Direct electrochemistry of recombinant tobacco peroxidase on gold

Direct electron transfer (DET) reactions of recombinant tobacco peroxidase (rTOP), namely direct electroreduction of Compound I/Compound II and heme Fe^3+/2+ conversion, were studied on gold electrodes. rTOP of wild type, non-glycosylated, was produced using an Escherichia coli expression system. At pH 5.0, the redox potential for direct electrochemical transformation of the Fe^3+/2+ of the peroxidase heme was −143 mV vs. AgAgCl, and 0.26 ± 0.07 pmol of the adsorbed rTOP were in DET contact with the gold electrode. The total amount of the adsorbed rTOP estimated from QCM data was 53 ± 5 pmol/cm² or 1.67 pmol when referred to the surface area of the electrodes used for electrochemical measurements. Of 1.67 pmol of adsorbed rTOP, only 0.76 pmol were catalytically active. DET between Au and the enzyme was also studied in the reaction of the bioelectrocatalytic reduction of H₂O₂ by cyclic voltammetry and amperometric detection of H₂O₂ at +50 mV with rTOP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Maximal bioelectrocatalytic current response of the rTOP-modified gold electrodes to H₂O₂ was observed at pH 5.0 and stemmed from its bioelectrocatalytic reduction based on DET between Au and the active site of rTOP. Kinetic analysis of the DET reactions gave 52% of the adsorbed rTOP molecules active in DET reactions (0.4 pmol of adsorbed catalytically active rTOP, correspondingly), which correlated well with the non-catalytic-voltammetry data. DET was characterised by a heterogeneous ET rate constant of 13.2 s⁻¹, if one takes into account the QCM data, and 19.6 s⁻¹, if the amount of rTOP estimated from the data on DET transformation of Fe^3+/2+ couple of rTOP is considered. The sensitivity for H₂O₂ obtained for the rTOP-modified Au electrodes was 0.7 ± 0.1 A M⁻¹ cm⁻². These are the first ever-reported data on DET reactions of anionic plant peroxidases on bare gold electrodes.     

Direct Heterogeneous Electron Transfer Reactions of Bacillus halodurans Bacterial Blue Multicopper Oxidase

Direct electron transfer reactions of Bacillus halodurans bacterial multicopper oxidase on bare spectrographic graphite, as well as bare and thiol‐modified gold electrodes were studied using cyclic voltammetry, potentiometry, amperometry, and spectroelectrochemistry. The redox potential of the T1 site of the enzyme was measured using mediatorless redox titration and found to be 325 mV±10 mV vs. NHE. From measurements with a mercaptopropionic acid‐modified gold electrode under aerobic conditions a midpoint potential of 360 mV vs. NHE for the T2/T3 copper cluster is deduced. Differing from most other characterized laccases of fungal and plant origins this bacterial enzyme exhibits bioelectrocatalytic activity at neutral pH and tolerates high chloride concentrations (200 mM), conditions that usually strongly inhibit catalysis. Moreover, it has the very high affinity towards molecular oxygen both in solution and in the adsorbed state (K_M≤50 μM).     

Direct electrochemical conversion of bilirubin oxidase at carbon nanotube-modified glassy carbon electrodes

We report on direct electron transfer reactions of bilirubin oxidase at multi-walled carbon nanotube (MWCNT) modified glassy carbon electrodes (GCE). The bioelectrocatalytic oxygen reduction was recorded using linear sweep voltammetry (LSV) with BOD in solution, adsorbed and covalently linked to the nanotubes. The MWCNT modification of GC electrodes strongly enhances the oxygen reduction compared to the signals at unmodified GCE. Under anaerobic conditions with a high protein concentration in solution a pair of redox peaks with a formal potential of 450 ± 15 mV vs Ag/AgCl, 1 M KCl (pH 7.4) was found with cyclic voltammetry. The redox conversion is indicated to be surface-controlled and pH-dependent (54.5 mV/pH). The quasi-reversible redox reaction might be attributed to the trinuclear T2/T3 cluster of BOD.     

