Electron transfer between redox proteins and

The first reports on direct electrochemistry of a redox active protein were published in 1977 by Hill [49] and Kuwana [50], They independently reported that cytochrome c (cyt c) exhibited virtually reversible electrochemistry on gold and tin doped indium oxide (ITO) electrodes as revealed by cyclic voltammetry, respectively. Unlike using specific promoters to realize direct electrochemistry of protein in the earlier studies, recently a novel approach that only employed specific modifications of the electrode surface without promoters was developed. From then on, achieving reversible, direct electron transfer between redox proteins and electrodes without using any mediators and promoters had made great accomplishments. 

Schuhmann has reviewed amperometric biosensors and indicated that these sensors can be categorized as devices employing 1) direct electron transfer between redox proteins and electrodes modified with self-assembled monolayers 2) anisotropic orientation of redox proteins at monolayer-modified electrodes ... 

An exciting method for accelerating the electron transfer between redox proteins and amperometric electrodes has been described by Heller and Degani (1987), who modified oxidoreductases with electrontunneling relays. The same mediators as are used in chemically modified electrodes are directly bound to groups of the protein molecule. The... 

Nowadays, the construction of electrochemical biosensors based on the use of gold nanoparticles constitutes an intensive research area because of the unique advantages that this nanomaterial lends to biosensing devices. So, gold nanoparticles provide a stable surface for immobilization of biomolecules with no loss of their biological activity. Moreover, they facilitate direct electron transfer between redox proteins and electrode materials, and constitute useful interfaces for the electrocatalysis of redox processes of molecules such as H202 or NADH involved in many biochemical reactions (1, 2). 

Frew, J.E. and Hill, H.A.O. (1988) "Direct and Indirect Electron Transfer between Redox Proteins and Electrodes", Eur. J. Biochem., 172, 261-9. 

Direct electron transfer between redox proteins and metal electrodes has many advantages with respect to analytical applications. Hill and coworkers have pointed out the similarities between heterogeneous electron transfer reactions of proteins at electrodes and catalysis. The sequence of events at the electrode include 1) diffusion of reactant protein to the electrode surface 2) adsorption of the protein in an orientation suitable for electron transfer 3) electron transfer 4) dissociation of the protein from the electrode surface and 5) diffusion of the protein away from the surface. If all these requirements are not met, well behaved redox activity will not be observed. There have been various approaches to accomplishing reversible redox reactions in proteins based upon these requirements. Hill and coworkers have focused on the second step and have shown by their elegant promoter studies that the correct orientation of the protein at the electrode is crucial for rapid electron transfer. Others have utilized mediator-type electrodes or chemically modified proteins. ... 

However, because of the mostly very slow electron transfer rate between the redox active protein and the anode, mediators have to be introduced to shuttle the electrons between the enzyme and the electrode effectively (indirect electrochemical procedure). As published in many papers, the direct electron transfer between the protein and an electrode can be accelerated by the application of promoters which are adsorbed at the electrode surface [27], However, this type of electrode modification, which is quite useful for analytical studies of the enzymes or for sensor applications is in most cases not stable and effective enough for long-term synthetic application. Therefore, soluble redox mediators such as ferrocene derivatives, quinoid compounds or other transition metal complexes are more appropriate for this purpose. 

It is still uncertain whether any of these three-iron clusters have any physiological role at all. It is evident that 3Fe and [4Fe-4S] clusters are interconverted readily, and a 3Fe cluster may lie on the biosynthetic route that leads to the formation of [4Fe-4S] clusters. There is some evidence that the 3Fe cluster of Fdll can participate in electron transfer between redox proteins, but again this has no ultimate significance, as a variety of added mediator complexes are able to carry out such a function. It has been suggested recently that the 3Fe centre in aconitase is capable of promoting partial catalytic activity of the enzyme.813... 

In principle, glucose oxidase could be oxidized directly at the electrode, which would be the ultimate electron acceptor. However, direct electron transfer between redox enzymes and electrodes is not possible because the FADH2/FAD redox centers are buried inside insulating protein chains (Heller, 1990). If it were not the case, various membrane redox enzymes with different standard potentials would equalize their potentials on contact, thus effectively shorting out the biological redox chains. The electron transfer rate is strongly dependent on the distance x between the electron donor and the electron acceptor. 

The direct electrochemistry of redox proteins has developed significantly in the past few years. Conditions now exist that permit the electrochemistry of all the proteins to be expressed at a range of electrodes, and important information about thermodynamic and kinetic properties of these proteins can be obtained. More recently, direct electron transfer between redox enzymes and electrodes has been achieved due to the more careful control of electrode surfaces. The need for biocompatible surfaces in bioelectrochemistry has stimulated the development of electrode surface engineering techniques, and protein electrochemistry has been reported at conducting polymer electrodes 82) and in membranes 83, 84). Furthermore, combination of direct protein electrochemistry with spectroscopic methods may offer 85) a novel way of investigating structure-function relationships in electron transport proteins. 

Studies of electrochemical reactions of redox proteins have attracted widespread interest and attention. Such studies can yield important information about not only intrinsic thermodynamic and kinetic properties of redox proteins, but also structural properties, such as binding characteristics of proteins at specific types of electrode surfaces and the orientational requirements for electron transfer between the protein and the electrode. The results are useful for the development of biosensors, biofuel cells, and biocatalysts. In addition, the information obtained from these studies can contribute to an understanding of the physiological implications of biological electron transfer reactions, because many electron transfer proteins are located at, or close to, charged membranes and are thus subject to large electric field effects that are similar to those near an electrode surface. 

Nanoparticle films provide a stable microenvironment for redox proteins and facilitate the direct electron transfer between those proteins and underlying electrodes. The simplest way to prepare such films is by composite deposition. Either a protein-nanoparticle or nanoparticle dispersion is deposited on electrodes. In the latter case, the protein is absorbed from solution. Clay [25-28], Ti02 [29-31], Sn02 [32], Zr02 [33], and Fe304 [34] were utilized to construct protein-nanoparticle films in this way, and direct electrochemistry of the proteins in these films was realized. 

Achieving fast electron transfer to enzyme active sites need not be complicated. As mentioned above, many redox enzymes incorporate a relay of electron transfer centers that facilitate fast electron transfer between the protein surface and the buried active site. These may be iron-sulfur clusters, heme porphyrin centers, or mononuclear... 

The electron-transfer rate between large redox protein and electrode surface is usually prohibitively slow, which is the major barricade of the electrochemical system. The way to achieve efficient electrical communication between redox protein and electrode has been among the most challenging objects in the field of bioelectrochemistry. In summary, two ways have been proposed. One is based on the so-called electrochemical mediators, both natural enzyme substrates and products, and artificial redox mediators, mostly dye molecules and conducted polymers. The other approach is based on the direct electron transfer of protein. With its inherited simplicity in either theoretical calculations or practical applications, the latter has received far greater interest despite its limited applications at the present stage. 

Fe-protein, the unique, highly specific electron donor to MoFe-protein, mediates coupling between ATP hydrolysis and electron transfer to MoFe-protein and also participates in the biosynthesis and insertion of FeMoco into MoFe-protein. Fe-protein contains one ferredoxin-like [Fe4S 4 2 /1+ cluster as its redox center. There is now evidence for an [Fe4S4]° super-reduced state in which four high-spin iron(II) (S= 2) sites are postulated. These were previously discussed in Section 6.3 and illustrated in Table 6.1.16 The [Fe4S4] cluster in this state bridges a dimer of... 

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