Crossing the Solubility Rubicon: 15-Crown-5 Facilitates the Preparation of Water-Soluble Sulfo-NHS Esters in Organic Solvents.

The Sulfo-NHS ester is a mainstay reagent for facilitating amide bond formation between carboxylic acids and amine functionalities in water. However, the preparation of Sulfo-NHS esters currently requires hydrophobic carboxylic acids, which are poorly water-soluble, to first be reacted with the N-hydroxysulfosuccinimide sodium salt, which is insoluble in organic solvents. The mutually incompatible solvation requirements thus complicate the synthesis of Sulfo-NHS esters. As a simple, rapid, and cost-effective solution to this problem, we report that the use of 15-crown-5 to complex the sodium cation of N-hydroxysulfosuccinimide sodium salt circumnavigates these solvation incompatibility issues by rendering the N-hydroxysulfosuccinimide salt soluble in organic solvents, resulting in a cleaner esterification reaction and thus improved yields of activated ester product. We also demonstrate that the resultant "crowned" Sulfo-NHS-ester remains water-soluble and is no less reactive than its classic "uncrowned" Sulfo-NHS counterpart when used in bioconjugation reactions between protein amine-functionalities and hydrophobic carboxylic acids.

Site-Selective Aryl Diazonium Installation onto Protein Surfaces at Neutral pH using a Maleimide-Functionalized Triazabutadiene.

Aryl diazonium cations are versatile bioconjugation reagents due to their reactivity towards electron-rich aryl residues and secondary amines, but historically their usage has been hampered by both their short lifespan in aqueous solution and the harsh conditions required to generate them in situ. Triazabutadienes address many of these issues as they are stable enough to endure multiple-step chemical syntheses and can persist for several hours in aqueous solution, yet upon UV-exposure rapidly release aryl diazonium cations under biologically-relevant conditions. This paper describes the synthesis of a novel maleimide-functionalized triazabutadiene suitable for site-selectively installing aryl diazonium cations into proteins at neutral pH; we show reaction with this molecule and a surface-cysteine of a thiol disulfide oxidoreductase. Through photoactivation of the site-selectively installed triazabutadiene motifs, we generate aryl diazonium functionality, which we further derivatize via azo-bond formation to electron-rich aryl species, showcasing the potential utility of this strategy for the generation of photoswitches or protein-drug conjugates.

Comparing the Catalytic and Structural Characteristics of a 'Short' Unspecific Peroxygenase (UPO) Expressed in Pichia†pastoris and Escherichia†coli.

Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an 'artificial' peroxygenase (artUPO), close in sequence to the 'short' UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)-alcohols, also given by a variant of the 'long' UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related'short' UPOs.

Mapping the protonation states of the histidine brace in an AA10 lytic polysaccharide monooxygenase using CW-EPR spectroscopy and DFT calculations.

The active site of the polysaccharide-degrading lytic polysaccharide monooxygenase (LPMO) enzyme features a single copper ion coordinated by a histidine brace. The primary coordination sphere of the copper contains several ligating atoms which are bonded to ionisable protons (e.g. OH2, NH2), the pKas of which are unknown. Using a combination of CW-EPR X-band spectroscopy over a range of pH values and DFT calculations, we show that the active site of a chitin-active AA10 LPMO can exist in three different protonation states (pKa1 = 8.7, pKa2 ∼ 11.5), representing the ionisation of the coordinating groups. The middle pH species (fully formed at pH ∼ 10.5) is proposed to be Cu(II)(His)2(OH)2 (N2O2 coordination) with a decoordinated R-NH3+ group at the amino terminus. This species also sees a rotation of the SOMO equatorial plane from the canonical histidine brace plane, whereby the nominal Cu d(x2 - y2)-orbital has rotated some 45° along the His-Cu(II)-His axis, driven by the elongation and decoordination of the amino group. The highest pH species (>12) is proposed to exist as a Cu(II)-azanide, in which the NH2 of the amino terminus has been deprotonated. The high pH means that this species is unlikely to be biologically relevant in the catalytic cycle of AA10 LPMOs.

Electrochemical and Solution Structural Characterization of Fe(III) Azotochelin Complexes: Examining the Coordination Behavior of a Tetradentate Siderophore.