Comparative Spectroelectrochemical Studies of Lyophilized and Nonlyophilized Laccases from Cerrena unicolor Basidiomycete

The electrochemical, spectroelectrochemical, and kinetic investigations of two preparations of Cerrena unicolor laccase, lyophilized (LLAC) and nonlyophilized frozen enzymes (FLAC), were performed. It was found that the value of the redox potential of the T1 site of C. unicolor laccase is ca. 750 vs. NHE. It was also shown that one of the redox potentials of the T2/T3 cluster of C. unicolor laccase is close to 400 mV, as was previously confirmed for other blue multicopper oxidases, such as trees and fungal laccases, ascorbate oxidase, and bilirubin oxidase. Furthermore, the poor stability of both preparations, but especially of LLAC, in their reduced state was confirmed using mediated and mediatorless spectroelectrochemical studies. DET‐based biocatalytic reduction of O₂ by C. unicolor laccase was only obtained, when FLAC was directly adsorbed on a spectrographic graphite electrode. Moreover, only low values of the steady‐state potentials of gold and graphite electrodes modified by C. unicolor laccase were also found. Heterogeneity of the 3‐D structures of laccase molecules, conformational changes, and partial denaturation of the enzyme, which appeared after enzyme isolation, purification, and especially lyophilization, were found to be the reasons for the low bioelectrocatalytic current, the high K_M‐value towards O₂, and the unusual electrochemical behavior of C. unicolor laccase used in the present study. In spite of the comparable specific activity and long‐term stability of both preparations in homogeneous solution, the stability of immobilized LLAC was found to be inadmissibly low for both fundamental studies and possible electrochemical applications. Indeed, FLAC is a much better source of enzyme than its lyophilized counterpart.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in hybrid organic-inorganic film of chitosan/sol-gel/carbon nanotubes

A hybrid organic-inorganic nanocomposite film of chitosan/sol-gel/multi-walled carbon nanotubes was constructed for the immobilization of horseradish peroxidase (HRP). This film was characterized by scanning electron microscopy. Direct electron transfer (DET) and bioelectrocatalysis of HRP incorporated into the composite film were investigated. The results indicate that the film can provide a favorable microenvironment for HRP to perform DET on the surface of glassy carbon electrodes with a pair of quasi-reversible redox waves and to retain its bioelectrocatalytic activity toward H₂O₂.     

肌红蛋白在碳纳米管修饰电极上的直接电化学和电催化性能

将肌红蛋白(Mb)通过吸附的方法固定在碳纳米管(CNT)表面, 用AFM、XPS、UV-Vis和FTIR对其进行了表征, 研究了CNT对Mb直接电子转移反应的促进作用. 循环伏安结果表明, Mb在CNT表面能进行有效和稳定的直接电子转移反应, 其循环伏安曲线上表现出一对良好的、几乎对称的氧化还原峰; 在20−160 mV•s−1的扫速范围内, 式量电位E0′几乎不随扫速而变化, 其平均值为(−0.343±0.001) V (vs SCE, pH 7.0); Mb在CNT表面直接电子转移的表观速率常数为(3.11±0.98) s−1; 式量电位E0′与溶液pH的关系表明, Mb的直接电化学过程是一个有H+参与的电极过程. 进一步的实验结果显示, 固定在CNT表面的Mb能保持其对H2O2和O2还原的生物电催化活性.     

Direct Electron Transfer at a Glucose Oxidase–Chitosan-Modified Vulcan Carbon Paste Electrode for Electrochemical Biosensing of Glucose

This article describes the investigation of direct electron transfer (DET) between glucose oxidase (GOD) and the electrode materials in an enzyme-catalyzed reaction for the development of improved bioelectrocatalytic system. The GOD pedestal electrochemical reaction takes place by means of DET in a tailored Vulcan carbon paste electrode surfaces with GOD and chitosan (CS), allowing efficient electron transfer between the electrode and enzyme. The key understanding of the stability, biocatalytic activity, selectivity, and redox properties of these enzyme-based glucose biosensors is studied without using any reagents, and the properties are characterized using electrochemical techniques like cyclic voltammogram, amperometry, and electrochemical impedance spectroscopy. Furthermore, the interaction between the enzyme and the electrode surface is studied using ultraviolet–visible (UV–Vis) and Fourier transform infrared (FTIR) spectroscopy. The present glucose biosensor exhibited better linearity, limit of detection (LOD?=?0.37?±?0.02 mol/L) and a Michaelis–Menten constant of 0.40?±?0.01 mol/L. The proposed enzyme electrode exhibited excellent sensitivity, selectivity, reproducibility, and stability. This provides a simple “reagent-less” approach and efficient platform for the direct electrochemistry of GOD and developing novel bioelectrocatalytic systems.     

