Direct and Mediated Electron Transfer

When discussing the transfer of electrons from the enzyme active site to the electrode surface, thus generating catalytic current, there are two types of electron transfer mechanisms mediated electron transfer (MET) and direct electron transfer (DET) [13]. Most oxidoieductase enzymes that have been commonly used in BFC development are unable to promote the transfer of electrons themselves because of the long electron transfer distance between the enzyme active site and the electrode surface as a result, DET is slow. In such a case, a redox-active compound is incorporated to allow for MET. In this approach, a small molecule or redox-active polymer participates directly in the catalytic reaction by reacting with the enzyme or its cofactor to become oxidized or reduced and diffuses to the electrode surface, where rapid electron transfer takes place [14]. Frequently, this redox molecule is a diffusible coenzyme or cofactor for the enzyme. Characteristic requirements for mediator species include stability and selectivity of both the oxidized and reduced forms of the species. The redox chemistry for the chosen mediator is to be reversible and with minimal overpotential [15]. 

Mediators can be polymerized on the electrode surface prior to enzyme immobilization, co-immobilized with enzyme, or simply added to the fuel solution. Common mediators used in BFC applications include low molecular weight, polymerizable, organic dyes such as methylene green, phenazines, and azure dyes, along with other redox-active compounds such as ferrocene, ferrocene derivalives, and conductive salts [14]. These mediators are often required for nicotinamide adenine dinucleotide (NAD )- and flavin adenine dinucleotide (FAD)-dependent enzymes, such as ADH, ALDH, and GOx. MET has been achieved at both cathodic and anodic interfaces through solution-phase mediators and mediators immobilized in various ways with or near the enzymes themselves [16,17]. However, these mediated systems do have drawbacks in that the species used to assist electron transfer are often not biocompatible, have short lifetimes themselves, or cause large potential losses. Table 5.1 lists common enzyme cofactors that can mediate or undergo DET with an enzyme on the electrode. 

A mediator would not be needed if the enzymes used in BFCs are capable of DET via the active site of the enzyme. Several enzymes capable of DET have been reported [18,19]. Many of these enzymes contain redox-active metal centers, such as iron-sulfur groups, heme groups, and metallic centers, that perform the catalytic transfer of electrons. These enzymes convert the chemical signal directly to an electrical signal through the transfer of charge to the redox center, which is in mm 

TABLE 5.1 List of Cofactor/Coenzyme Names and Abbreviations 

Nicotinamide adenine dinucleotide phosphate NADP+ (NADPH) 

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A. Lindgren, T. Ruzgas, L. Gorton, E. Csoregi, G. Bautista Ardila, I.Y. Sakharov and I.G. Gazaryan, Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer, Biosens. Bioelectron., 15 (2000) 491-497. 

Borgmann, S., Hartwich, G., Schulte, A., and Schuhmann, W. (2006) Amperometric enzyme sensors based on direct and mediated electron transfer, in Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics (eds E. Palecek, F. Scheller, and J. Wang), Elsevier, Amsterdam, pp. 599-655. 

Direct and Mediated Electron Transfer Between Proteins... 

F.-D. Munteanu, L. Gorton, A. Lindgren, T. Ruzgas, 1. Emneus, E. Csoregi, 1. G. Gazaryan, 1. V. Ouporov, E. A. Mareeva, L. M. Lagrimini, Direct and Mediated Electron Transfer Catalyzed by Anionic Tobacco Peroxidase. Effect of Calcium Ions. Appl. Biochem. Biotech., 84 (2000) 1-13. 

SCHEME 1. Schematic presentation of direct (non-mediated) electron transfer (A) and mediated electron transfer (B) from enzyme to electrode. 

The prerequisites for a DET can be derived from Marcus Theory [27,28]. The highly specific and directional protein-mediated electron transfer in biological systems is governed by factors such as the distance and the bonds between the redox centres, the redox-potential difference between donor and acceptor, an appropriate association of the redox couple and protein-structure dynamics coupled with electron transfer [24,27,29]. 

First wave. corresponds to mediated electron transfer, the second to both direct unmediated and mediated electron transfer. The second wave for both cases is governed by the differential equations and boundary conditions presented in Table 2.6. The general case is solved via numerical resolution of differential equations presented at the start of the table. 

