Redox proteins electrical contact

Willner B, Katz E, Willner 1. 2006. Electrical contacting of redox proteins by nanotechnological means. Curr Opin Biotech 17 589-596. 

More recently, nanotechnology has faciUtated progress in miniaturizing redox enzyme electrodes and extending their application. In order to achieve contact between the active site of the redox enzyme where electron transfer takes place, usually buried within the protein structure, and the electrode electrical contact, cofactor-functionaUzed nanomaterials have been developed [75]. Diffusible cofactors such as FAD can be used as the relay system for carrying electrons to electrical... 

The electrical contact of redox proteins is one of the most fundamental concepts of bioelectronics. Redox proteins usually lack direct electrical communication with electrodes. This can be explained by the Marcus theory16 that formulates the electron transfer (ET) rate, ket, between a donor-acceptor pair (Eq. 12.1), where d0 and d are the van der Waals and actual distances separating the donor-acceptor pair, respectively, and AG° and X correspond to the free energy change and the reorganization enery accompanying the electron transfer process, respectively. 

Another method for the analysis of aptamer-protein complexes involved the use of a positively charged ferrocene-tethered polythiophene, (19), as redox label reporting unit (Fig. 12.19). The antithrombin aptamer was immobilized on an electrode surface, and the electrostatic binding of the redox polymer (19) to the aptamer monolayer resulted in a supramolecular complex that revealed electrical contact between the polymer and the electrode.74 The formation of the aptamer-thrombin complex removed the polymer from the surface and blocked the electrical contact between the polymer label and the electrode. As a result, higher concentrations of thrombin increased the surface coverage of the aptamer-thrombin complex on the electrode, and this decreased the amperometric responses of the sensing device. 

Methods to electrically wire redox proteins with electrodes by the reconstitution of apo-proteins on relay-cofactor units were discussed. Similarly, the application of conductive nanoelements, such as metallic nanoparticles or carbon nanotubes, provided an effective means to communicate the redox centers of proteins with electrodes, and to electrically activate their biocatalytic functions. These fundamental paradigms for the electrical contact of redox enzymes with electrodes were used to develop amperometric sensors and biofuel cells as bioelectronic devices. 

Scheme 7 Electronic transduction of photo-switchable bioelectrocatalytic functions of proteins, (A) by the tethering of photoisomerizable units to the protein (R is a diffusional electron mediator that electrically contacts the redox...

A further approach to controlling electrical communication between redox proteins and their electrode support through a photo-command interface includes photo stimulated electrostatic control over the electrical contact between the redox enzyme and the electrode in the presence of a diffusional electron mediator (Scheme 12).[58] A mixed monolayer, consisting of the photoisomerizable thiolated nitrospiropyran units 30 and the semi-synthetic FAD cofactor 25, was assembled on an Au electrode. Apo-glucose oxidase was reconstituted onto the surface FAD sites to yield an aligned enzyme-layered electrode. The surface-reconstituted enzyme (2 x 10-12 mole cm-2) by itself lacked electrical communication with the electrode. In the presence of the positively charged, protonated diffusional electron mediator l-[l-(dimethylamino)ethyl]ferrocene 29, however, the bioelectrocatalytic functions of the enzyme-layered electrode could be activated and controlled by the photoisomerizable component co-immobilized in the monolayer assembly (Figure 12). In the... 

While an inereased loading of an electron mediator on a protein enhances the effectiveness of electrical contacting, the enzyme activity suffers owing to changes in its structure. The chemical modification of redox proteins with synthetic... 

Iron relay units provide a route for electron hopping between the electrode and the active redox center of the protein, and thus contribute to the electrical contacting of the enzyme layer with the electrode [88],... 

The site-specific modification of enzymes with a single electron-relay group located near to the redox cofactor and providing efficient electrical contact with the conductive support has been achieved by the reconstitution of enzymes with cofactors covalently linked to redox groups. Affinity interactions between enzymes and their cofactors at the electrode interface can allow the efficient electrical contacting of aligned proteins. 

For practical photoinduced synthetic biocatalyzed transformations, it is important to integrate biocatalysts in immobilized matrices that allow the recycling of the photosystems. The fact that bipyridinium sites act as electron mediators for various redox enzymes was used to develop two paradigms for the electrical contacting and photoactivation of the biocatalyst (Figure 39). By one approach, the bipyridinium electron relays are tethered by covalent bonds to the protein backbone (Figure 39A). These electron relays act as oxidative quenchers of the excited state of the dye and, upon photoreduction of the electron acceptor units, they act as electron carriers that activate the reductive functions of the enzyme. As an example, the... 

All these bioelectrocatalytic functions of redox proteins are based on the control and enhancement of the electrical communication between the redox sites of the proteins and the electrode support. This is accomplished by the nano-engineering of the surfaces with covalently anchored proteins, the structural aligmnent of the proteins on the electrodes and the chemical modification of the proteins with redox-active units. Preliminary results suggest that two approaches will play important roles in the future development of bioelectronic systems (i) protein mutagenesis with specific functional amino acid residues that can align the protein on the electrode surface and control the electrical contact with the electrode (ii) the synthesis of de novo proteins with tailored bioelectronic and electrobiocatalytic functions. 

Redox-proteins usually lack direct electrical contact with electrodes because the redox site is embedded in the protein. This situation insulates the electrical communication between the redox-site and the electrode by the spatial separation of the protein redox-site from the electrode support. Suitable func-... 

FIG. 7.19 Electronic transduction of phocoswitchable bioelectrocacalytic functions of enzymesf proteins by the application of a photoisomerizable command Interfece that controis the electrical contact between die redox enzyme/protain and the electrode. 

For biosensors of the third generation DET to small redox proteins is of particular interest as they show interaction with reactive (oxygen) species, while enzymes in direct electric contact are suitable for reagentless metabohte measurement. Peroxidase, catalase and superoxide dismutase are also relevant to the determination of reactive oxygen species and their scavengers. 

The following sections will concentrate on the analytical application of redox proteins and redox enzymes for biosensing. The biomolecule will be briefly introduced, the major route for its direct electric contact to electrodes outlined and the analytical application discussed. The bioelectrochemical studies on structure-function relationship and their role in biological redox processes will not be covered in detail in this review. 

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