that has been covalently modified with naphthyl-2-carboxylate functionalities by diazonium coupling. Modified electrodes coated with bilirubin oxidase show electrocatalytic voltammograms for the direct, four-electron reduction of O(2) by bilirubin oxidase with up to four times the current density of an unmodified PGE electrode. Electrocatalytic voltammograms measured with a rapidly rotating electrode (to remove effects of O(2) diffusion limitation) have a complex shape (an almost linear dependence of current on potential below pH 6) that is similar regardless of how PGE is chemically modified. Importantly, the same waveform is observed if bilirubin oxidase is adsorbed on Au(111) or Pt(111) single-crystal electrodes (at which activity is short-lived). The electrocatalytic behavior of bilirubin oxidase, including its enhanced response on chemically-modified PGE, therefore reflects inherent properties that do not depend on the electrode material. The variation of voltammetric waveshapes and potential-dependent (O(2)) Michaelis constants with pH and analysis in terms of the dispersion model are consistent with a change in rate-determining step over the pH range 5-8: at pH 5, the high activity is limited by the rate of interfacial redox cycling of the Type 1 copper whereas at pH 8 activity is much lower and a sigmoidal shape is approached, showing that interfacial electron transfer is no longer a limiting factor. The electrocatalytic activity of bilirubin oxidase on Pt(111) appears as a prominent pre-wave to electrocatalysis by Pt surface atoms, thus substantiating in a single, direct experiment that the minimum overpotential required for O(2) reduction by the enzyme is substantially smaller than required at Pt. At pH 8, the onset of O(2) reduction lies within 0.14 V of the four-electron O(2)/2H(2)O potential.

Designer laccases: a vogue for high-potential fungal enzymes?

Laccases are blue multicopper oxidases that catalyse the four-electron reduction of O(2) to water coupled with the oxidation of small organic substrates. Secreted basidiomycete white-rot fungal laccases orchestrate this with high thermodynamic efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-of-play regarding expression of high reduction potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biology, particularly in unmasking structure-function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases.

A highly active single-mutation variant of P450BM3 (CYP102A1).

The power of proline: Bold amino acid substitutions in sensitive protein regions are frequently unproductive, while more subtle mutations can be sufficient to bring about dramatic changes. But introducing proline at the residue next to the sulfur ligand in P450(BM3) (CYP102A1) has the unexpected and desirable effect of enhancing the activity of this fatty acid hydroxylase with a broad range of non-natural substrates, as illustrated by the figure.