Thermodynamic and electro-kinetic analyses of direct electron transfer (DET) and mediator-involved electron transfer (MET) with the help of a redox electron mediator

In this report, we conceptually distinguish direct electron transfer (DET) from mediator-involved (mediated) electron transfer (MET) in a glucose/oxygen-based fuel cell (FC) using an electrode potential/Fermi energy diagram. The anodic and cathodic overvoltages deviating from the equilibrium potential (the Fermi energy of redox electrons) were taken into account for the organic/inorganic redox couple and the mental experiments were performed during the trip of redox electrons through the interface between the anodic/cathodic organic/inorganic active mass and electrodes to propose electron transfer pathway. The proposed schema (inequality (MET) and equality in Fermi energy (DET)) should be experimentally corroborated by measurement of the electromotive force (emf). The MET is of technological significance in the presence of an electron mediator of the redox couple, despite a slightly narrower emf estimated between two electrodes by roughly 1 to 2 mV at most than the DET, in view of the thermodynamic and electro-kinetic viewpoints.

     

Bioelectrocatalytic Reduction of Oxygen in the Presence of Laccase Adsorbed on Carbon Electrodes

The kinetics of electroreduction of molecular oxygen on isotropic pyrocarbon with adsorbed laccase or a laccase–Nafion composite is studied. Kinetic parameters thus obtained are compared with those determined previously for electrodes of carbon black with adsorbed laccase. The closeness of kinetic parameters of the reaction of bioelectrocatalytic reduction of oxygen by laccase adsorbed on smooth (pyrocarbon) and disperse (carbon black) carbon materials led to a refined reaction mechanism. The slow stage of the reaction of bioelectrocatalytic reduction of oxygen is a synchronous transfer of two first electrons onto the oxygen molecule, similar to the mechanism of enzymatic catalysis by laccase.     

Direct Electrochemistry of Multi‐Copper Oxidases at Carbon Nanotubes Noncovalently Functionalized with Cellulose Derivatives

This study describes the direct electron transfer of multi‐copper oxidases, i.e., laccase (from Trametes versicolor) and bilirubin oxidase (BOD, from Myrothecium verrucaria) at multiwalled carbon nanotubes (MWNTs) noncovalently functionalized with biopolymers of cellulose derivatives, i.e., hydroxyethyl cellulose (HEC), methyl cellulose (MC), and carboxymethyl cellulose (CMC). The functionalization of the MWNTs with the cellulose derivatives is found to substantially solubilize the MWNTs into aqueous media and to avoid their aggregation on electrode surface. Under anaerobic conditions, the redox properties of laccase and BOD are difficult to be defined with cyclic voltammetry at either laccase/MWNT‐modified or BOD/MWNT‐modified electrodes. The direct electron transfer properties of laccase and BOD are thus studied in terms of the bioelectrocatalytic activities of the laccase/MWNT‐modified and BOD/MWNT‐modified electrodes toward the reduction of oxygen and found to be facilitated at the functionalized MWNTs. The possible application of the laccase‐catalyzed O₂ reduction at the laccase/MWNT‐modified electrode is illustrated by constructing a CNT‐based ascorbate/O₂ biofuel cell with the MWNT‐modified electrode as the anode for the oxidation of ascorbate biofuel.     

Laccase–gold nanoparticle assisted bioelectrocatalytic reduction of oxygen

It was found that homogeneous activity of Trametes hirsuta laccase is considerably diminished in the presence of gold nanoparticles (Au-NPs). Heterogeneous electron transfer studies revealed that Au-NPs facilitate direct electron transfer (DET) between the T1 copper site of the laccase and the surface of Au-NP modified electrodes. DET was characterized by the standard heterogeneous ET constant of 0.5 ± 0.6 s^?1 at Au-NPs with an average diameter of 50 nm. As a consequence of this a well pronounced DET based bioelectrocatalytic oxygen reduction with current densities of 5–30 µA cm^?2 has been achieved at the laccase–Au-NP modified electrodes.     

Direct and Cytochrome c Mediated Electrochemistry of Bilirubin Oxidase on Gold

Direct electron transfer (DET) of bilirubin oxidase from Myrothecium verrucaria (BOD) was established on promoter‐modified gold electrodes. The electrochemical behavior of the enzyme in solution was studied by means of cyclic voltammetry evaluating the biocatalytic reduction of dioxygen. The reaction of BOD at Au electrodes was shown to be efficient only at low pH. In addition, a novel interaction between BOD and cytochrome c (cyt.c) was found. It was shown that BOD efficiently accepts cyt.c as an electron donor in both cases when cyt.c is in solution and electrostatically adsorbed. The results suggest that cyt.c can play the role of a mediator facilitating electron transfer in a pH range where no DET could be observed between the enzyme and the electrode. For the interaction between cyt.c and BOD in solution the reaction kinetics has been studied electrochemically and spectrophotometrically.     

