Reconstitution of apo-glucose oxidase

Scheme 12 Surface reconstitution of apo-glucose oxidase on a mixed monolayer associated with an electrode consisting of an FAD cofactor and photoisomerizable nitrospiropyran units, and reversible photoswitching of the bioelectrocatalytic functions of the enzyme electrode.

Willner I, Blonder R, Katz E. Reconstitution of apo-glucose oxidase with a nitrospiro-pyran-modified FAD cofactor yields a photoswitchable biocatalysts for amperometric transduction of recorded optical signals. J Am Chem Soc 1996 118 5310-11. 

Figure 19. (A) The reconstitution of apo-glucose oxidase on a PQQ-FAD monolayer assembled on an Au electrode. (B) Cyclic voltammograms of the PQQ-FAD-reconstituted glucose oxidase on an Au electrode (a) in the absence of glucose (b) with glucose, 80 mM. Recorded in 0.1 M phosphate buffer, pH 7.0, under Ar, at 35°C potential scan rate, 5 mV s. Inset calibration curve corresponding to the amperometric responses (measured by chronoamperometry, = 0.2 V vs. SCE) of the PQQ-FAD-reconstituted glucose oxidase enzyme electrode at different concentrations of glucose.

Blonder, R., Willner, I., and Buckmann, A. F. Reconstitution of apo-glucose oxidase on nitrospiropyran and FAD mixed monolayers on gold electrodes Photostimulation of bioelectrocatalytic features of the biocatalyst. /. Am. Chem. Soc. 1998, 120, 9335-9341. 

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.

Figure 3-32. (A) The assembly of an electro switchable, electrically-contacted, glucose oxidase electrode by the reconstitution of apo-glucose oxidase, apo-GOx, on PQQ-FAD units linked to a polyacrylic acid fihn that includes Cu -ion attached to the fihn. The conductivity of the film and the electrical contacting of the enzyme is accomplished by applying a potential of -0.5 V vs. SCE and the generation of Cu-clusters in the film. Reproduced with permission from ref. 89. Copyright 2003 American Chemical Society.

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... 

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... 

Figure 3-10. The assembly of an Au-nanoparticle (1.4 nm) electrically contacted glucose oxidase electrode by (a) the primary reconstitution of apo-GOx on the FAD-functionalized Au-nanoparticle, and the immobilization of the enzyme-nanoparticle hybrid on an electrode surface (b) the primary immobilization of the FAD-modified Au-nanoparticle on tire electrode surface and the surface reconstitution of apo-GOx on the functionalized electrode. Reproduced with permission from ref. 41. Copyright 2003, AAAS.

The reconstitution method was suggested as a means to introduce the photoisomerizable unit into the vicinity of the biocatalytic redox-center, thereby generating a light-switchable bioelectrocatalyst that operates between fiilly switched ON and OFF states. Apo-glucose oxidase, apo-GOx, was reconstituted with the nitrospiropyran-FAD cofactor unit, (20), Fig. 3-30. 

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