Photoswitching bioelectrocatalytic

To optimize the photoswitchable bioelectrocatalytic features of the protein, site-specific functionalization or mutation of the active site microenvironment is essential. This was accomplished by a semisynthetic approach involving the reconstitution of the flavoenzyme-glucose oxidase with a semisynthetic photoisomerizable FAD cofactor (Scheme 9).1511 The photoisomerizable nitrospiropyran carboxylic acid (24) was covalently coupled to N6-(2-aminoethyl)-FAD (25), to yield the synthetic photoisomerizable nitrospiropyran-FAD cofactor 26a (Scheme 9(A)). The native FAD cofactor was removed from glucose oxidase, and the synthetic photoisomeriz-able-FAD cofactor 26a was reconstituted into the apo-glucose oxidase (apo-GOx), to yield the photoisomerizable enzyme 27a (Scheme 9(B)). This reconstituted protein... 

The photoisomerizable enzyme monolayer electrode also revealed photoswitchable bioelectrocatalytic activity (Figure 7.10). In the presence of ferrocene carboxylic acid (5) as a diffusional electron transfer mediator, the nitrospiropyran-tethered GOx (4a) revealed a high bioelectrocatalytic activity, reflected by a high electrocatalytic anodic current. The protonated nitromerocyanine-GOx (4b) exhibited a two-fold lower activity, as reflected by the decreased bioelectrocatalytic current. By the reversible photoisomerization of the enzyme electrode between the 4a- and 4b-states, the current responses are cycled between high and low values (Figure 7.10, inset). 

Although the tethering of photoisomerizable units to the protein leads to photoswitchable bioelectrocatalytic properties, the OFF state of the photoisomerizable GOx exhibits residual bioelectrocatalytic activity as the structural distortion of the protein in the 4b-state is not optimized. 

FIG. 7.8 Electronic transduction of photoswitchable bioelectrocatalytic functions of redox>enzymes by the tethering of photolsomerizable units to the protein. (R is a diifuslonal electron mediator that electrically contacts the redox site of the protein with the electrode support.)... 

A photoswitchable bioelectrocatalytic device based on a similar azo-SAM with a PAA-g-CD coating was designed, able to catalyze the oxidation of glucose by glucose oxidase upon inclusion of ferrocene-methanol (Fc), as electron mediator, into the available free CD units of the PAA-g-CD film. Photoreversible activation and deactivation of the enzyme could be obtained by UV/Vis light irradiation. The immobilization and release of the redox polymer was driven by the trans-cis photoisomerization of the azobenzene units in the SAM. ... 

Fig. 31a). The native FAD cofactor was extracted from GOx and the semisynthetic FAD cofactor was reconstituted into the apo-GOx (apo-GOx) (Fig. 31b). This reconstituted enzyme includes a photoisomerizable unit directly attached to the redox center of the enzyme, and hence, the enzyme is predisposed for optimized photoswitchable bioelectrocatalytic properties. The photoisomerizable enzyme was assembled on an Au-electrode as described in Fig. 31(c). The bioelectrocatalytic oxidation of glucose was stimulated in the presence of ferrocene carboxylic acid as a diffusional electron-transfer mediator. The (28a)-state of the reconstituted GOx was inactive for the bioelectrocatalytic transformation, whereas photoisomerization of the enzyme to the (28b)-state activated the system (Fig. 32). By the cyclic photoisomerization of the enzyme mono-layer between (28a) and (28b) states, the bioelectrocatalyzed oxidation of glucose was cycled between the off and on states, respectively (Fig. 32, inset). It was also found that the direction of the photo-bioelectrocatalytic switch of the (28a/28b)-FAD-reconstituted GOx is controlled by the electrical properties of the diffusional electron-transfer mediator [385]. With ferrocene dicarboxylic acid as a diffusional electron-transfer mediator, the enzyme in the (28a)-state was found to correspond to the switched off biocatalyst, while the (28b)-state exhibits switched on behavior. In the presence of the protonated 1-[1-(dimethylamino)ethyl]ferrocene, the direction of the photobioelectrocatalytic switch is reversed. This control of the photoswitch direction of the photoisomerizable GOx was attributed to electrostatic interactions between the diffusional electron-transfer mediator and the photoisomerizable unit linked to the FAD. The (28b)-state attracts the oxidized negatively charged... 

Scheme 8 Assembly of a photoisomerizable glucose oxidase monolayer electrode and the reversible photoswitchable activa-tion/deactivation of the bioelectrocatalytic functions of the enzyme electrode.

Figure 3-30. Organization of a photoswitchable glucose oxidase electrode for the bioelectrocatalyzed oxidation of glucose (A) The synthesis of the photoisomerizable nitrospiropyran-FAD cofactor. (B) The reconstitution of apo-glucose oxidase, apo-GOx, with the photoisomerizable FAD-cofactor (20a). (C) The assembly of the reconstituted photoisomerizable GOx on an electrode surface and the photoswitching of the bioelectrocatalytic function of the enzyme electrode in the presence of ferrocene carboxylic acid (21) as mediator.

The bioelectrocatalyzed oxidation of glucose in this system originates from the primary oxidation of the ferrocene carboxylic acid, (21), to the respective ferrocenylium cation that mediates the oxidation of the enzyme s redox center and its activation towards the oxidation of glucose. Photoisomerization of the enzyme monolayer to the MRH-GO state switched-off the bioelectrocatalytic functions of the protein monolayer, and only the electrical response of the diffusional electron mediator was observed, Fig. 3-31, curves (b) and (d). By the cyclic photoisomerization of the enzyme-monolayer electrode between the SP-GOx and MRlT-GOx states, the reversible photoswitching of the enzyme activity between ON and OFF states was demonstrated, Fig. 3-31 (inset). 

Fig. 33 Coupling of the photoswitchable interactions between Cyt c and a (29a)-pyridine mixed monolayer with (a) the reduction of O2 by COx and (c) the oxidation of lactate by LDH (b) when the electrode is in the cationic merocyanine state (29b), repulsive interactions disallow the functioning of bioelectrocatalytic processes.

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