Redox bioelectrocatalysis

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

Koper MTM, Heering HA. Comparison of Electrocatalysis and Bioelectrocatalysis, of Hydrogen and Oxygen Redox Reactions. In Wieckowski A, Norskov JK, editors. Fuel cell science. Hoboken, NJ Wiley-VCH 2010. Chapter 2. 

The first reports on a reversible DET between redox proteins and electrodes were published in 1977 showing that cytochrome c is reversibly oxidized and reduced at tin-doped indium oxide [30] and gold in the presence of 4,4 -bipyridyl [31]. Only shortly after these publications appeared, papers were published describing the DET between electrode and enzyme for laccase and peroxidase [32,33]. It was observed that the overpotential for oxygen reduction at a carbon electrode was reduced by several hundred millivolts compared to the uncatalyzed reduction when laccase was adsorbed. This reaction could be inhibited by azide. The term bioelectrocatalysis was introduced for such an acceleration of the electrode process by... 

SIGNAL-TRIGGERED SWITCHABLE BIOELECTROCATALYSIS BY RECONSTITUTION OF REDOX-PROTEINS... 

Coupling between a biologically catalyzed reaction and an electrochemical reaction, referred to as bioelectrocatalysis, is the constructional principle for enzyme-based electrochemical biosensors. This means that the flow of electrons from a donor through the enzyme to an acceptor must reach the electrode in order for the corresponding current to be detected. In case a direct electron transfer between the active site of an enzjane and an electrode is not possible, a small molecular redox active species, e.g. hydrophobic ferrocene, meldola blue and menadione as well as hydrophilic ferricyanide, can be used as an electron transfer mediator. This means that the electrons from the active site of the enzyme reduce the mediator molecule, which, in turn, can diffuse to the electrode, where it donates the electrons upon oxidation. When these mediator molecules are employed for coupling of an enzymatic redox reaction to an electrode at a constant potential, the resulting application can be referred to as mediated amperometry or mediated bioelectrocatalysis. 

This section treats the theory of homogeneous mediated bioelectrocatalysis in a quiet solution. An empirical equation explaining the catalytic current is presented, which is conveniently used for the determination of kinetic parameters of the enzyme reaction. A novel method of protein redox potential measurements is also described using a mediated continuous-flow column electrolytic spectroelectrochemical technique. 

Cyclic voltammetry is a method frequently used to measure 7s,i ni. Mediated bioelectrocatalysis yields cyclic voltammograms (CVs) of different shapes as illustrated in Fig. 2, depending on the measuring conditions [11]. Curve (a) is the wave for a reversible electrode reaction of an Mox/Mred redox couple. Bioelectrocatalysis mediated with the Mqx/ Mred redox couple produces a sigmoidal catalytic wave as curve (c) under the conditions [Mred] - M and [S] Ks. When [Mred] is increased to higher concentrations, an anodic peak of the diffusion current of Mred rnay be overlapped on the catalytic current as depicted by curve (d) the current, however, becomes steady state after appropriate periods... 

FIG. 15 Schematic representation of direct bioelectrocatalysis. Electrons are transferred from substrate to an electrode through an intramolecular electron transfer from redox center A to redox center B in the enzyme molecule adsorbed on the electrode surface. 

An important application of CyD complexes is in the field of mediated electrocatalysis and bioelectrocatalysis. Detection of a target analyte using a biosensor based on a redox enzyme is well recognized as a more convenient solution than one based on the electrochemistry of reaction products. An ideal mediator for electrocatalysis should be soluble in water or easily anchored on an electrode surface. CyDs have often been employed in solubilizing hydrophobic molecules used either as mediators in catalysis or occurring as the products of catalytic reactions. In the latter case, their role was to avoid fouling of the electrode surface. 

The structure and physicochemical properties of the enzymes which have been used to date to promote electrochemical reactions are briefly outlined. Methods of their immobilization are described. The status of research on redox transformations of proteins and enzymes at the electrode-electrolyte interface is discussed. Current concepts on the ways of conjugation of enzymatic and electrochemical reactions are summarized. Examples of bioelectrocatalysis in some electrochemical reactions are described. Electrocatalysis by enzymes under conditions of direct mediatorless transport of electrons between the electrode and the enzyme active center is considered in detail. Lastly, an analysis of the status of work pertaining to the field of sensors with enzymatic electrodes and to biofuel cells is provided. 