We report an electrochemical setup comprising a boron-doped diamond (BDD) working electrode for the electrochemical study of iron(III) catecholate siderophores. We demonstrate its successful application in the voltammetric investigation of iron(III) azotochelin, an iron complex of a bis(catecholate) siderophore. Cyclic voltammetry results, when complemented by UV-vis and native electrospray ionization-mass spectrometry (ESI-MS) characterization, reveal the formation of a coordinatively unsaturated tetracoordinate 1:1 complex of Fe:azotochelin (M1:L1) at neutral pH, contrary to iron(III) tetradentate siderophore complexes of other classes which favor the hexacoordinate environment of an M2:L3 species. A notable effect of pH and buffer composition on the reduction potential of iron(III) azotochelin is demonstrated. Lower pH values and buffers encompassing primary or secondary amines facilitate a positive potential shift of up to +290 mV and +250 mV vs Ag/AgCl 3 M NaCl, respectively. The study was extended to the investigation of the iron(III) complexes of hexadentate siderophores. For tris(catecholate) siderophores, enterobactin and protochelin, the reduction potentials were found to lie beyond the potential window accessible to the BDD electrode; however, we were successful in observing the electrochemical behavior of a tris(hydroxamate) siderophore, ferricrocin.

Ultrafast 2D-IR spectroscopy of [NiFe] hydrogenase from E. coli reveals the role of the protein scaffold in controlling the active site environment.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)2 unit of the active site. Measurements on as-isolated EcHyd-1 probed a mixture of active site states including two, which we assign to Nir-SI/II, that have not been previously observed in the E. coli enzyme. Explicit assignment of carbonyl (CO) and cyanide (CN) stretching bands to each state is enabled by 2D-IR. Energies of vibrational levels up to and including two-quantum vibrationally excited states of the CO and CN modes have been determined along with the associated vibrational relaxation dynamics. The carbonyl stretching mode potential is well described by a Morse function and couples weakly to the cyanide stretching vibrations. In contrast, the two CN stretching modes exhibit extremely strong coupling, leading to the observation of formally forbidden vibrational transitions in the 2D-IR spectra. We show that the vibrational relaxation times and structural dynamics of the CO and CN ligand stretching modes of the enzyme active site differ markedly from those of a model compound K[CpFe(CO)(CN)2] in aqueous solution and conclude that the protein scaffold creates a unique biomolecular environment for the NiFe site that cannot be represented by analogy to simple models of solvation.

C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.

Using Purely Sinusoidal Voltammetry for Rapid Inference of Surface-Confined Electrochemical Reaction Parameters.

Alternating current (AC) voltammetric techniques are experimentally powerful as they enable Faradaic current to be isolated from non-Faradaic contributions. Finding the best global fit between experimental voltammetric data and simulations based on reaction models requires searching a substantial parameter space at high resolution. In this paper, we estimate parameters from purely sinusoidal voltammetry (PSV) experiments, investigating the redox reactions of a surface-confined ferrocene derivative. The advantage of PSV is that a complete experiment can be simulated relatively rapidly, compared to other AC voltammetric techniques. In one example involving thermodynamic dispersion, a PSV parameter inference effort requiring 7,500,000 simulations was completed in 7 h, whereas the same process for our previously used technique, ramped Fourier transform AC voltammetry (ramped FTACV), would have taken 4 days. Using both synthetic and experimental data with a surface confined diazonium substituted ferrocene derivative, it is shown that the PSV technique can be used to recover the key chemical and physical parameters. By applying techniques from Bayesian inference and Markov chain Monte Carlo methods, the confidence, distribution, and degree of correlation of the recovered parameters was visualized and quantified.

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).

Methodologies for "Wiring" Redox Proteins/Enzymes to Electrode Surfaces.

The immobilization of redox proteins or enzymes onto conductive surfaces has application in the analysis of biological processes, the fabrication of biosensors, and in the development of green technologies and biochemical synthetic approaches. This review evaluates the methods through which redox proteins can be attached to electrode surfaces in a "wired" configuration, that is, one that facilitates direct electron transfer. The feasibility of simple electroactive adsorption onto a range of electrode surfaces is illustrated, with a highlight on the recent advances that have been achieved in biotechnological device construction using carbon materials and metal oxides. The covalent crosslinking strategies commonly used for the modification and biofunctionalization of electrode surfaces are also evaluated. Recent innovations in harnessing chemical biology methods for electrically wiring redox biology to surfaces are emphasized.

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme.

A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer. With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized "as-isolated" Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide. Based on comparison with earlier studies and our UV-vis redox titration data, we assign the reversible one-proton and one-electron reduction process centered around +174 mV vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conversion but ascribe the two-proton and two-electron transition occurring at negative potential to the organic pyranopterin ligand system. We predict that a dihydro-to-tetrahydro transition is needed to generate the catalytically active state of the enzyme. This is a previously unidentified mechanism, suggested by the structural simplicity of YedY, a protein in which Mo is the only metal site.

