Christopher F. Blanford's research while affiliated with The University of Manchester and other places

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.

The yellow rust of wheat (caused by Puccinia striiformis f. sp. tritici) is a devastating fungal infection that is responsible for significant wheat yield losses. The main challenge with the detection of this disease is that it can only be visually detected on the leaf surface between 7 and 10 days after infection, and by this point, counter measures such as the use of fungicides are generally less effective. The hypothesis of this study is to develop and use a compact electrochemical-based biosensor for the early detection of P. striiformis, thus enabling fast countermeasures to be taken. The biosensor that was developed consists of three layers. The first layer mimics the wheat leaf surface morphology. The second layer consists of a sucrose/agar mixture that acts as a substrate and contains a wheat-derived terpene volatile organic compound that stimulates the germination and growth of the spores of the yellow rust pathogen P. s. f. sp. tritici. The third layer consists of a nonenzymatic glucose sensor that produces a signal once invertase is produced by P. striiformis, which comes into contact with the second layer, thereby converting sucrose to glucose. The results show the proof that this innovative biosensor can enable the detection of yellow rust spores in 72 h.

A new route to single‐step and non‐covalent immobilization of proteins on graphene is exemplified with the first biosensor for nitriles based on a graphene field‐effect transistor (GFET). The biological recognition element is a fusion protein consisting of nitrile reductase QueF from Escherichia coli with an N‐terminal self‐assembling and graphene‐binding dodecapeptide. Atomic force microscopy and analysis using a quartz crystal microbalance show that both the oligopeptide and the fusion protein incorporating it form a single adlayer of monomeric enzyme on graphene. The fusion protein has a 6.3‐fold increase in binding affinity for benzyl cyanide (BnCN) versus wild‐type QueF and a 1.4‐fold increase for affinity for the enzyme's natural substrate preQ0. Density functional theory analysis of QueF's catalytic cycle with BnCN shows similar transition‐state barriers to preQ0, but differences in the formation of the initial thioimidate covalent bonding (∆G‡ = 19.0 kcal mol⁻¹ for preQ0 vs 27.7 kcal mol⁻¹ for BnCN) and final disassociation step (∆G = −24.3 kcal mol⁻¹ for preQ0 vs ∆G = +4.6 kcal mol⁻¹ for BnCN). Not only do these results offer a single‐step route to GFET modification, but they also present new opportunities in the biocatalytic synthesis of primary amines from nitriles.

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 full 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 diameter of 190 ± 50 nm were fabricated by double layers 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 polymerization was assessed by hard X-ray photoelectron spectroscopy (HAXPES). The mechanical properties of electrospun SF and IPNs mats were characterized by 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 has the potential to be combined with electrical stimulation treatment as a further step for the effective regeneration of nerves.

This study compares the enzymatic activity of nanoscale arrays bearing the oxidase CotA that are produced by two lithographic methods: polymer pen lithography (PPL), a scanning probe lithography for small‐area fabrication (≈1 cm²); and stepper photolithography, a large‐scale method (>300 cm²) used in the microelectronics sector. In both cases, arrays of 600 nm gold features are produced and functionalized with CotA through a bioconjugation method that gives uniform protein orientation. The enzyme activity of these arrays is then quantified over 100 days. The enzyme arrays produced by photolithography give higher oxidation activities immediately after fabrication but degrade more rapidly when compared to those produced by PPL. This result is due to the poorer passivation on the bulk surface of photolithographically produced arrays, resulting in a greater amount of non‐specifically adsorbed enzymes. However, once the results are adjusted to account for the differences in passivation and surface area, it is found that the enzymes immobilized on the gold features give essentially the same activity regardless of the lithographic method used. Thus, these results suggest PPL is a suitable method for prototyping biodevices prior to scale‐up, provided that due consideration is given to the design of the fabricated features.

