Enzymatic enzyme electron transfer

In the course of an enzymatic reaction electron transfer takes place between the electrode and the active center either with the aid of low-molecular weight, easily diffused carriers (mediators) or by way of direct electrochemical oxidation or reduction of an active site in the enzymes, i.e., by direct electron exchange between the enzyme and the electrode. 

The important criterion thus becomes the ability of the enzyme to distort and thereby reduce barrier width, and not stabilisation of the transition state with concomitant reduction in barrier height (activation energy). We now describe theoretical approaches to enzymatic catalysis that have led to the development of dynamic barrier (width) tunneUing theories for hydrogen transfer. Indeed, enzymatic hydrogen tunnelling can be treated conceptually in a similar way to the well-established quantum theories for electron transfer in proteins. 

Nowadays, studies of direct electrochemistry of redox proteins at the electrodesolution interface have held more and more scientists interest. Those studies are a convenient and informative means for understanding the kinetics and thermodynamics of biological redox processes. And they may provide a model for the study of the mechanism of electron transfer between enzymes in biological systems, and establish a foundation for fabricating new kinds of biosensors or enzymatic bioreactors. 

While cytochrome P-450 catalyzes the interaction with substrates, a final step of microsomal enzymatic system, flavoprotein NADPH-cytochrome P-450 reductase catalyzes the electron transfer from NADPH to cytochrome P-450. As is seen from Reaction (1), this enzyme contains one molecule of each of FMN and FAD. It has been suggested [4] that these flavins play different roles in catalysis FAD reacts with NADPH while FMN mediates electron... 

For enzymatic reductions with NAD(P)H-dependent enzymes, the electrochemical regeneration of NAD(P)H always has to be performed by indirect electrochemical methods. Direct electrochemical reduction, which requires high overpotentials, in all cases leads to varying amounts of enzymatically inactive NAD-dimers generated due to the one-electron transfer reaction. One rather complex attempt to circumvent this problem is the combination of the NAD+ reduction by electrogenerated and regenerated potassium amalgam with the electrochemical reoxidation of the enzymatically inactive species, mainly NAD dimers, back to NAD+ [51]. If one-electron... 

In MET, a low-molecular-weight, redox-active species, referred to as a mediator, is introduced to shuttle electrons between the enzyme active site and the electrode.In this case, the enzyme catalyzes the oxidation or reduction of the redox mediator. The reverse transformation (regeneration) of the mediator occurs on the electrode surface. The major characteristics of mediator-assisted electron transfer are that (i) the mediator acts as a cosubstrate for the enzymatic reaction and (ii) the electrochemical transformation of the mediator on the electrode has to be reversible. In these systems, the catalytic process involves enzymatic transformations of both the first substrate (fuel or oxidant) and the second substrate (mediator). The mediator is regenerated at the electrode surface, preferably at low overvoltage. The enzymatic reaction and the electrode reaction can be considered as separate yet coupled. 

In DET, the enzymatic and electrode reactions are coupled by direct (mediatorless) electron transfer. In this case, the electron is transferred directly from the electrode to the substrate molecule (or vice versa) via the active site of the enzyme. In such a system, the coupled overall process is the redox transformation of the substrate(s), which can be considered as an enzyme-catalyzed electrode process. According to this mechanism, the electrode surface acts as the enzyme cosubstrate, and the enzymatic and electrode reactions cannot be considered as separate, but as formal stages of the bioelectrocatalytic reaction mechanism. The catalytic effect of the enzyme is the... 

Several factors may limit the overall rate of enzymatic reductive reactions. First, the electron transfer to the reactive metal (e.g., Co, Fe, or Ni) may be limiting. It is also possible that access of the organic substrates to the reduced metals contained within enzyme microenvironments may be limited. Mass transfer limitation is even more important in intact bacterial cells. For example, Castro et al. (1985) found that rates of heme-catalyzed reductive dehalogenations were independent of the heme content of the cells. 

The enzyme can be incorporated into an amperometric sensor in a thick gel layer, in which case the depletion region due to the electrochemical reaction is usually confined within this layer. Alternatively, enzyme can be immobilized at the surface of the electrode or even within the electrode material itself, in which case the depletion region extends into the solution in the same way as it would for an unmodified electrode. In the latter case, the enzyme can then be seen as a true electrocatalyst that facilitates the interfacial electron transfer, which would otherwise be too slow. The general principles of the design and operation of these biosensors is illustrated on the example of the most studied enzymatic sensor, the glucose electrode (Fig. 2.14, Section 2.3.1). 

One of the promising potentials of SECM for biosensor research is the possibility to investigate immobilized enzymes independent of the communication to the electrode onto which they are immobilized. In fact, not too seldom, the immobilization of proteins onto electrode surfaces inhibits fast electron transfer reactions. SECM can be used to probe the enzymatic activity from the solution side of an immobilized enzyme film with a UME that is free of any cover layer. When designing an SECM experiment for the investigation of immobilized enzymes, one should consider the following guidelines. 

If the enzymes are immobilized on an electrode surface, generally only the GC mode can be used for their investigation, because the signal is independent of the nature of the support (Fig. 37.5c). This was demonstrated by Wittstock and Schuhmann [41]. If ox-idoreductases are immobilized on conducting surfaces, the feedback can result from a heterogeneous electron transfer at the electrode or the enzymatic reaction (Fig. 37.5b). Kranz et al. [33] isolated the contribution of the enzymes by carefully designed control experiments. In most cases such an approach is extremely... 

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