Surface‐Tuned Electron Transfer and Electrocatalysis of Hexameric Tyrosine‐Coordinated Heme Protein

Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine‐coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self‐assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl‐terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this “disc” orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi‐reversible redox behavior with rate constant k_s values between 0.93 and 2.86 s^?1 and apparent formal potentials ${E{{0{^{\prime }}\hfill \atop {\rm app}\hfill}}}$ between ‐131.1 and ‐249.1 mV. On the MUA/MU‐modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.     

Gold sputtered electrode surfaces enhance direct electron transfer reactions of human cytochrome P450s

We immobilized human cytochrome P450 (CYP), a membrane-bound enzyme, onto both smooth and nanostructured surfaces of gold electrodes via a naphthalene thiolate monolayer film. Rapid electron transfer of CYP with an electrode as a redox partner took place when the enzyme was immobilized onto an electrode surface with nanostructures. This structure was easily prepared by conventional sputtering techniques. A well-defined pair of peaks was observed at ? 0.175 V (vs. SHE) with the largest heterogeneous electron transfer rate constant of 340 s^? 1 for human CYP. The positive redox potential shift of 45 mV upon drug (testosterone) binding was clearly detected, which corresponded to a change in the spin states of heme iron in CYP. The present study showed that gold sputtered surfaces are very useful for direct electron transfer reactions of human CYP isoforms.     

高分子聚合物-多壁碳纳米管复合物固定漆酶及其在玻碳电极上的直接电子迁移

采用不同结构的高分子聚合物与纯化的多壁碳纳米管(MWCNTs)共混的方法,制备得到聚合物非共价功能化多壁碳管复合物,测定了这些载体对漆酶(lac)的担载量、固定漆酶的比活力及稳定性.以固定漆酶的复合物修饰玻碳(GC)电极后,采用循环伏安法研究这些电极在无氧磷酸盐缓冲液(PBS)中的直接电化学行为及催化氧还原活力,粗略地测定了固定漆酶与电极间电子转移的速率常数.实验结果表明,当聚合物中含亲漆酶基团或能与漆酶活性中心发生相互作用的官能团时利于直接电子转移,而且复合物固定漆酶保持了游离漆酶的天然构象.这些电极中,lac/NIPAM-co-BPCP-M WCNTs/GC(NIPAM-co-BPCP:N-烯丙基-1-苯甲酰基-3-苯基-4,5-2H-4-甲酰胺基吡唑-co-N-异丙基丙烯酰胺)在无氧PBS中发生直接电子转移的式电位(605mV)更接近漆酶活性中心的式电位(580mV),具有较快的异相电子转移速率(0.726s-1),较高的漆酶担载量(103.5mg/g)和固定漆酶比活力(1.68U/mg),较高的催化氧还原能力(氧还原起始电位820mV,在650mV时的催化峰电流为85.5μA)以及良好的重复使用性和长期使用性.     

Direct electron transfer--a favorite electron route for cellobiose dehydrogenase (CDH) from Trametes villosa. Comparison with CDH from Phanerochaete chrysosporium

This paper presents some functional differences as well as similarities observed when comparing the newly discovered cellobiose dehydrogenase (CDH) from Trametes villosa (T.v.) with the well-characterized one from Phanerochaete chrysosporium (P.c.). The enzymes were physically adsorbed on spectrographic graphite electrodes placed in an amperometric flow through cell connected to a flow system. In the case of T.v.-CDH-modified graphite electrodes, a high direct electron transfer (DET) current was registered at the polarized electrode in the presence of the enzyme substrate reflecting a very efficient internal electron transfer (IET) process between the reduced FAD-cofactor and the oxidized heme-cofactor. In the case of P.c.-CDH-modified graphite electrodes, the DET process is not as efficient, and the current will greatly increase in the presence of a mediator (mediated electron transfer, MET). As a consequence, when comparing the two types of enzyme-modified electrodes an inverted DET/MET ratio for T.v.-CDH is shown, in comparison with P.c.-CDH. The rates of the catalytic reaction were estimated to be comparable for both enzymes, by measuring the combined DET + MET currents. The inverted DET/MET ratio for T.v.-CDH-modified electrodes might suggest that probably there is a better docking between the two domains of this enzyme and that the linker region of P.c.-CDH might have an active role in modulating the rate of the IET (by changing the interdomain distance), with respect to pH. Based on the new properties of T.v.-CDH emphasized in the present study, an analytical application of a third-generation biosensor for lactose was recently published.     

