Direct electron transfer of glucose oxidase promoted by carbon nanotubes

A stable suspension of carbon nanotubes (CNT) was obtained by dispersing the CNT in a solution of surfactant, such as cetyltrimethylammonium bromide (CTAB, a cationic surfactant). CNT (dispersed in the solution of 0.1% CTAB) has promotion effects on the direct electron transfer of glucose oxidase (GOx), which was immobilized onto the surface of CNT. The direct electron transfer rate of GOx was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of GOx, with a midpoint potential of about -0.466 V (vs SCE (saturated calomel electrode)) in the phosphate buffer solution (PBS, pH 6.9). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (ks) and the value of midpoint potential (E1/2) were estimated. The dependence of E1/2 on solution pH indicated that the direct electron transfer reaction of GOx is a two-electron-transfer coupled with a two-proton-transfer reaction process. The experimental results also demonstrated that the immobilized GOx retained its bioelectrocatalytic activity for the oxidation of glucose, suggesting that the electrode may find use in biosensors (for example, it may be used as a bioanode in biofuel cells). The method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

Direct electron transfer from glucose oxidase immobilized on polyphenanthroline-modified glassy carbon electrode

This study reports direct electron transfer (DET) from immobilized glucose oxidase (GOx) via grafted and electropolymerized 1,10-phenanthroline monohydrate (PMH). The layer of poly-1,10-phenanthroline (PPMH) was gained via electrochemical deposition, which was used to create the PPMH-modified GC-electrode (PPMH/GC-electrode). Further, the GOx was immobilized on the PPMH/GC-electrode. The effect of surface-modification by the PPMH on the electron-transfer between enzyme and electrode-surface and some other electrochemical/analytical-parameters of newly designed enzymatic-electrode were evaluated. The PPMH/GC-electrode showed superior DET to/from flavine adenine dinucleotide cofactor of GOx, while some redox-compounds including ferrocene and K(3)[Fe(CN)(6)] were completely electrochemically inactive on the PPMH/GC-electrode. It was also found that the resulting GOx/PPMH/GC-electrode functioned as a "direct response type" glucose-biosensor. The biosensor showed excellent selectivity towards glucose and demonstrated good operational-stability. According to our best knowledge, this study is the first scientific report on electrochemical-polymerization of PMH on the GC-electrode in non-aqueous media followed by its application in the design of glucose-biosensor.     

Biocatalysis of theophylline oxidation by microbial theophylline oxidase in the presence of non-physiological electron acceptors

Microbial theophylline oxidase (ThOx) is a redox enzyme catalysing 8-hydroxylation of theophylline to form 1,3-dimethyluric acid. In this work, ThOx has been characterized as a fragile haem-containing protein complex composed of several non-covalently bound dynamic domains with molecular weights of around 60 and 210 kDa, and capable of formation of 1.5 MDa assemblies as well. The rate of theophylline oxidation by ThOx with the non-physiological electron acceptor ferricyanide was 0.17 s^?1, approaching that with cytochrome c, 0.33 s^?1. The apparent catalytic constant depended on the electron acceptor concentration. At concentrations lower than 0.2 mM the reaction did not fit the Michaelis–Menten scheme, and some non-catalytic processes dominated in the overall reaction. The kinetics of ThOx catalysis were also studied at electrodes modified with self-assembled monolayers (SAM) of hydroxyl- and amine-terminated alkanethiols. Different compositions of the SAM provide different orientations of ThOx on these layers. Depending on the orientation of ThOx onto the SAM-modified electrodes, the heterogeneous electron transfer (ET) constant, k_s, which characterizes the ET reaction between the electrodes and the haem of ThOx (E^o/ of 87 mV (NHE)) was 0.4 s^?1 and 3.2 s^?1. Only the low-ET-rate orientation appeared to be productive for the electrocatalytic function of ThOx, giving a reaction similar to that with ferricyanide and cytochrome c. The apparent efficiency of ThOx bioelectrocatalysis in the absence of mediators was substantially lower than that mediated by ferricyanide or cytochrome c. This lower efficiency is consistent with a correspondingly lower amount of ThOx being in direct ET contact with the electrodes and thus involved in electrocatalysis.     

