Direct electron transfer from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase

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Abstract

Direct electron transfer (DET) from bare spectrographic graphite (SPGE) or 3-mercaptopropionic acid-modified gold (MPA-gold) electrodes to Trachyderma tsunodae bilirubin oxidase (BOD) was studied under anaerobic and aerobic conditions by cyclic voltammetry and chronoamperometry. On cyclic voltammograms nonturnover Faradaic signals with midpoint potentials of about 700 mV and 400 mV were clearly observed corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively. The immobilized BOD was differently oriented on the two electrodes and its catalysis of O2-electroreduction was also massively different. On SPGE, where most of the enzyme was oriented with the T1 copper site proximal to the carbon with a quite slow ET process, well-pronounced DET-bioelectroreduction of O2 was observed, starting already at > 700 mV vs. NHE. In contrast, on MPA-gold most of the enzyme was oriented with its T2/T3 copper cluster proximal to the metal. Indeed, there was little DET-based catalysis of O2-electroreduction, even though the ET between the MPA-gold and the T2/T3 copper cluster of BOD was similar to that observed for the T1 site at SPGE. When BOD actively catalyzes the O2-electroreduction, the redox potential of its T1 site is 690 mV vs. NHE and that of one of its T2/T3 copper centers is 390 mV vs. NHE. The redox potential of the T2/T3 copper cluster of a resting form of BOD is suggested to be about 360 mV vs. NHE. These values, combined with the observed biocatalytic behavior, strongly suggest an uphill intra-molecular electron transfer from the T1 site to the T2/T3 cluster during the catalytic turnover of the enzyme.

Abbreviations

BOD
bilirubin oxidase
NI
native intermediate
PI
peroxy intermediate
FR
fully reduced BOD
RF
resting form
DET
direct electron transfer
ET
electron transfer
IET
intra-molecular electron transfer
CV
cyclic voltammogram
Em
midpoint redox potential
ET1, ET2, ET3, and ET2/T3
redox potentials of the T1, T2, T3 sites and the T2/T3 cluster, respectively
ΔEp
peak separation between anodic and cathodic peaks
k0
standard electron transfer rate constant
kDET
heterogeneous DET rate constant
Γ
surface concentration
jcat
biocatalytic current density
jcatmax
maximum biocatalytic current density
Δjcat(+ F−)
differences in biocatalytic current densities in the absence and presence of F
EDC
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
NHS
N-hydroxysuccinimide
AMTP
4-aminothiophenol
MHOL
6-mercapto-1-hexanol
MPA
3-mercaptopropionic acid
DT
1-decanethiol
SPGE
spectrographic graphite electrode
MPA-gold
3-mercaptopropionic acid-modified gold electrode

Keywords

Bilirubin oxidase
Bioelectrocatalysis
O2-electroreduction
Electron transfer kinetics

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