Fig. 5 Direct electron transfer of laccase (A) and mediated electron transfer of formate dehydrogenase (B) onto the 96 screen-printed electrodes. The electrodes are made of a carbon working electrode and of a silver/silver chloride reference electrode.

In order to screen mutants with improved direct electron transfer, it is necessary to use an electrochemical screening system. Currently, only a few electrochemical screening methods were described in literature such as the system developed by the Bartlett group used to screen NADH electro-oxidation. This system uses a multichannel potentiostat with sixty electrodes to screen zinc(n) or ruthenium(ii) complexes bearing the redox phenidione as a mediator for NADH oxidation. It allows the complete evaluation of the electrochemical kinetic constants of the mediators and the immobilization procedure. Unfortunately, this system could only be used with a single electrolyte solution for all the electrodes (e.g., when a single reaction condition or enzyme is assayed), and it requires mL-scale reaction volumes. Recently, another system was described which makes it possible to screen bioelectrocatalytic reactions on 96 independent electrodes screen-printed onto a printed-circuit-board. It showed the possibility to screen direct or mediated electron transfer between oxidoreductases and electrode by intermittent pulse amperometry at the pL-scale (Fig. 6). The direct electron transfer assay was validated with laccase and unmodified electrodes.As an example of the mediated electron transfer assay, the 96 carbon electrodes were modified by phenazines to sereen libraries of a formate dehydrogenase obtained by directed evolution. ... 

UPS spectra of clean Ar+ sputtered and in Vacuo carbon-contaminated surfaces are shown in Figure 4. On the clean, sputtered surface a filled state due to Ti3+ lies 0.6 eV below the conduction band.(22) Carbon-induced filled states lie in a broad peak with considerable intensity between the valence band edge and the Ti + peak. Frank et al.lQ) reported evidence that a state lying about 1.2 eV above the valence band mediates electron transfer from Ti02 electrodes. Although these carbon states are as of now poorly defined and have not been directly implicated in any aqueous photochemistry, their nearly ubiquitous presence should be considered in discussions of charge transfer at real oxide surfaces. 

Demonstrating that a redox transformation of a contaminant involves mediated electron transfer requires meeting several criteria (i) the overall reaction must be energetically favorable, (ii) the mediator must have a reduction potential that lies between the bulk donor and the terminal acceptor so that both steps in the electron transfer chain will be energetically favorable, and (iii) both steps in the mediated reaction must be kinetically fast relative to the direct reaction between bulk donor and terminal acceptor. Most evidence for involvement of mediators in reduction of contaminants comes from studies with model systems, because natural reducing media (such as anaerobic sediments) consist of more redox couples than can be characterized readily. Although this is an active area of research, we can identify a variety of likely mediator half-reactions (see Table 16.5). 

Like the various forms of iron, NOM apparently serves as both bulk reductant and mediator of reduction as well as bulk reductant (recall section 2.2.2). NOM also can act as an electron acceptor for microbial respiration by iron reducing bacteria (26), thereby facilitating the catabolism of aromatic hydrocarbons under anaerobic conditions (103). In general, it appears that NOM can mediate electron transfer between a wide range of donors and acceptors in environmental systems (104,105). In this way, NOM probably facilitates many redox reactions that are favorable in a thermodynamic sense but do not occur by direct interaction between donor and acceptor due to unfavorable kinetics. 

Daasbjerg and Lund provided an interesting probe of the borderline between direct and mediated reactions192. Rate constants ( r) for the S 2 reaction of superoxide (02 ) with alkyl halides (e.g. chlorobutane) were compared with the expected rate constants ( ET) for electron transfer reaction between the same alkyl halide and an aromatic anion radical of... 

The mechanism and theory of bioelectrocatalysis is still under development. Electron transfer and variation of potential in the electrodeenzyme-electrolyte system has therefore to be investigated. Whether the enzyme is soluble and the electron transfer process occurs through a mediator, or whether there is direct enzyme immobilization on the electrode surface, the homogeneous process in the enzyme active centre has to be described by the laws of enzyme catalysis, and the heterogeneous processes on the electrode surface by the laws of electrochemical kinetics. Besides this there are other aspects outside electrochemistry or... 

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