基于直接接触的微生物胞外电子传递

微生物电子传递在微生物的代谢繁殖和物质的生物地球化学循环中发挥着关键作用。其中基于直接接触的微生物胞外电子传递(Direct extracellular electron transfer,DEET)已成为微生物学、地球化学和生物物理学等学科共同关注的焦点,并在近几年取得了一系列重要发现和理论突破,包括微生物纳米导线、电缆细菌、微生物种间DEET等。伴随着这些新进展,更多的问题也需要研究者们在进一步的研究中解决,包括DEET的分子机制及其相关功能微生物种群等。不同学科理论和技术的交叉是进一步揭示DEET过程的关键。     

Xanthine oxidase/laponite nanoparticles immobilized on glassy carbon electrode: Direct electron transfer and multielectrocatalysis

In this work, colloidal laponite nanoparticles were further expanded into the design of the third-generation biosensor. Direct electrochemistry of the complex molybdoenzyme xanthine oxidase (XnOx) immobilized on glassy carbon electrode (GCE) by laponite nanoparticles was investigated for the first time. XnOx/laponite thin film modified electrode showed only one pair of well defined and reversible cyclic voltammetric peaks attributed to XnOx–FAD cofactor at about −0.370 V vs. SCE (pH 5). The formal potential of XnOx–FAD/FADH₂ couple varied linearly with the increase of pH in the range of 4.0–8.0 with a slope of −54.3 mV pH⁻¹, which indicated that two-proton transfer was accompanied with two-electron transfer in the electrochemical reaction. More interestingly, the immobilized XnOx retained its biological activity well and displayed an excellent electrocatalytic performance to both the oxidation of xanthine and the reduction of nitrate. The electrocatalytic response showed a linear dependence on the xanthine concentration ranging from 3.9 × 10⁻⁸ to 2.1 × 10⁻⁵ M with a detection limit of 1.0 × 10⁻⁸ M based on S/N = 3.     

Bioelectrocatalytic detection of theophylline at theophylline oxidase electrodes

Bioelectrocatalytic oxidation of theophylline was studied at gold and graphite electrodes modified with microbial theophylline oxidase (ThOx), a multi-cofactor redox enzyme capable of selective oxidation of theophylline. Gold electrodes were additionally modified with self-assembled monolayers (SAMs) of (-OH)- and (-NH(2))-terminated alkanethiols of different chain lengths, to achieve compatibility between ThOx and the electrode surface. On graphite, ThOx was either physically co-adsorbed with a surfactant didodecyldimethylammonium bromide (DDAB), or entrapped within an Os-redox-polymer film. At all electrodes, ThOx was bioelectrocatalytically active; direct electrochemistry of ThOx in the absence of theophylline was followed only at the SAM-modified gold electrodes. Direct electrochemistry of ThOx correlated with redox transformations of the heme domain of ThOx, with a E(o/)of -110+/-2 mV versus Ag|AgCl, at pH 7. Bioelectrocatalytic oxidation of theophylline was optimal at mixed (-OH)/(-NH(2))-terminated SAMs; co-adsorption of ThOx with DDAB improved the bioelectrocatalytic performance of the ThOx-electrode. In both cases, the response to theophylline was within the mM range. Alternatively, a reagentless ThOx-electrode based on ThOx cross-linked within the Os-redox-polymer matrix demonstrated a linear response to theophylline within the physiologically important 0.02-0.6mM (3.6-72 mg l(-1)) concentration range with a sensitivity of 52.1+/-7.8 mA cm(-2)M(-1).     

