5.2 Enzyme Trimming
97
It is worthy of note that an FDH variant lacking 199 amino acid residues, including
the heme 1c and 2c moieties, (1c2c_FDH) showed clear DET-type catalytic activity
and its electron transfer occurred at a more negative potential than that observed
for r_FDH and 1c_FDH (Fig. 5.2A right and C) [32]. In the case of this mutant
(1c2c_FDH), the electron during the DET-type bioelectrocatalysis is transferred
from heme 3c to the electrode, and the interfacial electron transfer kinetics was
drastically improved, most likely as a consequence of the shortening of the distance
between the electrode surface and heme 3c resulting from downsizing of the enzyme
(Fig. 5.2A). However, due to the mentioned downsizing of the enzyme, the catalytic
activity and the limiting catalytic current density of 1c2c_FDH decreased compared
to r_FDH (Fig. 5.2B) [32].
5.3 Site-Directed Mutation
Site-direct mutantion of selected amino acids in the active site or in close proximity
to redox-active canters is a successful concept and has been well explored to improve
the electrical communication between an electrodes and biocatalysts.
An example of site-direct mutations affecting DET-type bioelectrocatalysis is
shown in multi-coper oxidase (MCO), a family of redox protein for 4-electron reduction of O 2 . The active site of MCOs contains 4 Cu atoms which are divided into three
types based on their spectroscopic and magnetic properties: type I (T1), type II (T2),
and type III (T3) coppers. The T1 Cu oxidizes electron donators and transfers the
electron to the trinuclear center composed of one T2 Cu and two T3 Cu atoms where
O 2 is reduced to H 2 O. In a DET-type bioelectrocatalytic reaction, the T1 site accepts
electrons from electrodes. DET-type bioelectrocatalytic property of several mutations of CueO, namely M510L, M510Q, and D439A was examined [33]. M510L
and M510Q were prepared by replacing the Met510, the axial ligand of the T1 site
of CueO, with Leu and with Gln, respectively. D439A was prepared by replacing
the Asp439 with Ala, Asp439 forming a hydrogen bond with His443 coordinating to
the T1 Cu. It has been found that M510L and D439A exhibit a positive shift, while
M510Q exhibited a negative shift, in the onset potential of the DET-type bioelectrocatalytic waves, compare to the wild-type CueO. The result is accordance with a
report related to BOD, in which substitution of the Met coordinated to the T1 Cu at the
axial position with Gln decreased the redox potential of T1 from 0.46 to 0.23 V [34].
In addition to single-site mutation, Zhang et al. recently constructed a series
of CueO mutants by direct evolution method and found that a double substitution mutant, D439T/L502K, significantly increased the onset potential up to 0.54 V
versus Ag|AgCl [35] (Fig. 5.3). The positions, D439 and L502, are located in the
second coordination spheres of the T1 Cu and form hydrogen bonds with coordinated
ligands, H443 and C500, respectively. The substitutions are assumed to have altered
the donating ability of His443 and C500 to the T1 Cu. Combination the double
substitution CueO mutant-based biocathode with a GDH-based bioanode realized a
97
It is worthy of note that an FDH variant lacking 199 amino acid residues, including
the heme 1c and 2c moieties, (1c2c_FDH) showed clear DET-type catalytic activity
and its electron transfer occurred at a more negative potential than that observed
for r_FDH and 1c_FDH (Fig. 5.2A right and C) [32]. In the case of this mutant
(1c2c_FDH), the electron during the DET-type bioelectrocatalysis is transferred
from heme 3c to the electrode, and the interfacial electron transfer kinetics was
drastically improved, most likely as a consequence of the shortening of the distance
between the electrode surface and heme 3c resulting from downsizing of the enzyme
(Fig. 5.2A). However, due to the mentioned downsizing of the enzyme, the catalytic
activity and the limiting catalytic current density of 1c2c_FDH decreased compared
to r_FDH (Fig. 5.2B) [32].
5.3 Site-Directed Mutation
Site-direct mutantion of selected amino acids in the active site or in close proximity
to redox-active canters is a successful concept and has been well explored to improve
the electrical communication between an electrodes and biocatalysts.
An example of site-direct mutations affecting DET-type bioelectrocatalysis is
shown in multi-coper oxidase (MCO), a family of redox protein for 4-electron reduction of O 2 . The active site of MCOs contains 4 Cu atoms which are divided into three
types based on their spectroscopic and magnetic properties: type I (T1), type II (T2),
and type III (T3) coppers. The T1 Cu oxidizes electron donators and transfers the
electron to the trinuclear center composed of one T2 Cu and two T3 Cu atoms where
O 2 is reduced to H 2 O. In a DET-type bioelectrocatalytic reaction, the T1 site accepts
electrons from electrodes. DET-type bioelectrocatalytic property of several mutations of CueO, namely M510L, M510Q, and D439A was examined [33]. M510L
and M510Q were prepared by replacing the Met510, the axial ligand of the T1 site
of CueO, with Leu and with Gln, respectively. D439A was prepared by replacing
the Asp439 with Ala, Asp439 forming a hydrogen bond with His443 coordinating to
the T1 Cu. It has been found that M510L and D439A exhibit a positive shift, while
M510Q exhibited a negative shift, in the onset potential of the DET-type bioelectrocatalytic waves, compare to the wild-type CueO. The result is accordance with a
report related to BOD, in which substitution of the Met coordinated to the T1 Cu at the
axial position with Gln decreased the redox potential of T1 from 0.46 to 0.23 V [34].
In addition to single-site mutation, Zhang et al. recently constructed a series
of CueO mutants by direct evolution method and found that a double substitution mutant, D439T/L502K, significantly increased the onset potential up to 0.54 V
versus Ag|AgCl [35] (Fig. 5.3). The positions, D439 and L502, are located in the
second coordination spheres of the T1 Cu and form hydrogen bonds with coordinated
ligands, H443 and C500, respectively. The substitutions are assumed to have altered
the donating ability of His443 and C500 to the T1 Cu. Combination the double
substitution CueO mutant-based biocathode with a GDH-based bioanode realized a