Relay-cofactor

The reconstitution of apo-enzymes on relay-cofactor monolayer-functionalized electrodes was used to align redox enzymes and to establish electrical contact... 

Figure 12.2 (a) Reconstitution of an apo-enzyme on a relay-cofactor monolayer for the... 

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. 

Figure 3-2. Methods for the assembly of reconstituted relay-cofactor enzyme assembhes (A) by the surface reconstitution of the apo-enzyme (B) by the attachment of the prereconstituted enzyme on the electrode.

By one method a relay-cofactor dyad is assembled on the electrode, and the respective apo-protein is reconstituted on the surface to yield an aligned protein that is linked to the conductive surface by the relay component. The second method involves the synthesis of the relay-cofactor unit and the reconstitution of the apo-protein in solution. The specific immobilization of the enzyme on the electrode by the relay unit provides the structurally organized enzyme electrodes. While the first method is technically easier, the second methodology that involves tedious synthetic and separation steps, permits the fundamental structural characterization of the reconstituted protein. In the two configurations, the redox enzymes are anticipated to be electrically contacted with the electrode by means of the relay, a conductive... 

Interfaces Modified with Electroactive Biological Species, Fig. 1 Electrical wiring of redox enzymes, (a) Optimal configuration for the electrical contacting of a redox enzyme with the electrode, (b) Reconstitution of an apo-enzyme on a relay-cofactor monolayer for the... 

To summarize, squalene epoxidase is a flavoprotein capable of catalyzing the insertion of oxygen into the 2,3-double bond of squalene to give 2,3-oxidosqualene, with the second oxygen atom from 02 being reduced to water. The reducing equivalents necessary for this transformation are relayed from NADPH through NADPH-cytochrome c reductase to the flavin cofactor of the epoxidase. 

FIGURE 6-9 Electrical contact of a flavoenzyme by its reconstitution with a relay-FAD semisynthetic cofactor. Fc = ferrocene. (Reproduced with permission from reference 2.)... 

Under conditions of copper deficiency, some methanotrophs can express a cytosolic, soluble form of MMO (sMMO) (20-23), the properties of which form the focus of the present review. The sMMO system comprises three separate protein components which have all been purified to homogeneity (24,25). The hydroxylase component, a 251 kD protein, contains two copies each of three subunits in an a 82y2 configuration. The a subunit of the hydroxylase houses the dinuclear iron center (26) responsible for dioxygen activation and for substrate hydroxylation (27). The 38.6 kD reductase contains flavin adenine dinucleotide (FAD) and Fe2S2 cofactors (28), which enable it to relay electrons from reduced nicotinamide adenine dinucleotide (NADH) to the diiron center in the... 

FIGURE 33 Electrical contacting of a flavoenzyme by its reconstitution with a relay-FAD semisynthetic cofactor. 

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... 

Tetranuclear iron-sulfur clusters are key relay stations in the electron flow in photosynthesis. Photosystem I comprises three subunits, PsaA, PsaB and PsaC. The latter contains two [Fe4S4] centres FA and FB. The core subunits PsaA and B, respectively, house a [Fe4S4] centre denoted FX in addition to other, organic cofactors. The role of this latter cluster was probed in preparations partially devoid of PsaC. It was concluded from the results that FX has a major role in controlling the electron transport through PS I.236 Since the final acceptor of the electrons in PS I is a ferredoxin with a [Fe2S2] cluster it was of interest to study a... 

Zayats M, Katz E, Willner I. Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor. Journal of the American Chemical Society 2002, 124, 2120-2121. 

Mother nature has resolved the various limitations involved in multi-electron processes. Unique assemblies composed of cofactors and enzymes provide the microscopic catalytic environments capable of activating the substrates, acting as multi-electron relay systems and inducing selectivity and specificity. Artificially tailored heterogeneous and homogeneous catalysts as well as biocatalysts (enzymes and cofactors) are, thus, essential ingredients of artificial photosynthetic devices. 

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. 

Enzymes reconstituted with semi-artificial cofactors linked to redox relays... 

Photogenerated cofactors can be employed to drive biocatalytic enzyme cascades including the photosynthetic carbon dioxide fixation process [184] (Figure 35). Photogenerated NADPH provides a two-electron relay for the insertion of CO2 into a-ketoglutaric acid (23) and pyruvic acid (21) in the presence of isocitrate dehydrogenase (IcitDH) and malic enzyme (MalE), respectively. In these photosystems, Ru(bpy)3 " acts as a photosensitizer, as a primary electron acceptor... 

The need to improve the electrical communication between redox proteins and electrodes, and the understanding that the structural orientation at the molecular level of redox proteins and electroactive relay units on the conductive surfaces is a key element to facilitate ET, introduced tremendous research efforts to nano-engineer enzyme electrodes with improved ET functionalities. The present chapter addresses recent advances in the assembly of structurally aligned enzyme layers on electrodes by means of surface reconstitution and surface crosslinking of structurally oriented enzyme/cofactor complexes on electrodes. The ET properties of the nano-structured interfaces is discussed, as well as the possible application of the systems in bioelectronic devices such as biosensors, biofuel cell elements or optical and electrical switches. 

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