Peroxidase activity of de novo heme proteins immobilized on electrodes

Abstract

De novo proteins from designed combinatorial libraries were bound to heme terminated gold electrodes. The novel heme proteins were shown to possess peroxidase activity, and this activity was compared to that of horseradish peroxidase and bovine serum albumin when immobilized in a similar fashion. The various designed proteins from the libraries displayed distinctly different levels of peroxidase activity, thereby demonstrating that the sequence and structure of a designed protein can exert a substantial effect on the peroxidase activity of immobilized heme.

Fig. 1

Schematic representation of reconstitution of protein on heme modified electrodes. Initially the gold electrode was coated with mixed thiol monolayer consisting of a 1:5 ratio of longer alkyl-thiols bearing an activated functional group and short unsubstituted alkyl-thiols. Incubation was in DMSO for 90 min at room temperature. The coated gold electrode was then reacted with 20 mM 1,12 diaminododecane which, reacts only with the thiols bearing activated functional group. The resulting electrode has amine terminated groups which are covalently coupled to the carboxyl groups on Heme (Fe(III) protoporphyrin IX), using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling agent. Finally, the heme terminated electrode is reconstituted with the protein to form heme protein on surface.

Fig. 2

Cyclic voltammogram (CV) of the heme electrode reconstituted with protein. Note that the peaks of the heme modified electrode (solid dark line) decrease on treatment with protein S-824 (inner dashed line).

Fig. 3

The chronocoulometric response (charge versus time response to an applied potential step) obtained for the immobilized heme-HRP system with successive addition of hydrogen peroxide to the solution. The arrows point towards the charge value corresponding to each addition of peroxide.

Fig. 4

The net electrocatalytic reduction charge (after background subtraction of charge at 0 μM of peroxide) is plotted against the concentration of peroxide at heme modified electrodes coated with different proteins. The potential is stepped from 0 mV to −500 mV and the pulse width is 500 ms. The mediator used for the HRP and the de novo proteins is hydroxyquinone. Table 1 lists the net electrocatalytic charge (ΔQ) due to peroxidase activity of the proteins at 200 μM concentration of peroxide.

Fig. 5

Peroxidase activity of the de novo proteins in solution. Purified protein was used in the assay. The typical reaction mixture contained 50 μM purified protein, 4 μM heme chloride, 10 μM TMB (3,3,5,5′ tetramethyl benzidine), and 0.00075% H₂O₂. Absorbance at 450 nm was recorded and was normalized by dividing it with concentration of protein determined from absorbance at 280 using extinction coefficient of 6990 cm⁻¹.

Scheme 1

P and Q are the oxidized and reduced forms of a mediator, which is a redox cosubstrate that shuttles electrons from the electrode to the redox center in the protein.