Construction of a Novel Stable Electron Transfer Protein for the Creation of Artificial Direct Electron Transfer Enzymes
Authors: Inukai Niina , Takamura Eiichiro , Sakamoto Hiroaki , Satomura Takenori , Suye Shinichiro 
Published: December 2024

Abstract
In recent years, wearable sensors aiming to enable continuous measurement for disease prevention and diagnosis have been the focus of much research. Wearable sensors are often used inside the user's body or on biological surfaces. To reduce the burden on the user, these devices must be safe and compact. Lithium has been used as the conventional power source, but lithium batteries are not biocompatible and are not safe. Therefore, biofuel cells (BFCs) that use enzymes as electrodes are expected as a power source. In conventional BFCs, mediated electron transfer (MET) -type enzymes, in which a low molecular weight redox substance (mediator) mediates the electron transfer between the electrode and the enzyme, have been the mainstream. However, the low long-term stability and toxicity of the mediator used has been a challenge. Direct electron transfer (DET) -type enzymes, which do not require a mediator for electron transfer between the electrode and enzyme, are therefore attracting attention. However, there are few reports on DET-type enzymes, and research is being conducted to convert existing MET-type enzymes to DET-type enzymes. Most of the currently reported artificial DET-type enzymes have heme proteins as electron transfer subunits. However, heme proteins have low long-term stability. In the present study, we focused on the electron transfer domain of multicopper oxidase (McoP) from hyperthermophilic archaeon, Pyrobaculum aerophilum, which has excellent long-term stability. McoP has four copper atoms, which can be divided into three types according to their spectroscopic and magnetic properties. Type 1 (T1) Cu makes the enzyme blue and shows strong optical absorption at a wavelength of 610 nm due to d-d transition. Type 2 (T2) Cu is colorless and can be detected by electro paramagnetic resonance spectroscopy (EPR). The T3 type is distinguished by an optical absorption band at a wavelength of 330 nm. T1Cu has a copper atom bonded to two histidine residues and one cysteine, forming a distorted triangular pyramidal structure. T1Cu in McoP also has one methionine axially present, which affects the redox potential of the enzyme and stabilizes it. T1Cu and all ligands are present in domain 3. Therefore, we hypothesized that domain 3 alone could function as an electron-transfer subunit with excellent long-term stability.
Experiment
Plasmid vector for expression of domain 3 of McoP was prepared from McoP expression vector using In-Fusion clonig. Subsequently, the protein was expressed in E. coli BL21-Codon Plus (DE3) RIPL. The purified protein was then evaluated and confirmed. Spectroscopic observation showed a peak around 600 nm derived from T1Cu as well as McoP. Cyclic voltammetry (CV) was performed for electrochemical evaluation of domain 3. The prified protein (1 mg/ml) was modified onto a gold electrode by physisorption. Domain 3 modified electrode was used as working electrode. CVs were performed in a three-electrode system using a platinum counter electrode and an Ag/AgCl reference electrode. The CV perfprmed under O2 or N2 saturated condition. The domain 3 modified electrode showed redox peaks. These results indicate that domain 3 can be expressed and functions as an electron transfer subunit. In the future, the present protein will be used as an electron transfar subunit to convert MET-type enzymes to DET-type enzymes.

Figure 1