Another aspect of bioelectrocatalysis is the application of electrochemical methods to study aspects of the mechanism of the enzymatic effect and, in particular, of the relationship between conformational transformations of proteins, the redox potential value at the active center, and the electron transfer rate. Understanding the specificities of biocatalysts opens up a possibility for developing synthetic models of enzymes on the basis of complex compounds of a nonprotein nature. 

It is frequently the case that enzymes undergo slow heterogeneous electron transfer with an electrode surface. Such slow rates of electron transfer can largely be attributed to insulation of the redox active site by the bulk of the protein matrfac. This is especially true for en mes that have a non-dissociable redox cofactor, such as the flavin adenine dinucleotide (FAD) within the active site of glucose oxidase. In cases where an enzyme active site is physically inaccessible to the electrode surface, an artificial intermediate redox species is used to shuttle electrons between the enzyme and electrode. It should be noted at the outset that much of the early research in mediated bioelectrocatalysis was performed on homogeneous mixtures of substrate, enzyme and mediator. However, the vast majority of literature since the late 1980s has dealt exclusively with systems in which the enzyme and mediator are immobilized in some capacity at the electrode surface. Thus the majority of the topies eovered here are focused on immobilized systems to reflect the dominant trend in bioeleetrocatalysis. 

In bioelectrocatalysis, oxidases and dehydrogenases are the most frequently used enzymes for hioelectrode design. The redox centers of these enzymes are usually huried in the protein core and operate with... 

E ) bioelectrocatalysis, respectively. In the process of shuttling charge between the redox-center and the electrode, the mediator is cycled between its oxidized and reduced states. The mediator should be stable in both the reduced and oxidized forms and any side reactions between the mediator redox states and the enzyme or the environment should be eliminated. To be effective in its role, the mediator must often compete with the enzyme s natural substrate (e.g. molecular oxygen in case of oxidases), effectively and efficiently diverting the flow of electrons to and from the electrode. An efficient mediator should provide rapid reaction with the redox enzyme, effectively oxidizing or reducing the enzyme-active center. A mediator should also exhibit reversible electrochemistry (a large rate constant (fcet) for the interfacial electron-transfer at the electrode surface). 

NAD (P)+-dependent enzymes, electrically contacted with electrode surfaces, can provide efhcient bioelectrocatalysis for the NAD(P)H oxidation. For example, diaphorase (DI) was applied to oxidize NADFl, using a variety of quinone compounds, several kinds of flavins, or viologens as mediators between the enzyme and electrode [217, 218]. The bimolecular reaction rate constants between the enzyme and mediators whose redox potentials are more positive than -0.28 V at pH 8.5 can be as high as 10 s , suggesting that the reac-... 

Specific redox characteristics of a catalyst derived from CV scans are also used to confirm an enzyme s ability for bioelectrocatalysis by either direct electron transfer (DET) or mediated electron transfer (MET) to the electrode. DET and MET are two distinct mechanisms of bioelectrocatalysis. MET has the advantage of being compatible with almost all naturally occurring oxidoreductase enzymes and coenzymes, but it requires additional components (either smaU-molecule redox mediators or redox polymers) because the enzymes cannot efficiently transfer electrons to the electrode. These additional components make the system more complex and less stable [8]. The vast majority of oxidoreductase enzymes that require MET to an electrode are nicotinamide adenine dinucleotide (NAD" ) dependent. Two of the most commonly encountered NAD -dependent enzymes in BFC anodes are glucose dehydrogenase (GDH) and alcohol dehydrogenase (ADH). These enzymes have been thoroughly characterized in respect to half-cell electrochemistry and have been demonstrated for operation in BFC. More information about MET can be found in Chapter 9. 

To demonstrate the previously described BP as a good candidate for direct bioelectrocatalysis, in 2011 Hussein et al. immobilized MCOs on a BP electrode via physisorption [35]. MCOs are often apphed as oxygen reduction catalysts in BFCs [5,40,46-52]. By using BP as an electrode material, DET from the conductive surface to the T1 redox site is achieved and ehminates the need for mediators, thus simplifying design [53,54]. Compared with MCO-coated CNT aggregates, BP cathodes fabricated in this manner exhibited superior performance when normalized to the average mass of the CNT and BP electrodes, respectively [55]. 

Bioelectrocatalysis is a unique combination of electrochemical and biochemical reactions, which rests not only on the ability to control at will the oxidizing and reducing ability of the electrode by changing its Fermi level, but on the specificity and selectivity of biochemical catalysis. Endeavors have been made to develop bioelectrocatalysis with enzymes that catalyze redox reactions. 

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