Vanadium haloperoxidases are one of the few enzymes in nature that utilize a vanadium center and catalyze the halogenation of substrates through the biosynthesis of hypohalite. Vanadium chloroperoxidases (VCPOs) bind and activate hydrogen peroxide and in a reaction with chloride convert it into hypochlorite as a precursor for a substrate chlorination reaction. Despite the fact that these enzymes have been studied extensively, surprisingly little is known on their catalytic cycle and particularly on the function of the vanadium atom in the reaction mechanism. In order to gain insight into the intricate details of the catalytic cycle of VCPOs, we performed an extensive computational study using large cluster model complexes, where we tested many possible pathways and active-site protonation states. Our work establishes that the biosynthesis of hypochlorite proceeds in two steps: H2O2 activation on the vanadium center to form an end-on V(V)-hydroperoxo complex, followed by OH+ transfer from hydroperoxo to chloride on the vanadium center to form hypochlorite. We show that the initial reaction starts with a proton transfer from H2O2 to the equatorial OH group of the VV(O)2(OH)2- active site, followed by hydroperoxo binding and water release to form the highly stable vanadium-hydroperoxo-dioxo-hydroxo complex. A further proton transfer from an active-site His or Lys residue can lead to the vanadium-peroxo-hydroxo-oxo complex, which we assign as a dead-end complex unable to react further to hypochlorite products. The mechanisms were considered under various protonation state, and it is shown to be the most effective with His404 singly protonated. The work shows that vanadium is a spectator ion that does not change its oxidation state during the reaction mechanism but holds and positions the H2O2 substrate and guides its proton-relay steps through its oxo and hydroxo ligands. The effect of the protonation state of first- and second-coordination sphere residues and ligands was tested and shows that the reaction is highly sensitive to local changes in the protonation state. Finally, the computations show that the oxygen atom of HOCl exclusively derives from H2O2.

This work presents a general method for producing edge-modified graphene using electrophilic aromatic substitution. Five types of edge-modified graphene were created from graphene/graphite nanoplatelets sourced commercially and produced by ultrasonic exfoliation of graphite in N-methyl-2-pyrrolidone. In contrast to published methods based on Friedel–Crafts acylation, this method does not introduce a carbonyl group that may retard electron transfer between the graphene sheet and its pendant groups. Graphene sulphonate (G–SO3⁻) was prepared by chlorosulphonation and then reduced to form graphene thiol (G–SH). The modifications tuned the graphene nanoparticles’ solubility: G–SO3⁻ was readily dispersible in water, and G–SH was dispersible in toluene. The synthetic utility of the directly attached reactive moieties was demonstrated by creating a “glycographene” through radical addition of allyl mannoside to G–SH. Chemical modifications were confirmed by FT-IR and XPS. Based on XPS analysis of edge-modified GNPs, G–SO3⁻ and G–SH had a S:C atomic ratio of 0.3:100. XPS showed that a significant amount of carbon sp² character remained after functionalisation, indicating little modification to the conductive basal plane. The edge specificity of the modifications was visualised on edge-modified samples of graphene produced by chemical vapour deposition (CVD): scanning electron microscopy of gold nanoparticles attached to G–SH samples, epifluorescence microscopy of a glycographene bioconjugate with a fluorescently tagged lectin, and quenched stochastic optical reconstruction microscopy (qSTORM) of thiol-reactive fluorophores on CVD G–SH samples. Microelectrochemistry of unmodified CVD graphene and dye-modified CVD G–SH showed no statistically significant difference in interfacial electron transfer rate (k⁰). This platform synthesis technology can allow pristine graphene, rather than graphene oxide or its derivatives, to be used in applications that require the superior mechanical or electronic properties of pristine graphene, including theranostics and tissue engineering. Graphical Abstract Electrophilic aromatic substitution produces edge-specific modifications to CVD graphene and graphene nanoplatelets that are suitable for specific attachment of biomolecules

Multicopper oxidases (MCO), such as laccases and bilirubin oxidases, are catalysts of wide interest to a range of biotechnological applications, especially bioremediation and energy transduction. Many of these processes take place in the presence of dissolved species, particularly halides, at concentrations high enough to inhibit the catalytic activity of MCOs. Despite this, MCO inhibition is rarely considered the focus of reviews on the topic. This review critically analyses the scientific literature on modes of MCO inhibition and current hypotheses on the structural origins of inhibition. It provides a comprehensive overview of what is known from the different techniques applied to the study MCO inhibition. These techniques include solution-based enzymatic assays, electrochemical methodologies, various spectroscopic approaches, X-ray crystallography and computational analyses. This review highlights gaps in the literature, identifies nine challenges to accurate benchmarking MCO inhibition and gives recommendations to address these challenges and to interpret published data on the subject.