Direct electron transfer reactions of laccases from different origins on carbon electrodes

Electrochemical studies of laccases from basidiomycetes, i.e., Trametes hirsuta, Trametes ochracea, Coriolopsis fulvocinerea, Cerrena maxima, and Cerrena unicolor, have been performed. Direct (mediatorless) electrochemistry of laccases on graphite electrodes has been investigated with cyclic voltammetry, square wave voltammetry as well as potentiometry. For all mentioned high potential laccases direct electron transfer (DET) has been registered at spectrographic graphite and highly ordered pyrolytic graphite electrodes. The characteristics of DET reactions of the enzymes were analysed under aerobic and anaerobic conditions. It is shown that the T1 site of the laccase is the primary electron acceptor, both in solution (homogenous case) and at surface of the graphite electrode (heterogeneous case). A mechanism of ET for the process of the electro-reduction of oxygen at the laccase-modified graphite electrodes is proposed and the similarity of this heterogeneous process to the laccase catalysed oxygen reduction homogeneous reaction is concluded.     

Stabilization role of a phenothiazine derivative on the electrocatalytic oxidation of hydrogen via Aquifex aeolicus hydrogenase at graphite membrane electrodes

The [NiFe] membrane-bound hydrogenase from the microaerophilic, hyperthermophilic Aquifex aeolicus bacterium (Aa Hase) presents oxygen, carbon monoxide, and temperature resistances. Since it oxidizes hydrogen with high turnover, this enzyme is thus of particular interest for biotechnological applications, such as biofuel cells. Efficient immobilization of the enzyme onto electrodes is however a mandatory step. To gain further insight into the parameters governing the interfacial electron process, cyclic voltammetry was performed combining the use of a phenothiazine dye with a membrane electrode design where the enzyme is entrapped in a thin layer. In the absence of the phenothiazine dye, direct electron transfer (DET) for H(2) oxidation is observed due to Aa Hase adsorbed onto the PG electrode. An unexpected loss of the catalytic current with time is however observed. The effect of toluidine blue O (TBO) on the catalytic process is first studied with TBO in solution. In addition to the expected mediated electron transfer process (MET), TBO is demonstrated to reconnect directly some Aa Hase molecules possibly released from the electrode but still entrapped in the thin layer. On adsorbed TBO the two same processes occur demonstrating the ability of the TBO film to connect Aa Hase via a DET process. Loss of activity is however observed due to the poor stability of adsorbed TBO at high temperatures. Aa Hase immobilization is then studied on electropolymerized TBO (pTBO). The effect of film thickness, temperature, presence of inhibitors and pH is evaluated. Given a film thickness less than 20 nm, H(2) oxidation proceeds via a mixed DET/MET process through the pTBO film. A high and very stable H(2) oxidation activity is reached, showing the potential applicability of the bioelectrode for biotechnologies. Finally, the multifunctional roles of TBO-based matrix are underlined, including redox mediator, Aa Hase anchor, but also buffering and ROS scavenger capabilities to drive pH local changes and avoid oxidative damage.     

Direct Electron Transfer Reaction of Ascorbate Oxidase Immobilized by a Self‐Assembled Monolayer and Polymer Membrane Combined System

This paper reports a novel enzyme‐immobilization method for the direct electron transfer (DET) reaction of ascorbate oxidase from Acremonium sp. HI‐25 (ASOM). ASOM was adsorbed onto a gold electrode modified with a self‐assembled monolayer (SAM) of alkanethiol derivatives and immobilized by a cationic polymer membrane and anionic ω‐carboxyalkanethiol combined system. The redox responses of the immobilized ASOM were investigated by cyclic voltammetry. We found that the DET reaction of ASOM was facilitated by this novel immobilization. On the other hand, the redox responses of poly(ethylene oxide) (PEO)‐modified ASOMs were also investigated. ASOM was modified with two types of PEO which possess straight chain‐shaped (PEO₂₀₀₀) or comb‐shaped conformation (PM₆₇). As a result, the DET reactions of PEO‐modified ASOMs were also facilitated by this immobilization method. We concluded that this immobilization method is effective for promoting the DET reaction of ASOMs.     

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design

Copper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at −250 mV (up to 6.13 mA cm⁻²). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications.