Redox Centers Of Enzyme Research Articles

Fast electron transfer between glucose oxidase and electrodes via phenothiazine mediators with poly(ethylene oxide) spacers attached to the enzyme surface

A series of glucose oxidase hybrids capable of a direct electrical communication with electrodes is synthesized by covalently bonding phenothiazine mediators to surface lysine residues via poly(ethylene oxide) spacers with different lengths. The hybrid with the optimum length of spacer chain exhibits the fast electron transfer between the redox center of enzyme and electrodes. This fast electron transfer in the hybrids realizes a large catalytic current comparable to that for the corresponding freely diffusing mediator systems.

Bioinorganic Composites for Enzyme Electrodes

Sparingly soluble redox salts were combined with a model enzyme, glucose oxidase, in a host matrix of a biopolymer chitosan to form bioinorganic composite films on the surface of glassy carbon electrodes. Four redox salts, each containing the Ru(NH3)6(3+) cation and a selected anion, such as Ru(CN)6(4-), Fe(CN)6(4-), Co(CN)6(3-) or IrCl6(3-), were studied. The composition and catalytic properties of such composite materials toward glucose oxidation were investigated by spectroscopic and electrochemical methods. The composite films provided an oxygen-independent electrical communication between the enzyme's redox centers and a glassy carbon surface at a potential as low as -0.10 V vs Ag/AgCl(3 M Cl-). The nature of the electrical communication is discussed in terms of redox mediation by the Ru(NH3)6(3+)-containing ion pairs formed inside the biocomposites. The kinetic significance of the mediator's charge is considered by postulating that neutral ion pairs are more efficient redox mediators of the enzymatic reaction than those negatively charged. The low operating potential of enzyme electrodes based on the bioinorganic composites allows for an interference-free determination of glucose. The design of the biocomposites is generic and can incorporate oxidoreductase enzymes other than glucose oxidase to provide a host of biosensors for biologically and environmentally important analytes.

Determination of the midpoint potential of the FAD and FMN flavin cofactors and of the 3Fe-4S cluster of glutamate synthase.

Glutamate synthase is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of the L-glutamine amide group to C(2) of 2-oxoglutarate, forming two molecules of L-glutamate. The bacterial enzyme is an alphabeta protomer, which contains one FAD (on the beta subunit, approximately 50 kDa), one FMN (on the alpha subunit, approximately 150 kDa), and three different Fe-S clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters at an unknown location). To address the problem of the intramolecular electron pathway, we have measured the midpoint potential values of the flavin cofactors and of the 3Fe-4S cluster of glutamate synthase in the isolated alpha and beta subunits and in the alphabeta holoenzyme. No detectable amounts of flavin semiquinones were observed during reductive titrations of the enzyme, indicating that the midpoint potential value of each flavin(ox)/flavin(sq) couple is, in all cases, significantly more negative than that of the corresponding flavin(sq)/flavin(hq) couple. Association of the two subunits to form the alphabeta protomer does not alter significantly the midpoint potential value of the FMN cofactor and of the 3Fe-4S cluster (approximately -240 and -270 mV, respectively), but it makes that of FAD some 40 mV less negative (approximately -340 mV for the beta subunit and -300 mV for FAD bound to the holoenzyme). Binding of the nonreducible NADP(+) analogue, 3-aminopyridine adenine dinucleotide phosphate, made the measured midpoint potential value of the FAD cofactor approximately 30-40 mV less negative in the isolated beta subunit, but had no effect on the redox properties of the alphabeta holoenzyme. This result correlates with the formation of a stable charge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated beta subunit, but not in the alphabeta holoenzyme. Binding of L-methionine sulfone, a glutamine analogue, had no significant effect on the redox properties of the enzyme cofactors. On the contrary, 2-oxoglutarate made the measured midpoint potential value of the 3Fe-4S cluster approximately 20 mV more negative in the isolated alpha subunit, but up to 100 mV less negative in the alphabeta holoenzyme as compared to the values of the corresponding free enzyme forms. These findings are consistent with electron transfer from the entry site (FAD) to the exit site (FMN) through the 3Fe-4S center of the enzyme and the involvement of at least one of the two low-potential 4Fe-4S centers, which are present in the glutamate synthase holoenzyme, but not in the isolated subunits. Furthermore, the data demonstrate a specific role of 2-oxoglutarate in promoting electron transfer from FAD to the 3Fe-4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxoglutarate is indeed consistent with the recently determined three-dimensional structure of the glutamate synthase alpha subunit, in which several polypeptide stretches are suitably positioned to mediate communication between substrate binding sites and the enzyme redox centers (FMN and the 3Fe-4S cluster) to tightly control and coordinate the individual reaction steps [Binda, C., et al. (2000) Structure 8, 1299-1308].

Electrochemistry of reconstituted glucose oxidase on carbon paste electrodes

Flavine Adenine Dinucleotide (FAD) was covalently immobilised onto glassy carbon matrix using a 13-carbon atom long spacer arm. FAD modified electrodes offer a convenient handle for immobilising glucose oxidase (GOD) enzyme for direct electron transfer. The electrochemical characteristics of immobilised FAD were compared with free FAD in solution at blank paste electrode surface. The prominent peaks at −475 mV and −601 mV indicate the positive coupling of FAD onto the carbon matrix. In our case we observed that reduction peaks are well separated from the oxidation and the immobilised FAD shows more reversible behavior compared to the free FAD in solution. GOD apoenzyme has been prepared by acidification of GOD solution (5 mg/ml) in buffer (100 mM sodium acetate) using ammonium sulfate solution at pH 1.4. The apoenzyme was coupled to the FAD modified matrix by incubating the matrix with the solution of apoenzyme for 4–12 h. The paste electrode with reconstituted GOD was investigated for its electrochemical characteristics and for its response with substrate (glucose solution). Our major finding is that the reconstituted enzyme shows better electron transfer rates compared to normal enzyme. The reason for this can be attributed to the long spacer arm holding the electroactive FAD which facilitates better electron transfer between enzyme redox center and electrode surface. The above technique appears to be a promising approach to be used in biosensor application.

Polyacrylamide-based redox polymer for connecting redox centers of enzymes to electrodes.

Enzyme electrodes based on complexing a water-soluble copolymer of acrylamide and vinylimidazole with [Os(dmebpy)2C1]+/2+ (dmebpy = 4,4'-dimethyl-2,2'-bipyridine) and cross-linking with oxidases by water-soluble cross-linkers are described. The potential of the polyacrylamide-based redox polymer is +55 mV (SCE), a typical electron diffusion coefficient (De) in the redox hydrogel that results from its cross-linking is (1.3 +/- 0.1) x 10(-9) cm2/s. The properties of the enzyme electrodes formed when this redox hydrogel "wired" horseradish peroxidase (HRP), lactate oxidase (LOx) or glucose oxidase (GOx) depended on the thickness of the hydrogel film, the chemistry of their cross-linking, and their enzyme content. At the wired HRP electrodes, H2O2 was electrocatalytically reduced to water at 0.0 V (SCE). Lactate and glucose were electrocatalytically oxidized at 0.16 V (SCE). The GOx electrodes, when made with 140 micrograms/cm2 thick polymer films, were selective for glucose in the presence of physiological concentrations of urate and ascorbate.

"Wired" enzyme electrodes for amperometric determination of glucose or lactate in the presence of interfering substances.

Glucose oxidase (GOX) or lactate oxidase (LOX) were immobilized in an osmium-based three-dimensional redox hydrogel that electrically connected the enzyme's redox centers to electrodes. The enzyme "wiring" hydrogel was formed by cross-linking poly(1-vinylimidazole) (PVI) complexed with Os-(4,4'-dimethylbpy)2Cl (termed PVI15-dmeOs) with poly(ethylene glycol) diglycidyl ether (peg). Glucose and lactate sensors exhibited typical limiting current densities of 250 and 500 microA/cm2, respectively. When the electrodes were poised at 200 mV (SCE), the currents resulting from electrooxidation of ascorbate, urate, acetaminophen, and L-cysteine were negligible. When a Nafion film was employed, the linear range was extended from 6 to 30 mM glucose and from 4 to 7 mM lactate. The redox potential of the gel-forming polymer was 95 mV (SCE). Glucose and lactate measurements performed in bovine calf serum correlated well with a substrate calibration in phosphate buffer.

Electrical connection of enzyme redox centers to electrodes

Abstract : Electrically insulating proteins can be made redox-conducting through incorporation of a high density of electron relaying redox centers. Electrons diffuse in the resulting redox conductors by self-exchange between identical and electron transfer between different relaying centers. When the self-exchange rate of the relays and their density are high, the flux of electrons through a 1 micrometer thick film of a 3-dimensional macromolecular network can match or exceed the rate of supply of electrons to or from the ensemble of enzyme molecules covalently bound to it. The network now molecularly wires the enzyme molecules to the electrode and the current measures the turnover of the wired enzyme molecules. When the enzyme turnover is substrate-flux, i.e. concentration limited, the current increases with the concentration of the substrate.

Electrical Communication Between Graphite Electrodes and Glucose Oxidase/Redox Polymer Complexes

Abstract Redox polymers can fold along the glycoproteins of glucose oxidase (MW 160,000) at low electrolyte concentrations and thereby penetrate the enzyme. Upon penetration, the distance between the redox centers of the polymer and the FADH2 centers of the reduced enzyme is reduced sufficiently for electrons to be transferred and, therefore, for the mediated electro-oxidation of glucose on conventional electrodes. At high (1M) electrolyte (NaCl) concentrations the redox polymers coil. Such coiling prevents the penetration of the enzyme by the redox polymers. Consequently, electron transfer does not take place and glucose is not electro-oxidized. When an appropriate polycationic redox polymer is covalently bound to the enzyme, the electro-oxidation of glucose occurs even at high electrolyte concentrations. Electron transfer from the enzyme's FADH2 centers to copolymers of poly(N-methyl-4-vinylpyridinium) chloride with either poly(vinylferrocene), E° = 0.25V (SCE), or with poly(4-vinylpyridine) complexes of Os(bpy)2Cl, E° = 0.25V (SCE), or of Os(4,4′-dimethylbpy)2Cl, E° = 0.15V (SCE) is rapid. The polycationic poly(4-vinylpyridine) complex of Os(bpy)2Cl can be covalently bound to glucose oxidase by preparing the terpolymer with 4-aminostyrene, diazotization and reaction of the diazonium cations with tyrosine or tryptophane residues of the enzyme. Glucose electrodes made with the redox polymer modified enzyme are relatively stable and sensitive. Furthermore, simple and fast amperometric glucose electrodes can be made by adsorbing either non-quaternized or quaternized poly(vinylpyridine) complexes of [Os(bipyridine)2Cl]2+ (MW 300,000) on graphite, and adsorbing on these polymer films glucose oxidase. These electrodes do not require diffusing redox mediators or membranes to contain the enzyme and the redox polymer. The redox polymer is shown to electrically “wire” the enzyme's redox centers to the electrode. The glucose response of the electrodes is faster than 1 sec; their current increases linearly with glucose concentration through the 0–10 mM range. At 60 mM glucose, their current density is about 20 μA/cm2.

Direct electrical communication between chemically modified enzymes and metal electrodes. I. Electron transfer from glucose oxidase to metal electrodes via electron relays, bound covalently to the enzyme

Glucose-reduced glucose oxidase does not directly transfer electrons to conventional electrodes because the distance between its redox centers and the electrode surface exceeds, even on closest approach, the distance across which electrons are transferred at sufficient rates. Therefore, electrical communication between the redox centers of this enzyme and electrodes required either the presence, and diffusion to and from the enzyme's redox center, of O/sub 2/ and H/sub 2/O/sub 2/, or the presence of members of a redox couple, or the use of special electrodes like TTF/TCNQ. They show here that direct electrical communication between the redox center of a large enzyme molecule and a simple metal electrode can be established through chemical modification of the enzyme. When a sufficient number of electron-relaying centers are attached through covalent bonding to the protein of glucose oxidase, electrons are transferred from the enzyme's redox centers to relays that are closer to the periphery of the enzyme. Because some of the relays are located sufficiently close to the enzyme's surface, electrons are transferred at practical rates to the electrode. As a result, a glucose-concentration-dependent current flows in an electrochemical cell made with conventional electrodes when the electrolytic solution contains the relay-modified enzyme. Such amore » current does not flow when the solution contains the natural enzyme. Specifically, electrical communication is established between the FAD/FADH/sub 2/ centers of glucose oxidase and gold, platinum, or carbon electrodes through the covalent bonding of an average of 12 molecules of ferrocenecarboxylic acid per glucose oxidase molecule.« less