细菌与古菌之间的直接电子传递

冰川占地球陆地表面的11%,储存了约10⁴ Pg有机碳。随着冰川消融有机碳被释放至下游生态系统中,刺激海洋、湖泊和径流的初级生产力进而影响其生态系统。微生物参与的固碳过程决定了冰川有机碳储量及向下游输出碳量。研究冰川固碳微生物群落构成及其生态功能,可为估算冰川碳积累量和保护下游生态系统提供数据基础。本文综述了冰川碳储量和释放量、冰川生态系统主要固碳途径、固碳微生物群落组成、固碳速率以及影响固碳速率的环境因素。最后基于研究现状展望了冰川生态系统固碳微生物的未来研究和发展方向。     

Dynamics of electron transfer pathways in cytochrome C oxidase

Cytochrome c oxidase mediates the final step of electron transfer reactions in the respiratory chain, catalyzing the transfer between cytochrome c and the molecular oxygen and concomitantly pumping protons across the inner mitochondrial membrane. We investigate the electron transfer reactions in cytochrome c oxidase, particularly the control of the effective electronic coupling by the nuclear thermal motion. The effective coupling is calculated using the Green's function technique with an extended Huckel level electronic Hamiltonian, combined with all-atom molecular dynamics of the protein in a native (membrane and solvent) environment. The effective coupling between Cu(A) and heme a is found to be dominated by the pathway that starts from His(B204). The coupling between heme a and heme a(3) is dominated by a through-space jump between the two heme rings rather than by covalent pathways. In the both steps, the effective electronic coupling is robust to the thermal nuclear vibrations, thereby providing fast and efficient electron transfer.     

Ultrafast haem-haem electron transfer in cytochrome c oxidase.

Electron transfer between the redox centres is essential for the function of the haem-copper oxidases. To date, the fastest rate of electron transfer between the haem groups has been determined to be ca. 3 x 10(5) s(-1). Here, we show by optical spectroscopy that about one half of this electron transfer actually occurs at least three orders of magnitude faster, after photolysis of carbon monoxide from the half-reduced bovine heart enzyme. We ascribe this to the true haem-haem electron tunnelling rate between the haem groups.     

Direct electron transfer between copper-containing proteins and electrodes

The electrochemistry of some copper-containing proteins and enzymes, viz. azurin, galactose oxidase, tyrosinase (catechol oxidase), and the “blue” multicopper oxidases (ascorbate oxidase, bilirubin oxidase, ceruloplasmin, laccase) is reviewed and discussed in conjunction with their basic biochemical and structural characteristics. It is shown that long-range electron transfer between these enzymes and electrodes can be established, and the mechanistic schemes of the DET processes are proposed.     

Molecular mechanism of intramolecular electron transfer in dimeric sulfite oxidase

Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo^(IV) or Mo^(V) follow this path.     

Cytochrome oxidase: pathways for electron tunneling and proton transfer

 Electrons from cytochrome c, the substrate of cytochrome oxidase, a redox-linked proton pump, are accepted by Cu_A in subunit II. From there they are transferred to the proton pumping machinery in subunit I, cytochrome a and cytochrome a ₃–Cu_B. The reduction of the latter site, which is the dioxygen reducing unit, is coupled to proton uptake. Dioxygen reduction involves a peroxide and a ferryl ion intermediate, and it is the transition between these and back to the resting oxidized enzyme that are coupled to proton pumping. The X-ray structures suggest electron–transfer pathways that can account for the observed rates provided that the reorganization energies are small. They also reveal two proton-transfer pathways, and mutagenesis experiments have shown that one is used for proton uptake during the initial reduction of cytochrome a ₃–Cu_B, whereas the other mediates transfer of the pumped protons. Received: 23 March 1998 / Accepted: 11 May 1998     

The rate of internal heme-heme electron transfer in cytochrome C oxidase

Cytochrome c oxidase catalyzes the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of dioxygen to water. The process involves a number of intramolecular electron-transfer reactions, one of which takes place between the two hemes of the enzyme, hemes a and a3, with a rate of approximately 3 x 10(5) s(-1) (tau approximately 3 micros). In a recent report [Verkhovsky et al. (2001) Biochim. Biophys. Acta 1506, 143-146], it was suggested that the 3 x 10(5) s(-1) electron transfer may be controlled by structural rearrangements and that there is an additional electron transfer that is several orders of magnitude faster. In the present study, we have reinvestigated the spectral changes occurring in the nanosecond and microsecond time frames after photolysis of CO from the fully reduced and mixed-valence enzymes. On the basis of the differences between them, we conclude that in the bovine enzyme the microscopic forward and reverse rate constants for the electron-transfer reactions from heme a to heme a3 are not faster than approximately 2 x 10(5) and approximately 1 x 10(5) s(-1), respectively.     

Effect of solution viscosity on intramolecular electron transfer in sulfite oxidase

Our previous studies have shown that the rate constant for intramolecular electron transfer (IET) between the heme and molybdenum centers of chicken liver sulfite oxidase varies from approximately 20 to 1400 s(-1) depending upon reaction conditions [Pacheco, A., Hazzard, J. T., Tollin, G., and Enemark, J. H. (1999) J. Biol. Inorg. Chem. 4, 390-401]. These two centers are linked by a flexible polypeptide loop, suggesting that conformational changes, which alter the Mo-Fe distance, may play an important role in the observed IET rates. In this study, we have investigated IET in sulfite oxidase using laser flash photolysis as a function of solution viscosity. The solution viscosity was varied over the range of 1.0-2.0 cP by addition of either polyethylene glycol 400 or sucrose. In the presence of either viscosogen, an appreciable decrease in the IET rate constant value is observed with an increase in the solvent viscosity. The IET rate constant exhibits a linear dependence on the negative 0.7th power of the viscosity. Steady-state kinetics and EPR experiments are consistent with the interpretation that viscosity, and not other properties of the added viscosogens, is responsible for the dependence of IET rates on the solvent composition. The results are consistent with the role of conformational changes on IET in sulfite oxidase, which helps to clarify the inconsistency between the large rate constant for IET between the Mo and Fe centers and the long distance (approximately 32 A) between these two metal centers observed in the crystal structure [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983].     

Coupling of electron and proton transfer in the photosynthetic water oxidase

According to current estimates, the photosynthetic water oxidase functions with a quite restricted driving force. This emphasizes the importance of the catalytic mechanisms in this enzyme. The general problem of coupling electron and proton transfer is discussed from this viewpoint and it is argued that 'weak coupling' is preferable to 'strong coupling'. Weak coupling can be achieved by facilitating deprotonation either before (proton-first path) or after (electron-first path) the oxidation step. The proton-first path is probably relevant to the oxidation of tyrosine Y(Z) by P-680. Histidine D1-190 is believed to play a key role as a proton acceptor facilitating Y(Z) deprotonation. The pK(a) of an efficient proton acceptor is submitted to conflicting requirements, since a high pK(a) favors proton transfer from the donor, but also from the medium. H-bonding between Y(Z) and His, together with the Coulombic interaction between negative tyrosinate and positive imidazolium, are suggested to play a decisive role in alleviating these constraints. Current data and concepts on the coupling of electron and proton transfer in the water oxidase are discussed.     