Retuning the Catalytic Bias and Overpotential of a [NiFe]-Hydrogenase via a Single Amino Acid Exchange at the Electron Entry/Exit Site.

The redox chemistry of the electron entry/exit site in Escherichia coli hydrogenase-1 is shown to play a vital role in tuning biocatalysis. Inspired by nature, we generate a HyaA-R193L variant to disrupt a proposed Arg-His cation-π interaction in the secondary coordination sphere of the outermost, "distal", iron-sulfur cluster. This rewires the enzyme, enhancing the relative rate of H2 production and the thermodynamic efficiency of H2 oxidation catalysis. On the basis of Fourier transformed alternating current voltammetry measurements, we relate these changes in catalysis to a shift in the distal [Fe4S4]2+/1+ redox potential, a previously experimentally inaccessible parameter. Thus, metalloenzyme chemistry is shown to be tuned by the second coordination sphere of an electron transfer site distant from the catalytic center.

Analysis of HypD Disulfide Redox Chemistry via Optimization of Fourier Transformed ac Voltammetric Data.

Rapid disulfide bond formation and cleavage is an essential mechanism of life. Using large amplitude Fourier transformed alternating current voltammetry (FTacV) we have measured previously uncharacterized disulfide bond redox chemistry in Escherichia coli HypD. This protein is representative of a class of assembly proteins that play an essential role in the biosynthesis of the active site of [NiFe]-hydrogenases, a family of H2-activating enzymes. Compared to conventional electrochemical methods, the advantages of the FTacV technique are the high resolution of the faradaic signal in the higher order harmonics and the fact that a single electrochemical experiment contains all the data needed to estimate the (very fast) electron transfer rates (both rate constants ≥ 4000 s-1) and quantify the energetics of the cysteine disulfide redox-reaction (reversible potentials for both processes approximately -0.21 ± 0.01 V vs SHE at pH 6). Previously, deriving such data depended on an inefficient manual trial-and-error approach to simulation. As a highly advantageous alternative, we describe herein an automated multiparameter data optimization analysis strategy where the simulated and experimental faradaic current data are compared for both the real and imaginary components in each of the 4th to 12th harmonics after quantifying the charging current data using the time-domain response.

Redox-Tagged Carbon Monoxide-Releasing Molecules (CORMs): Ferrocene-Containing [Mn(C

This study describes the synthesis and characterization of a new class of ferrocene-containing carbon monoxide-releasing molecules (CORMs, 1-3). The ferrocenyl group is both a recognized therapeutically viable coligand and a handle for informative infrared spectroelectrochemistry. Deoxymyoglobin CO-release assays and in situ infrared spectroscopy confirm compounds 2 and 3 as photoCORMs and 1 as a thermal CORM, attributed to the increased sensitivity of the Mn-ferrocenyl bond to protonation in 1. Electrochemical and infrared spectroelectrochemical experiments confirm a single reversible redox couple associated with the ferrocenyl moiety with the Mn tetracarbonyl center showing no redox activity up to +590 mV vs Fc/Fc+, though no concomitant CO release was observed in association with the redox activity. The effects of linker length on communication between the Fe and Mn centers suggest that the incorporation of redox-active ligands into CORMs focuses on the first coordination sphere of the CORM. Redox-tagged CORMs could prove to be a useful mechanistic probe; our findings could be developed to use redox changes to trigger CO release.

Structural differences of oxidized iron-sulfur and nickel-iron cofactors in O2-tolerant and O2-sensitive hydrogenases studied by X-ray absorption spectroscopy.

The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ~0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ~0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases.

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH.

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins.

A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.

Selective Extraction and In Situ Reduction of Precious Metal Salts from Model Waste To Generate Hybrid Gels with Embedded Electrocatalytic Nanoparticles.

A hydrogel based on 1,3:2,4-dibenzylidenesorbitol (DBS), modified with acyl hydrazides which extracts gold/silver salts from model waste is reported, with preferential uptake of precious heavy metals over other common metals. Reduction of gold/silver salts occurs spontaneously in the gel to yield metal nanoparticles located on the gel nanofibers. High nanoparticle loadings can be achieved, endowing the gel with electrochemical activity. These hybrid gels exhibit higher conductances than gels doped with carbon nanotubes, and can be used to modify electrode surfaces, enhancing electrocatalysis. We reason this simple, industrially and environmentally relevant approach to conducting materials is of considerable significance.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase.

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.

X-ray crystallographic and computational studies of the O2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli.

The crystal structure of the membrane-bound O(2)-tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H(2)-reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O(2)-sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oε of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H(2)-reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O(2) to H(2)O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O(2) reduction, located near the active site.