Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone

Abstract

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b₅ (cyt b₅). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (V_max) was 1.64 ± 0.08 min^-1, and the Michaelis constant (K_M) was 78 ± 9 μM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b₅. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion.

Keywords: 6-hydroxychlorzoxazone; Bactosomes; CYP2E1; chlorzoxazone; screen-printed electrodes; voltammetry.

Scheme 1

Reaction converting chlorzoxazone into 6-hydroxychlorzoxazone, catalyzed by CYP2E1.

Figure 1

Cyclic voltammograms of SPEs modified with DDAB (▬) and immobilized Bactosomes (▬) registered in 100 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl saturated with argon (anaerobic conditions) (a) and cyclic voltammogram obtained after subtracting the cyclic voltammogram of DDAB-modified SPE from the cyclic voltammogram of DDAB-modified SPE with immobilized Bactosomes (b). The voltammograms were registered at a 50 mV/s scan rate.

Figure 2

Differential pulse voltammograms of SPEs modified with DDAB (cathodic curve (▬) and anodic curve (▬)) and with immobilized Bactosomes (cathodic curve (▬) and anodic curve (▬)) registered in 100 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl saturated with argon (anaerobic conditions). Modulation amplitude 20 mV, step potential 5 mV, interval time 500 ms, modulation time 50 ms.

Figure 3

Cyclic voltammograms of SPEs modified with DDAB (▬) and immobilized Bactosomes (▬) registered in 100 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl (aerobic conditions). The voltammograms were registered at a 50 mV/s scan rate.

Figure 4

Cyclic voltammograms of SPEs registered in a 100 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl (▬) and 50 μM chlorzoxazone (▬) or 50 μM 6-hydroxychlorzoxazone (▬), or a mixture of 50 μM chlorzoxazone and 50 μM 6-hydroxychlorzoxazone (▬). The voltammograms were registered at a 100 mV/s scan rate.

Figure 5

Cyclic voltammograms of SPEs registered in 100 mM potassium phosphate supporting solution, containing 50 mM NaCl, 1% ethanol (v/v), and 50 μM 6-hydroxychlorzoxazone, in the pH range of 4.21–10.78 (a). The dependence of 6-hydroxychlorzoxazone oxidation peak potential on pH (b). The values are the means from three experiments ± standard deviations. The voltammograms were registered at a 100 mV/s scan rate.

Scheme 2

The proposed mechanism of electrochemical oxidation for 6-hydroxychlorzoxazone.

Figure 6

Square-wave voltammograms of SPEs registered in 100 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl and different concentrations (0–1 μM) of 6-hydroxychlorzoxazone (a); and dependence of the oxidation peak current on concentrations of 6-hydroxychlorzoxazone (b). Mean values ± standard deviations from 3–5 independent experiments are presented. Frequency 25 Hz, amplitude 40 mV, step potential 5 mV.

Figure 7

Square-wave voltammograms of SPEs registered before (▬) or after the electrocatalytic reaction in 100 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl and 500 μM chlorzoxazone, for 60 min at a fixed potential of −0.55 V of a working electrode, modified with DDAB (▬) and with immobilized Bactosomes (▬). Frequency 25 Hz, amplitude 40 mV, step potential 5 mV.

Figure 8

The dependence of the initial rate of 6-hydroxychlorzoxazone formation by Bactosomes immobilized on SPEs on chlorzoxazone concentration. Mean values ± standard deviations from at least five independent experiments are presented.

Figure 9

The reduction potentials of the components of Bactosomes, recombinant CYP2E1, cyt b₅, and CPR, and the potentials applied to the working electrodes with immobilized Bactosomes during the electrocatalytic reactions towards chlorzoxazone (for the CPR/CYP2E1/cyt b₅-mediated electrocatalytic reaction and for the CYP2E1/cyt b₅-mediated electrocatalytic reaction).

Figure 10

Dependence of the initial reaction rate of 6-hydroxychlorzoxazone formation on the applied reduction potential for Bactosomes immobilized on the electrode. The electrocatalytic reaction was carried out in 100 mM potassium phosphate buffer, pH 7.4, containing 50 mM NaCl, 280 units/mL catalase, and 150 μM chlorzoxazone. Mean values ± standard deviations from 3–5 independent experiments are presented.