Direct methanol biocatalytic fuel cell--considerations of restraints on electron transfer

In this paper structure and operational principles of a novel type direct methanol biocatalytic fuel cell (DMBFC) system is introduced. In addition observed restraints in the energy generation are discussed. The operational principle of the biofuel cell is enzymatic breakdown of methanol by methanol dehydrogenase (MDH) from Methylobacterium extorquens at the anode. The terminal electron acceptor at the cathode is potassium permanganate. Performance characteristics of the system are the following: open circuit voltage 1.4 V, power density 0.25 mW/cm2 and current density 0.38 mA/cm2 at the operating voltage of 0.67 V, and a continuous operation time of 2 weeks. A biofuel cell usually requires an electrochemically active reagent, a mediator, to ensure effective transfer of the electrons from the activity centre of the enzyme to the electrode. Inactivation of the mediator was found to restrict the electron transfer. Moreover, the rate of inactivation was found to increase in fuel cell conditions. The half-life of TMPD was observed to be maximum 5 days compared to 10 days in normal conditions. Experiments showed that addition of 0.2% w/w of aluminium dioxide into the anodic graphite paste stabilized the mediator.     

Spectroelectrochemical determination of the heterogeneous electron transfer kinetics of soluble spinach ferredoxin

Heterogeneous electron transfer rate parameters for soluble spinach ferredoxin are reported using a recently developed single potential step spectroelectrochemical technique. The reductive kinetics were measured by monitoring the $\text{decrease}$ in absorbance as a function of time for several overpotential steps at methyl viologen modified optically transparent gold minigrid electrodes. These measurements yielded an average formal heterogeneous electron transfer rate constant (k_f,h^o′ = 6.5 (±1.3) × 10^?5 cm/s) and electrochemical transfer coefficient (α = 0.60 ± (0.16)) at pH 7.5. These results are the first heterogeneous electron transfer rate parameters reported for this species.     

Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer

A directed evolution protocol was developed for glucose oxidase (GOx) from Aspergillus niger that mimics applications conditions and employs a well-known mediator, oxidized ferrocenemethanol, in a medium throughput screen (96-well plate format). Upon reduction, oxidized ferrocenemethanol shows a color change from blue to pale yellow that can be recorded at 625 nm. Under optimized screening conditions, a CV of less than 20% was achieved in 96-well microtiter plates. For validating the screening system, two mutant libraries of GOx were generated by standard error-prone PCR conditions (0.04 mM MnCl(2)) and Saccharomyces cerevisiae was employed as host for secreted GOx expression. Two screening of approximately 2000 GOx mutants yielded a double mutant (T30S I94V) with improved pH and thermal resistance. Thermal resistance at a residual activity of 50% was increased from 58 degrees C (wild type, WT) to 62 degrees C (T30S I94V) and pH stability was improved at basic pH (pH 8-11). K(m) for glucose remained nearly unchanged (20.8 mM WT; 21.3 mM T30S I94V) and k(cat) increased (69.5/s WT; 137.7/s T30S I94V).     

Direct and indirect electron transfer between electrodes and redox proteins

The direct electrochemistry of redox proteins has been achieved at a variety of electrodes, including modified gold, pyrolytic graphite and metal oxides. Careful design of electrode surfaces and electrolyte conditions are required for the attainment of rapid and reversible protein-electrode interaction. The electron transfer reactions of more complex systems, such as redox enzymes, are now being examined. The 'well-behaved' electrochemistry of redox proteins can be usefully exploited by coupling the electrode reaction to enzymes for which the redox proteins act as cofactors. In systems where direct electron transfer is very slow, small electron carriers, or mediators, may be employed to enhance the rate of electron exchange with the electrode. The organometallic compound ferrocene and its derivatives have proved particularly effective in this role. A new generation of electrochemical biosensors employs ferrocene derivatives as mediators.     

Direct demonstration of electron transfer between tryptophan and tyrosine in proteins

With several proteins it has been shown that electrons can be transferred intramolecularly from tyrosine to electron-deficient tryptophan units. Rates vary from ～ 10²s^?1 (in lysozyme) to ～ 2×10⁴ s^?1 (in trypsin). For β-lactoglobulin the activation energy is 45kJ mol^?1. This is incompatible with charge conduction along the polypeptide chain and rules out any mechanism involving temperature-labile hydrogen bonds as the main pathway. It seems likely that the electron transfer proceeds directly between the aromatic groups, while they are maintained at a distance from each other.